AU725767B2 - Method for protecting plants - Google Patents

Description

WO71 nO ni pCTfFlP97IO77.il 8/53 7o METHOD FOR PROTECTING

PLANTS

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.

Systemic acquired resistance (SAR) is one component of the complex system plants use to defend themselves from pathogens (Hunt and Ryals, Crit Rev. in Plant Sci. 15, 583- 606 (1996), incorporated by reference herein in its entirety; Ryals et al., Plant Cell 8, 1809- 1819 (1996), incorporated by reference herein in its entirety). See also, U.S. Patent No.

5,614,395, incorporated by reference herein in its entirety. SAR is a particularly important aspect of plant-pathogen responses because it is a pathogen-inducible, systemic resistance against a broad spectrum of infectious agents, including viruses, bacteria, and fungi. When the SAR signal transduction pathway is blocked, plants become more susceptible to pathogens that normally cause disease, and they also become susceptible to some infectious agents that would not normally cause disease (Gaffney et al., Science 261, 754- 756 (1993), incorporated by reference herein in its entirety; Delaney et al., Science 266, 1247-1250 (1994), incorporated by reference herein in its entirety; Delaney et al., Proc.

Natl. Acad. Sci. USA 92, 6602-6606 (1995), incorporated by reference herein in its entirety; Delaney, Plant Phys. 113, 5-12 (1997), incorporated by reference herein in its entirety; Bi et al., Plant J. 8, 235-245 (1995), incorporated by reference herein in its entirety; Mauch-Mani and Slusarenko, Plant Cell8, 203-212 (1996), incorporated by reference herein in its entirety). These observations indicate that the SAR signal transduction pathway is critical for maintaining plant health.

Conceptually, the SAR response can be divided into two phases. In the initiation phase, a pathogen infection is recognized, and a signal is released that travels through the WO 98/29537 PCT/EP97/07253 -2phloem 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). Although it has been suggested that SA might serve as the systemic signal, this is currently controversial and, to date, all that is known for certain is that if SA cannot accumulate, then SAR signal transduction is blocked (Pallas et al., 1996; Shulaev et al., Plant Cell7, 1691-1701 (1995), incorporated by reference herein in its entirety; Vernooij et al., Plant Cell 6, 959-965 (1994), incorporated by reference herein in its entirety).

Recently, Arabidopsis has emerged as a model system to study SAR (Uknes et al., Plant Cell 4, 645-656 (1992), incorporated by reference herein in its entirety; Uknes et al., Mol. Plant-Microbe Interact. 6, 692-698 (1993), incorporated by reference herein in its entirety; Cameron et al., Plant J. 5, 715-725 (1994), incorporated by reference herein in its entirety; Mauch-Mani and Slusarenko, Mol. Plant-Microbe Interact. 7, 378-383 (1994), incorporated by reference herein in its entirety; Dempsey and Klessig, Bulletin de L'lnstitut Pasteur93, 167-186 (1995), incorporated by reference herein in its entirety). It has been demonstrated that 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. 8, 228-234 (1995), incorporated by reference herein in its entirety; Lawton et al., Plant J. 71-82 (1996), incorporated by reference herein in its entirety). Following treatment with either INA or BTH or pathogen infection, at least three pathogenesis-related (PR) protein genes, namely, PR-1, PR-2, and PR-5 are coordinately induced concomitant with the onset of resistance (Uknes et al., 1992, 1993). In tobacco, the best characterized species, treatment with a pathogen or an immunization compound induces the expression of at least WO 98/29537 PCT/EP97/07253 -3nine sets of genes (Ward et al., Plant Cell3, 1085-1094 (1991), incorporated by reference herein in its entirety). Transgenic disease-resistant plants have been created by transforming plants with various SAR genes Patent No. 5,614,395).

A number of Arabidopsis mutants have been isolated that have modified SAR signal transduction (Delaney, 1997) The first of these mutants are the so-called Isd (lesions simulating disease) mutants and acd2 (accelerated cell death) (Dietrich et al., Cell77, 565- 577 (1994), incorporated by reference herein in its entirety; Greenberg et al., Cell77, 551- 563 (1994), incorporated by reference herein in its entirety). These mutants all have some degree of spontaneous necrotic lesion formation on their leaves, elevated levels of SA, mRNA accumulation for the SAR genes, and significantly enhanced disease resistance. At least seven different Isd mutants have been isolated and characterized (Dietrich et al., 1994; Weymann et al., Plant Cell7, 2013-2022 (1995), incorporated by reference herein in its entirety). Another interesting class of mutants are cim (constitutive immunity) mutants (Lawton et al., "The molecular biology of systemic aquired resistance" in Mechanisms of Defence Responses in Plants, B. Fritig and M. Legrand, eds (Dordrecht, The Netherlands: Kluwer Academic Publishers), pp. 422-432 (1993), incorporated by reference herein in its entirety). See also, International PCT Application WO 94/16077, which is incorporated by reference herein in their entireties. Like Isd mutants and acd2, cim mutants have elevated SA and SAR gene expression and resistance, but in contrast to Isd or acd2, do not display detectable lesions on their leaves. cprl (constitutive expresser of PR genes) may be a type of cim mutant; however, because the presence of microscopic lesions on the leaves of cprl has not been ruled out, cprl might be a type of Isd mutant (Bowling et al., Plant Cell6, 1845-1857 (1994), incorporated by reference herein in its entirety).

Mutants have also been isolated that are blocked in SAR signaling. ndrl (non-racespecific disease resistance) is a mutant that allows growth of both Pseudomonas syringae containing various avirulence genes and also normally avirulent isolates of Peronospora parasitica (Century et al., Proc. Natl. Acad.Sci. USA 92, 6597-6601 (1995), incorporated by reference herein in its entirety). Apparently this mutant is blocked early in SAR signaling.

nprl (nonexpresser of PR genes) is a mutant that cannot induce expression of the SAR signaling pathway following INA treatment (Cao et al., Plant Cell 6, 1583-1592 (1994), incorporated by reference herein in its entirety). eds (enhanced disease susceptibility) mutants have been isolated based on their ability to support bacterial infection following inoculation of a low bacterial concentration (Glazebrook et al., Genetics 143, 973-982 V WO 98/29537 PCT/EP97/07253 (1996), incorporated by reference herein in its entirety; Parker et al., Plant Cell8, 2033- 2046 (1996), incorporated by reference herein in its entirety). Certain eds mutants are phenotypically very similar to nprl, and, recently, eds5 and eds53 have been shown to be allelic to nprl (Glazebrook et al., 1996). nim1 (noninducible immunity) is a mutant that supports P. parasitica causal agent of downy mildew disease) growth following INA treatment (Delaney et al., 1995; W094/16077). Although 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. nim 1 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).

Recently, two allelic Arabidopsis genes have been isolated and characterized, mutants of which are responsible for the nim and nprl phenotypes, respectively (Ryals et al., Plant Cell 9, 425-439 (1997), incorporated by reference herein in its entirety; Cao et al., Cell 88, 57-63 (1997), incorporated by reference herein in its entirety). The wild-type NIM1 gene product is involved in the signal transduction cascade leading to both SAR and gene-forgene disease resistance in Arabidopsis (Ryals et al., 1997). Ryals et al., 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-xB/IKB signal transduction pathways have been implicated in disease resistance responses in a range of organisms from Drosophila to mammals. In mammals, NF-iB/IKB signal transduction can be induced by a number of different stimuli including exposure of cells to lipopolysaccharide, tumor necrosis factor, interleukin 1 or virus infection (Baeuerle and Baltimore, Cell87, 13-20 (1996); Baldwin, Annu. Rev. Immunol. 14, 649-681 (1996)). The activated pathway leads to the synthesis of a number of factors involved in inflammation and immune responses, such as IL-2, IL-6, IL-8 and granulocyte/macrophagecolony stimulating factor (deMartin et al., Gene 152, 253-255 (1995)). In transgenic mouse studies, the knock-out of NF-iB/IKB signal transduction leads to a defective immune response including enhanced susceptibility to bacterial and viral pathogens (Beg and 11O\ OQI/"0911 PCTi Baltimore, Science 274, 782-784 (1996); Van Antwerp et al., Science 274, 787-789 (1996); Wang et al., Science 274, 784-787 (1996); Baeuerle and Baltimore (1996)). In Arabidopsis, SAR is functionally analogous to inflammation in that normal resistance processes are potentiated following SAR activation leading to enhanced disease resistance (Bi et al., 1995; Cao et al., 1994; Delaney et al., 1995; Delaney et al., 1994; Gaffney et al., 1993; Mauch-Mani and Slusarenko 1996; Delaney, 1997). Furthermore, inactivation of the pathway leads to enhanced susceptibility to bacterial, viral and fungal pathogens.

Interestingly, SA has been reported to block NF-KB activation in mammalian cells (Kopp and Ghosh, Science 265, 956-959 (1994)), while SA activates signal transduction in Arabidopsis. Bacterial infection of Drosophila activates a signal transduction cascade leading to the synthesis of a number of antifungal proteins such as cercropin B, defensin, diptericin and drosomycin (Ip et al., Ce1l75, 753-763 (1993); Lemaitre et al., Cell86, 973- 983 (1996)). This induction is dependent on the gene product of dorsal and dif, two NF-KB homologs, and is repressed by cactus, an IxB homolog, in the fly. Mutants that have decreased synthesis of the antifungal and antibacterial proteins have dramatically lowered resistance to infection.

Despite much research and the use of sophisticated and intensive crop protection measures, including genetic transformation of plants, losses due to disease remain in the billions of dollars annually. Therefore, there is a continuing need to develop new crop protection measures based on the ever-increasing understanding of the genetic basis for disease resistance in plants.

In view of the above, 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.

V WO 98/29537 PCT/EP97/07253 -6- 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. Similarly, 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. However, by concurrently applying a microbicide to an immunomodulated plant, the disease resistance is unexpectedly synergistically enhanced; the level of disease resistance is greater than the expected additive levels of disease resistance.

Accordingly, the present invention concems 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. Similarly, 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. However, by concurrently applying a microbicide to an immunomodulated plant, the control of pathogenic disease is unexpectedly synergistically enhanced; the level of disease control is greater than the expected additive levels of disease resistance.

In addition to greater 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. Furthermore, 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 r.njrcanauLouJ,,.,, WCC.OC-JsG UIIuU -7combined modes of action of pathogen control are completely different from one another, the threat of resistance developing is effectively prevented.

Thus the present invention relates to a method for protecting a plant from pathogen attack through synergistic disease resistance, comprising the steps of: obtaining an immunomodulated plant having a first level of disease resistance by applying to a plant INA or SA; or by selecting a plant based on constitutive expression of SAR genes and/or a disease-resistant phenotype; or by genetically engineering a plant by transforming it with one or more SAR genes; and applying to said immunomodulated plant at least one microbicide that confers a second level of disease resistance; whereby application of said microbicide to said immunomodulated plant confers a synergistically enhanced third level of disease resistance that is greater than the sum of the first and second levels of disease resistance.

Preferred is a method according to the invention, wherein said immunomodulated plant is a constitutive immunity (cim) mutant plant.

In particularly preferred is a method according to the invention, wherein said cim mutant plant is selected from a population of plants according to the following steps: evaluating the expression of SAR genes in uninfected plants that are phenotypically normal in that said uninfected plants lack a lesion mimic phenotype; and selecting uninfected plants that constitutively express SAR genes in theabsence of viral, bacterial, or fungal infection.

Also preferred is a method according to the invention, wherein said immunomodulated plant is a lesion mimic mutant plant.

In particularly preferred is a method according to the invention, wherein said lesion mimic mutant plant is selected from a population of plants according to the following steps: evaluating the expression of SAR genes in uninfected plants that have a lesion mimic phenotype; and selecting uninfected plants that constitutively express SAR genes in the absence of viral, bacterial, or fungal infection.

Also preferred is a method according to the invention, wherein said immunomodulated S plant is obtained by recombinant expression in a plant of an SAR gene.

In particularly preferred is a method according to the invention, wherein said SAR gene sdfunctional form of a NIM1 gene.

WO OQ/0917 PCITIEP97/n72j3 .wm^i~y i- -8- 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.

Especially preferred is a method according to the invention, wherein said NIM1 protein comprises the amino acid sequence set forth in SEQ ID NO:2.

Especially preferred is a method according to the invention, wherein said NIM1 gene hybridizes under the following conditions to the coding sequence set forth in SEQ ID NO:1: hybridization in 1%BSA; 520mM NaPO4, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55 0 C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 550C.

Especially preferred is a method according to the invention, wherein said NIM1 gene comprises the coding sequence set forth in SEQ ID NO:1 and all DNA molecules hybridizing therewith using moderate stringent conditions.

In particularly preferred is a method according to the invention, wherein said SAR gene encodes an altered form of a NIM1 protein that acts as a dominant-negative regulator of the SAR signal transduction pathway.

More preferred is method according to the invention, wherein said altered form of the NIM1 protein has alanines instead of serines in amino acid positions corresponding to positions 55 and 59 of SEQ ID NO:2.

Especially preferred is a method according to the invention, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO:8.

Especially preferred is a method according to the invention, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:7 and all DNA molecules hybridizing therewith using moderate stringent conditions.

Especially preferred is 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 min. (X3) 3XSSC for 15 min. (X1) at 550C.

More preferred is a method according to the invention, wherein 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.

Especially preferred is a method according to the invention wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID WO 98/29537 PCT/EP97/07253 -9- Especially preferred is a method according to the invention wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:9 and all DNA molecules hybridizing therewith using moderate stringent conditions.

Especially preferred is 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 min. (X3) 3XSSC for 15 min. (X1) at 550C.

Especially preferred is a method according to the invention, wherein said altered form of the NIM1 protein has a C-terminal truncation of amino acids corresponding approximately to amino acid positions 522-593 of SEQ ID NO:2.

Especially preferred is a method according to the invention, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO:12.

Especially preferred is a method according to the invention, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:11 and all DNA molecules hybridizing therewith using moderate stringent conditions.

Especially preferred is 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 0 C for 18-24h, and wash in 6XSSC for min. (X3) 3XSSC for 15 min. (X1) at 550C.

More preferred is a method according to the invention, wherein 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.

Especially preferred is a method according to the invention, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO:14.

Especially preferred is a method according to the invention, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:13 and all DNA molecules hybridizing therewith using moderate stringent conditions.

Especially preferred is 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 WO 98/29537 PCT/EP97/07253 salt; 1mM EDTA; 250 mM sodium chloride at 55 0 C for 18-24h, and wash in 6XSSC for min. (X3) 3XSSC for 15 min. (X1) at 55 0

C.

More preferred is a method according to the invention, wherein said altered form of the NIM1 protein consists essentially of ankyrin motifs corresponding approximately to amino acid positions 103-362 of SEQ ID NO:2.

Especially preferred is a method according to the invention, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO:16.

Especially preferred is a method according to the invention, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:15 and all DNA molecules hybridizing therewith using moderate stringent conditions.

Especially preferred is 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 0 C for 18-24h, and wash in 6XSSC for min. (X3) 3XSSC for 15 min. (X1) at 55 0

C.

Examples of 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 cane, tea, vines, hops, bananas and natural rubber plants, as well as omamentals (flowers, shrubs, broad-leaved trees and evergreens, such as conifers). This list does not represent any limitation.

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 WO 98/29537 PCT/EP97/07253 -11 Hemileia, Rhizoctonia, and Puccinia; Fungi imperfecti such as the genera Botrytis, Helminthosporium, Rhynchosporium, Fusarium F. monoliforme), Septoria, Cercospora, Altemaria, Pyricularia, and Pseudocercosporella P. herpotrichoides); Oomycete fungi such as from the genera Phytophthora P. parasitica), Peronospora P. tabacina), Bremia, Pythium, and Plasmopara; as well as other fungi such as Scleropthora macrospora, Sclerophthora rayissiae, Scierospora graminicola, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora sacchari 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 stewartit, insects such as aphids, e.g. Myzus persicae; and lepidoptera such as Heliothus spp.; and nematodes such as Meloidogyne incognita.

Obtaining Immunomodulated Plants All three of the following general routes for obtaining immunomodulated plants are related in that they all fit into the SAR signal transduction pathway model set forth in Ryals et al., (1996). Upon activating the SAR signal transduction pathway to achieve disease resistance, the same set of SAR genes is turned "on" and disease resistance results, regardless of which of the three below-described routes is taken. The differences among these three routes pertain only to which point in the pathway SAR is turned on; the end result is same among these three routes. Therefore, analyses and results observed with regard to immunomodulated plants attained through one route may be extrapolated and applied to immunomodulated plants attained through a different route.

A. Application of a Chemical Inducer of Systemic Acquired Resistance 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).

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 WO 98/29537 PCT/EP97/07253 -12- Joumal10(1), 61-70 (1996); Lawton et al., Plant Journal10(1), 71-82 (1996); and Gorlach et al., Plant Cell 8, 629-643 (1996), each of which is incorporated 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 2,6dichloroisonicotinic acid (INA) and the lower alkyl esters thereof, as well as salicylic acid compounds Examples of suitable INA and SA compounds are described in U.S.

Patent No. 5,614,395.

B. Breeding Constitutive Immunity (CIM) Mutant Plants 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 incorporated by reference. In Arabidopsis, 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.

To identify and select plants that constitutively express SAR genes, Northem analysis is performed to detect expression of SAR genes. Known SAR DNA sequences can be utilized in cross-hybridization experiments as described in Uknes et al. (1992). Methods for the hybridization and cloning of nucleic acid sequences are well known in the art. (See, for example, Molecular Cloning, A Laboratory Manual, 2nd Edition, Vol. 1-3, Sambrook et al. (eds.) Cold Spring Harbor Laboratory Press (1989) and the references cited therein). At least two classes of SAR signal transduction mutants that constitutively express SAR genes have been isolated. One class has been designated as "Isd' mutants (Isd lesion simulating disease), which are also referred to as "cim Class I" mutants. See WO 94/16077. sd(ci(m 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). A second class has been designated as "cinf' (cim constitutive immunity) mutants, which are also referred to as "cim Class II" mutants. See, WO 94/16077. cim mutants have all the characteristics of Isdmutants except spontaneous lesions.

That is, cim mutants are visibly phenotypically normal.

Once plants that constitutively express SAR genes are selected, they can be utilized in breeding programs to incorporate constitutive expression of the SAR genes and resistance to WO 98/29537 PCTIEP97/07253 -13pathogens 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 Isdmutants display lesion formation and necrosis, cim mutants with their normal phenotypes are preferable for use in such breeding programs and in the method of the present invention, although Isdmutants could be used if desired.

C. Transforming Plants with SAR Genes A third route for obtaining immunomodulated plants is by transforming plants with an SAR gene, preferably a functional form of the NIM1 gene.

1. Recombinant Expression of the Wild-Type NIM1 Gene Recombinant overexpression of the 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, incorporated 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. Preferably, 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.

The section below entitled "Recombinant DNA Technology" sets forth protocols that may be used to recombinantly express the wild-type NIM1 gene in transgenic plants at higher-thanwild-type levels. Alternately, plants can be transformed with the wild-type NPR1 gene to produce disease resistant plants as described in Cao, et al. (1997).

2. Recombinant Expression of an Altered Form of the NIM1 Gene 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-KB/IxB 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 IKB subclass a. See, co-pending PCT application "METHODS OF USING THE NIM1 GENE TO CONFER DISEASE RESISTANCE IN PLANTS" incorporated herein by reference.

WO 98/29537 PCT/EP97/07253 -14- 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-KB and IKB (Michaely and Bennett, Trends Cell Biol. 2, 127-129 (1992)). Pairwise visual inspections between the NIM1 protein (SEQ ID NO:2) and 70 known ankyrincontaining proteins were carried out, and striking similarities were found to members of the IKcBa class of transcription regulators (Baeuerle and Baltimore 1996; Baldwin 1996). As shown in Figure 1, the NIM1 protein (SEQ ID NO:2) shares significant homology with licBa proteins from mouse, rat, and pig (SEQ ID NOs: 3, 4, and 5, respectively). NIM1 contains several important structural domains of licBa throughout the entire length of the protein, including ankyrin domains (indicated by the dashed underscoring in Figure 2 aminoterminal serines (amino acids 55 and 59 of NIM1) a pair of lysines (amino acids 99 and 100 in NIM1) and an acidic C-terminus. Overall, NIM1 and IKBa share identity at 30% of the residues and conservative replacements at 50% of the residues. Thus, there is homology between IKBa and NIM1 throughout the proteins, with an overall similarity of One way in which IcBac protein functions in signal transduction is by binding to the cytosolic transcription factor NF-KB and preventing it from entering the nucleus and altering transcription of target genes (Baeuerle and Baltimore, 1996; Baldwin, 1996). The target genes of NF-KB regulate (activate or inhibit) several cellular processes, including antiviral, antimicrobial and cell death responses (Baeuerle and Baltimore, 1996). When the signal transduction pathway is activated, IKBa is phosphorylated at two serine residues (amino acids 32 and 36 of Mouse IKBa). This programs ubiquitination at a double lysine (amino acids 21 and 22 of Mouse IKBa). Following ubiquitination, the NF-KB/IB complex is routed through the proteosome where IKBa is degraded and NF-B is released to the nucleus.

The phosphorylated serine residues important in IkBa function are conserved in NIM1 within a large contiguous block of conserved sequence from amino acids 35 to 84 (Figure In contrast to IKBa, 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 IhBa in the serine/threonine rich carboxy terminal region which has been shown to be important in basal turnover rate (Sun et al., Mol. Cell. Biol. 16, 1058-1065 (1996)).

According to the present invention based on the analysis of structural homology and the WO 98/29537 PCT/EP9/072N3 presence of elements known to be important for lcBa function, NIM1 is expected to function like the ixBa, having analogous effects on plant gene regulation.

Plants containing the wild-type NIM1 gene when treated with inducer chemicals are predicted to have more NIM1 gene product (IKB homolog) or less phosphorylation of the NIM1 gene product (IKB homolog). In accordance with this model, 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. In the niml mutant plants, a non-functional NIM1 gene product is present. Therefore, in accordance with this model, the NF-KB homolog is free to go to the nucleus and repress resistance and SAR gene expression.

Consistent with this idea, animal cells treated with salicylic acid show increased stability/abundance of IKB and a reduction of active NF-B in the nucleus (Kopp and Ghosh, 1994). Mutations of IKB are known that act as super-repressors or dominant-negatives (Britta-Mareen Traenckner et al., EMBO 14: 2876-2883 (1995); Brown et al., Science 267: 1485-1488 (1996); Brockman et al., Molecular and Cellular Biology15: 2809-2818 (1995); Wang et al., Science 274: 784-787 (1996)). These mutant forms of I1B bind to NF-KB but are not phosphorylated or ubiquitinated and therefore are not degraded. NF-KB remains bound to the IKB and cannot move into the nucleus.

In view of the above, altered forms of NIM1 that act as dominant-negative regulators of the SAR signal transduction pathway can be created. 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. The section below entitled "Recombinant DNA Technology" sets forth protocols that may be used to recombinantly express the altered forms of the NIM1 gene in transgenic plants at higher-than-wild-type levels. Below are described several altered forms of the NIM1 gene that act as dominant-negative regulators of the SAR signal transduction pathway.

WO 98/29537 PCT/EP97/07253 -16a. Changes of Serine Residues 55 and 59 to Alanine Residues: Phosphorylation of serine residues in human IxBa is required for stimulus activated degradation of IBa thereby activating NF-KB. Mutagenesis of the serine residues (S32 and S36) in human IBa to alanine residues inhibits stimulus-induced phosphorylation, thus blocking IKBa proteosome-mediated degradation (Traenckner et al, 1995; Brown et a., 1996; Brockman et al., 1995; Wang et aL, 1996). This altered form of liBa can function as a dominant-negative form by retaining NF-KB in the cytoplasm thereby blocking downstream signaling events. Based on the amino acid sequence comparison between NIM1 and IB shown in Figure 1, serines 55 (S55) and 59 (S59) in NIM1 (SEQ ID NO:2) are homologous to S32 and S36 in human IxBa. To construct dominant-negative forms of NIM1, the serines at amino acid positions 55 and 59 are mutagenized to alanine residues. Thus, in a preferred embodiment, 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).

b. N-terminal Deletion: Deletion of amino acids 1-36 (Brockman et al., 1995; Sun et al., 1996) or 1-72 (Sun et al., 1996) of human IkBa, which includes ubiquination lysine residues K21 and K22 as well as phosphorylation sites S32 and S36, results in a dominant-negative IkBa phenotype in transfected human cell cultures. An N-terminal deletion of the first 125 amino acids of the NIM1 gene product will remove eight lysine residues that could serve as ubiquination sites as well as the putative phosphorylation sites at S55 and S59 discussed above. Thus, in a preferred embodiment, the NIM1 gene is altered so that the encoded product is missing approximately the first 125 amino acids compared to the native Arabidopsis NIM1 amino acid sequence (SEQ ID NO:2).

c. C-Terminal Deletion: Deletion of amino acids 261-317 of human IkBa may result in enhanced intrinsic stability by blocking constitutive phosphorylation of serine and threonine residues in the Cterminus. This altered form of liBa is expected to function as a dominant-negative form. A region rich in serine and threonine is present at amino acids 522-593 in the C-terminus of NIM1. Thus, in a preferred embodiment, the NIM1 gene is altered so that the encoded WO 98/29537 PCT/EP97/07253 -17product is missing approximately its C-terminal portion, including amino acides 522-593, compared to the native Arabidopsis NIM1 amino acid sequence (SEQ ID NO:2).

d. N-terminaVC-terminal Deletion Chimera and Ankyrin Domains Altered forms of the NIM1 gene product may also be produced as a result of Cterminal and N-terminal segment deletions or chimeras. In yet another embodiment of the present invention, constructs comprising the ankyrin domains from the NIM1 gene are provided.

3. Recombinant Expression of Other SAR Genes 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.

Recombinant DNA Technology 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 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 Xgtll, XgtlO 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 WO 98129537 PCT/EP97/07253 18system 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.

A. Construction of Plant Expression Cassettes 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 futher 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.

1. 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, 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.

a. Constitutive Expression, the CaMV 355 Promoter: Construction of the plasmid pCGN1761 is described in the published patent application EP 0 392 225 (Example 23), which is hereby incorporated by reference.

pCGN1761 contains the "double" CaMV 35S promoter and the tmltranscriptional terminator with a unique EcoRI site between the promoter and the terminator and has a pUC-type WO 98/29537 PCT/EP97/07253 -19backbone. A derivative of pCGN1761 is constructed which has a modified polylinker which includes Not and Xholsites in addition to the existing EcoRi site. This derivative is designated pCGN1761ENX. pCGN1761ENX is useful for the cloning of cDNA sequences or gene sequences (including microbial ORF sequences) within its polylinker for the purpose of their expression under the control of the 35S promoter in transgenic plants. The entire 35S promoter-gene sequence-tml terminator cassette of such a construction can be excised by Hindlll, Sphl, Sail, and Xbal sites 5' to the promoter and Xbal, BamHland Bgl! sites 3' to the terminator for transfer to transformation vectors such as those described below. Furthermore, 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, Notlor Xhol) for replacement with another promoter. If desired, 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. For example, pCGN1761ENX may be modified by optimization of the translational initiation site as described in Example 37 of U.S. Patent No. 5,639,949, incorporated herein by reference.

b. Expression under a Chemically/Pathogen Regulatable Promoter: The double 35S promoter in pCGN1761ENX may be replaced with any other promoter of choice that will result in suitably high expression levels. By way of example, 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 PRla promoter is cleaved from plasmid pCIB1004 (for construction, see example 21 of EP 0 332 104, which is hereby incorporated by reference) and transferred to plasmid pCGN1761ENX (Uknes et al., 1992). pCIB1004 is cleaved with Ncoland the resultant 3' overhang of the linearized fragment is rendered blunt by treatment with T4 DNA polymerase. The fragment is then cleaved with HindIll and the resultant PR-la promotercontaining fragment is gel purified and cloned into pCGN1761ENX from which the double promoter has been removed. This is done by cleavage with Xholand blunting with T4 polymerase, followed by cleavage with Hindlll and isolation of the larger vector-terminator WO 98/29537 PCT/EP97/07253 containing fragment into which the pCIB1004 promoter fragment is cloned. This generates a pCGN1761ENX derivative with the PR-la promoter and the tml terminator and an intervening polylinker with unique EcoRI and Notl sites. The selected coding sequence can be inserted into this vector, and the fusion products promoter-gene-terminator) can subsequently be transferred to any selected transformation vector, including those described infra. 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.

c. Constitutive Expression, the Actin Promoter: Several isoforms of actin are known to be expressed in most cell types and consequently the actin promoter is a good choice for a constitutive promoter. In particular, the promoter from the rice Acti 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. Furthermore, 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)). These incorporate the Actl-intron 1, Adhl 5' flanking sequence and Adhi-intron 1 (from the maize alcohol dehydrogenase gene) and sequence from the CaMV 35S promoter. Vectors showing highest expression were fusions of 35S and Actl intron or the Actl 5' flanking sequence and the Actl intron. Optimization of sequences around the initiating ATG (of the GUS reporter gene) also enhanced expression. The promoter expression cassettes described by McElroy et al. (Mol. Gen. Genet. 231: 150-160 (1991)) can be easily modified for gene expression and are particularly suitable for use in monocotyledonous hosts. For example, promotercontaining 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. In a separate report, the rice Act/ 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)).

WO 98/29537 PCT/EP97/07253 -21 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 sunflower Binet et al. Plant Science 79: 87-94 (1991) and maize Christensen et al. Plant Molec.

Biol. 12: 619-632 (1989)). The maize ubiquitin promoter has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926 (to Lubrizol) which is herein incorporated by reference. Taylor et al. (Plant Cell Rep. 12: 491-495 (1993)) describe 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.

e. Root Specific Expression: Another pattern of gene expression is root expression. A suitable root promoter is described by de Framond (FEBS 290:103-106 (1991)) and also in the published patent application EP 0 452 269, which is herein incorporated 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.

f. Wound-Inducible Promoters: Wound-inducible promoters may also be suitable for gene expression. Numerous such promoters have been described Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1:151-158 (1989), Rohrmeier Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), Warner et al. Plant J. 3: 191-201 (1993)) and all are suitable for use with the instant invention. Logemann et al.

describe the 5' upstream sequences of the dicotyledonous potato wuni gene. Xu et al.

show that a wound-inducible promoter from the dicotyledon potato (pin2) is active in the monocotyledon rice. Further, Rohrmeier Lehle describe the cloning of the maize Wipl cDNA which is wound induced and which can be used to isolate the cognate promoter using WO 98/29537 PCT/EP97/07253 -22standard techniques. Similar, Firek et al. and Warner et al. have described a woundinduced gene from the monocotyledon Asparagus officinalis, which is expressed at local wound and pathogen invasion sites. Using cloning techniques well known in the art, these promoters can be transferred to suitable vectors, fused to the genes pertaining to this invention, and used to express these genes at the sites of plant wounding.

g. Pith-Preferred Expression: Patent Application WO 93/07278, which is herein incorporated by reference, describes the isolation of the maize trpA gene, which is preferentially expressed in pith cells. The gene sequence and promoter extending up to -1726 bp from the start of transcription are presented. Using standard molecular biological techniques, this promoter, or parts thereof, can be transferred to a vector such as pCGN1761 where it can replace the promoter and be used to drive the expression of a foreign gene in a pith-preferred manner. In fact, fragments containing the pith-preferred promoter or parts thereof can be transferred to any vector and modified for utility in transgenic plants.

h. Leaf-Specific Expression: A maize gene encoding phosphoenol carboxylase (PEPC) 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.

2. Transcriptional Terminators A variety of 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 tmlterminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons.

3. Sequences for the Enhancement or Regulation of Expression Numerous 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.

WO 98/29537 PCT/EP97/07253 -23 Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, 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 Genes Develop. 1:1183-1200 (1987)). In the same experimental system, 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.

A number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells.

Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the "W-sequence"), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression Gallie et al. Nucl. Acids Res. 15: 8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990)).

4. Targeting of the Gene Product Within the Cell Various mechanisms for targeting gene products are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterized in some detail. For example, the targeting of gene products to the chloroplast is controlled by a signal sequence found at the amino terminal end of various proteins which is cleaved during chloroplast import to yield the mature protein Comai et a. J. Biol. Chem. 263: 15104-15109 (1988)). These signal sequences can be fused to heterologous gene products to effect the import of heterologous products into the chloroplast (van den Broeck, et al. Nature 313: 358-363 (1985)). 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.

Other gene products are localized to other organelles such as the mitochondrion and the peroxisome Unger et a. Plant Molec. Biol. 13: 411-418 (1989)). The 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 WO 98/29537 PCT/EP97/07253 -24- 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)).

In addition, sequences have been characterized which cause the targeting of gene products to other cell compartments. Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler Ho, Plant Cell 2:769-783 (1990)). Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).

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. For chloroplast targeting, for example, the chloroplast signal sequence from the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused in frame to the amino terminal ATG of the transgene. The signal sequence selected should include the known cleavage site, and the fusion constructed should take into account any amino acids after the cleavage site which are required for cleavage. In some cases this requirement may be fulfilled by the addition of a small number of amino acids between the cleavage site and the transgene ATG or, alternatively, replacement of some amino acids within the transgene sequence. Fusions constructed for chloroplast import can be tested for efficacy of chloroplast uptake by in vitro translation of in vitro transcribed constructions followed by in vitro chloroplast uptake using techniques described by Bartlett et al. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier pp 1081-1091 (1982) and Wasmann 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.

B. Construction of Plant Transformation Vectors Numerous transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts, and the genes pertinent to this WO 98/29537 PCT/EP97/07253 invention can be used in conjunction with any such vectors. The selection of vector will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptll gene, which confers resistance to kanamycin and related antibiotics (Messing Vierra.

Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bargene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res 18:1062 (1990), Spencer et al. Theor. Appl. Genet 79: 625-631 (1990)), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger Diggelmann, Mol Cell Biol 4: 2929- 2931), and the dhfrgene, which confers resistance to methatrexate (Bourouis et al., EMBO J. 1099-1104 (1983)), and the EPSPS gene, which confers resistance to glyphosate Patent Nos. 4,940,935 and 5,188,642).

1. Vectors Suitable for Agrobacterium Transformation Many vectors are available for transformation using Agrobacterium tumefaciens.

These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) and pXYZ. Below, the construction of two typical vectors suitable for Agrobacterium transformation is described.

a. pCIB200 and pCIB2001: 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. 164: 446-455 (1985)) allowing excision of the tetracycline-resistance gene, followed by insertion of an Acclfragment from pUC4K carrying an NPTII (Messing Vierra, Gene 19: 259-268 (1982): Bevan et al., Nature 304: 184-187 (1983): McBride et al., Plant Molecular Biology 14: 266-276 (1990)). Xhollinkers are ligated to the EcoRVfragment 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 Xholdigested fragment are cloned into Sall-digested pTJS75kan to create pCIB200 (see also EP 0 332 104, example 19). pCIB200 contains the following unique polylinker restriction sites: EcoRI, Sstl, Kpnl, Bglll, Xbal, and Sail. pCIB2001 is a derivative of pCIB200 created by the insertion into the polylinker of additional restriction sites. Unique restriction sites in the WO 98/29537 PCT/EP97/07253 -26polylinker of pCIB2001 are EcoRI, Sstl, Kpnl, Bglll, Xbal, Sail, Mlul, Bcll, 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-mediated transformation, the RK2-derived trfA function for mobilization between E. col and other hosts, and the OriTand OriVfunctions also from RK2. The pCIB2001 polylinker is suitable for the cloning of plant expressibn cassettes containing their own regulatory signals.

b. pCIB10 and Hygromycin Selection Derivatives thereof: The binary vector pCIB10 contains a gene encoding kanamycin resistance for selection in plants and T-DNA right and left border sequences and incorporates sequences from the wide host-range plasmid pRK252 allowing it to replicate in both E. co/land Agrobacterium. Its construction is described by Rothstein et al. (Gene 53:153-161 (1987)).

Various derivatives of pCIB10 are constructed which incorporate the gene for hygromycin B phosphotransferase described by Gritz et al. (Gene 25: 179-188 (1983)). These derivatives enable selection of transgenic plant cells on hygromycin only (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717).

2. Vectors Suitable for non-Agrobacterium Transformation Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Below, the construction of typical vectors suitable for non-Agrobacterium transformation is described.

a. pCIB3064: pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques in combination with selection by the herbicide basta (or phosphinothricin). The plasmid pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. coi 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 WO 98/29537 PCT/EP97/07253 -27 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 Sad, 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 Smalfragment containing the bargene from Streptomyces viridochromogenes is excised and inserted into the Hpal site of pCIB3060 (Thompson et al. EMBO J 6: 2519-2523 (1987)). This generated pCIB3064, which comprises the bargene under the control of the CaMV 35S promoter and terminator for herbicide selection, a gene for ampicillin resistance (for selection in E. coll) 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.

b. pSOG19 and is a transformation vector that utilizes the E. coligene dihydrofolate reductase (DFR) as a selectable marker conferring resistance to methotrexate. 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. colidihydrofolate 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 carry the pUC gene for ampicillin resistance and have Hindll, Sphl, Pstl and EcoRI sites available for the cloning of foreign substances.

C. Transformation 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. For example, Ti plasmid vectors have been utilized for the delivery of WO 98/29537 PCT/EP97/07253 -28 foreign DNA, as well as direct DNA uptake, liposomes, electroporation, micro-injection, and microprojectiles. In addition, 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.

1. Transformation of Dicotyledons Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium. Non- Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described by Paszkowski et al., EMBO J 3: 2717-2722 (1984), Potrykus et al., Mol. Gen.

Genet. 199:169-177 (1985), Reich et 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-mediated transformation is a preferred technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species. Agrobacterium transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest pCIB200 or pCIB2001) to an appropriate Agrobacterium strain which may depend of the complement of virgenes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally strain CIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5:159-169 (1993)). The transfer of the recombinant binary vector to Agrobacterium is accomplished by a triparental mating procedure using E. coli carrying the recombinant binary vector, a helper E. coi strain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Hafgen 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.

WO 98/29537 PCVIEP97/072 3 -29- 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 incorporation within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the desired gene. Alternatively, 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 dried yeast cells, dried bacterium or a bacteriophage, each containing DNA sought to be introduced) can also be propelled into plant cell tissue.

2. Transformation of Monocotyledons 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 cotransformation) and both these techniques are suitable for use with this invention. Cotransformation 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. However, a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al. Biotechnology 4:1093-1096 (1986)).

Patent Applications EP 0 292 435, EP 0 392 225, 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)) and Fromm et al. (Biotechnology 8: 833-839 (1990)) have published techniques for transformation of A188-derived maize line using particle bombardment. Furthermore, WO 93/07278 and Koziel et al. (Biotechnology 11: 194-200 (1993)) describe techniques for the transformation of elite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after pollination and a PDS-1000He Biolistics device for bombardment.

WO 98/29537 PCT/EP97/07253 Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment. Protoplast-mediated transformation has been described for Japonica-types and Indica-types (Zhang et al. Plant Cell Rep Z: 379-384 (1988); Shimamoto t al. Nature 338: 274-277 (1989); Datta et al. Biotechnology 8: 736-740 (1990)). Both types are also routinely transformable using particle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)).

Patent Application EP 0 332 581 describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Furthermore, wheat transformation has been described by Vasil et al. (Biotechnology 10: 667-674 (1992)) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al. (Biotechnology 11: 1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102: 1077-1084 (1993)) using particle bombardment of immature embryos and immature embryo-derived callus. A preferred technique for wheat transformation, however, involves the transformation of wheat by particle bombardment of immature embryos and includes either a high sucrose or a high maltose step prior to gene delivery. Prior to bombardment, 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/I 2,4-D for induction of somatic embryos, which is allowed to proceed in the dark. On the chosen day of bombardment, 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 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.

Approximately one month later 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/I methotrexate in the case of pSOG35). After approximately one month, developed shoots WO 98/29537 PCT/EP97/07253 -31are transferred to larger sterile containers known as 'GA7s" which contain half-strength MS, 2% sucrose, and the same concentration of selection agent.

More recently, tranformation of monocotyledons using Agrobacterium has been described. See, WO 94/00977 and U.S. Patent No. 5,591,616, both of which are incorporated herein by reference.

Breeding 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 agronomically 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 incorporated into plant lines through breeding. Breeding approaches and techniques are known in the art. See, for example, Welsh J. Fundamentals of Plant Genetics and Breeding, John Wiley Sons, NY (1981); Crop Breeding, Wood D. R. American Society of Agronomy Madison, Wisconsin (1983); Mayo The Theory of Plant Breeding, Second Edition, Clarendon Press, Oxford (1987); Singh, Breeding for Resistance to Diseases and Insect Pests, Springer-Verlag, NY (1986); and Wricke and Weber, Quantitative Genetics and Selection Plant Breeding, Walter de Gruyter and Co., Berlin (1986).

The genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in descendants plants. Generally said maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting. Specialized processes such as hydroponics or greenhouse technologies can also be applied. As the growing crop is vulnerable to attack and damages caused by insects or infections as well as to competition by weed plants, measures are undertaken to control weeds, plant diseases, insects, nematodes, and other adverse conditions to improve yield. These include mechanical measures such a tillage of the soil or removal of weeds and infected plants, as well as the application of agrochemicals such as herbicides, fungicides, gametocides, nematicides, growth regulants, ripening agents and insecticides.

WO 98/29537 PCT/EP97/07253 -32- Use of the advantageous genetic properties of the transgenic plants and seeds according to the invention can further be made in plant breeding, which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering. The various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate descendants plants. Depending on the desired properties, different breeding measures are taken. The relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc.

Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical, or biochemical means. Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines. Thus, 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. Alternatively new crops with improved stress tolerance can be obtained, which, due to their optimized genetic "equipment', yield harvested product of better quality than products that were not able to tolerate comparable adverse developmental conditions.

In seeds production, germination quality and uniformity of seeds are essential product characteristics, whereas germination quality and uniformity of seeds harvested and sold by the farmer is not important. As it is difficult to keep a crop free from other crop and weed seeds, to control seedborne diseases, and to produce seed with good germination, fairly extensive and well-defined seed production practices have been developed by seed producers, who are experienced in the, art of growing, conditioning and marketing of pure seed. Thus, it is common practice for the farmer to buy certified seed meeting specific quality standards instead of using seed harvested from his own crop. Propagation material to be used as seeds is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures thereof.

Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD"), metalaxyl (Apron"), and pirimiphos-methyl (Actellic*). if desired, these compounds are formulated together with further carriers, surfactants or application- WO 98/29537 PCT/EP97/07253 -33 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.

It is a further aspect of the present invention to provide new agricultural methods, such as the methods examplified above, which are characterized by the use of transgenic plants, transgenic plant material, or transgenic seed according to the present invention.

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. Examples of a suitable packaging material include paper, such as kraft paper, rigid or pliable plastic or other polymeric material, glass or metal. Desirably the bag, container, or vessel is comprised of a plurality of layers of packaging materials, of the same or differing type. In one embodiment the bag, container or vessel is provided so as to exclude or limit water and moisture from contacting the seed. in one example, the bag, container or vessel is sealed, for example heat sealed, to prevent water or moisture from entering. In another embodiment water absorbent materials are placed between or adjacent to packaging material layers. In yet another embodiment 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 NIM1 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.

Application Of A Microbicide To Immunomodulated Plants As described herein, 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 synergistically enhanced disease resistance.

WO 98/29537 WO 9829537PCT/EP97/07253 A. Conventional Microbicides According to the method of the present invention, any commercial or conventional microbicide may be applied to immunomodulated plants obtained through any of the three above-described routes. Examples of suitable microbicides include, but are not limited to, the following fungicides: 4-[3-(4-chlorophenyl)-3-(3,4-dimethoxyphenyl)acrytoyl]morpholine ("'dimethomorphm), (reference: C. Tomlin (Editor): The Pesticide Manual, 10Oth edition, Famhan, UK, 1994, pages 351-352); 5-methyl-i ,2,4-triazolo[3,4-b]l ,3]benzothiazole (otricyclazolew), (reference: C. Tomlin (Editor): The Pesticide Manual, 10th edition, Famham, UK, 1994, pages 1017-1 01 3-allyloxy-1 ,2-benzothiazole-11i-dioxide ("probonazole'), (reference: C. Tomlin (Editor): The Pesticide Manual, 10Oth edition, Farnham, UK, 1994, pages 8314832); n-2-(4chlorophenyl)ethyl]-a-(, ,1 -dimethylethyl)-1 H-i ,2,4-tiiazole-1 -ethanol, (tebuconazole"), (reference: EP-A-40 345);1 -[[3-(2-chlorophenyl)-2-(4-fluorophenyl)oxiran-2-yl]methyl]-1 H-i ,2,4triazole, (epoxyconazole*), (reference: EP-A-196 038); p-(4-chlorophenyl)-p- (i -cyclopropylethyl)-1 H-i,2,4-tiiazole-1 -ethanol, ("cyproconazole*), (reference: US-4 664 696); 5-(4-chlorobenzyl)-2,2-dimethy-1 -(1H-i ,2,4-triazol-1 -ymethyl)-cyclopentanol, (ometconazolem), (reference: EP-A-267 778); 2-(2,4-dichlorophenyl)-3-(l H-i ,2,4-trazol-1 -yl)-propyl-1 1 ,2,2tetrafluoroethyl-ether, (utetlraconazolen), (reference: EP-A-234 242); methyl-(E)-2-(2-[6-(2cyanophenoxy)pynmidin--4-yloxy~phenyl}-3-methoxyacrylate, ("ICI A 5504", mazoxystrobinm), (reference: EP-A-382 375); methyl-(E)-2-methoximino-2-[a--(o-tolyloxy)--o-tolyl]acetate, ("BAS 490 nkresoxime methyl"), (reference: EP-A-400 417); 2-(2-phenoxyphenyl)-(E)-2methoximino-N-methylacetamide, (reference: EP-A-398 692); [2-(2,5-dimethylphenoxymethyl)phenyl]-(E)--2-methoximino-N-methylacetamide, (reference: EP-A-398 692); (1 R,3S11 S,3R)-2,2dichloro-N-[(R)-1 -(4-chlorophenyl)ethyl-ethyl-3-methylcyclopropanecarboxamide, ("KTU 3616"), (reference: EP-A-341 475); manganese ethylenebis(dithiocarbamate)polymer-zinc complex, (mmancozebm), (reference: US 2 974 156); 1-[2-(2,4-dichlorophenyl)-4-propyl-1,3dioxolan-2-ylmethyl]-i H-i ,2,4-triazole, ("propiconazolem), (reference: GB-i 522657); 1 chloro-4-(4-chlorophenoxy)pheny]-4-methyl-1,3-dioxolan-2-ytmethy l)-1 H-i ,2,4-triazole, ("dilenoconazolem), (reference: GB-209860); 1 -[2-(2,4-dichlorophenyl)pentyl-1 H-I ,2,4-triazole, ("penconazole"), (reference: GB-i 589852); cis-4-[3-(4-tert-butylphenyl)--2-methylpropyl]-2,6dimethylmorpholine, ("fenpropimorphm), (reference: DE 2752135); 1-[3-(4-tert-butytphenyl)-2methylpropyl]-pipeddine, ("fenpropidin"), (reference: DE27521 35); 4-cyclopropyl-6-methyl4 jphenyl-2-pyrimidinamine ("cyprodinil") (reference: EP-A-31 0550); (RS)-N-(2,6-dimethylphenyt-N- WO 98/29537 PCT/EP97/07253 (methoxyacetyl)-alanine methyl ester ("metalaxyl"), (reference: GB-1500581); dimethylphenyl-N-(methoxyacetyl)-alanine methyl ester ('R-metalaxyl"), (reference: GB- 1500581); 1,2,5,6-tetrahydro-4H-pyrrolo[3,2,1-ij]quinolin-4-one ("pyroquilon"), (reference: GB- 1394373); ethyl hydrogen phosphonate ("fosetyl"), (reference: C. Tomlin (Editor): The Pesticide Manual, 10th edition, Famhan, UK, 1994, pages 530-532); and copper hydroxide (reference: C.

Tomlin (Editor): The Pesticide Manual, 10th edition, Famhan, UK, 1994, pages 229-230).

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 applicationpromoting 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 in rice culture) or if the microbicide is introduced in solid form into the soil, e.g. in the form of granules (soil application). In order to treat seed, 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. In addition, in special cases, 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 dilutable solutions, dilute emulsions, wettable powders, soluble powders, dusts, granules, or by encapsulation in e.g. polymer substances. As with the nature of the compositions, 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.iJha, preferably from 100 g to 1000 g a.iJha, especially from 150 g to 700 g a.iJha. In the case of the treatment of seed, 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.

WO 98/29537 PCT/EP97/072N3 -36 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 others 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-pyrrolidone, dimethyl sutfoxide or dimethylformamide, as well as vegetable oils or epoxidised vegetable oils, such as epoxidised coconut oil or soybean oil; or water.

The solid carriers used, e.g. for dusts and dispersible powders, are normally natural mineral fillers, such as calcite, talcum, kaolin, montmorillonite or attapulgite. In order to improve the physical properties it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. Suitable granulated adsorptive carriers are porous types, for example pumice, broken brick, sepiolite or bentonite, and suitable nonsorbent carriers are, for example, calcite or sand. In addition, a great number of pregranulated materials of inorganic or organic nature can be used, e.g. especially dolomite or pulverised plant residues.

Depending upon the nature of the microbicide, suitable surface-active compounds are non-ionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties. The term "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, phosphatidylserine, 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.

Whereas commercial products will preferably be formulated as concentrates, the end user will normally employ dilute formulations.

B. Plant Activating Microbicides If applied to immunomodulated plants obtained through the second or third abovedescribed route (selective breeding or genetic engineering), the microbicide may alternately 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 WO 98/29537 PCT/EP97/07253 -37 described in U.S. Patent Nos. 5,523,311 and 5,614,395. Hence, two methods of immunomodulation are concurrently employed. By applying plant activating microbicides to immunomodulated plants obtained through either a selective breeding route or a genetic engineering route, "extra-immunomodulation" results, and synergistically enhanced disease resistance is achieved.

As described below, transgenic immunomodulated plants overexpressing NIM1 responded much faster and to much lower doses of BTH, as shown by PR-1 gene expression and resistance to P. parasitica, than wild-type plants. See, Example 35 and the Northern blots in Figure 3. Synergistically enhanced disease resistance in NIM1overexpressors can be achieved with only 10pM 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. In addition, one can take advantage of the alteration of the time-course of SAR activation that occurs when SAR-inducing chemicals are applied to alreadyimmunomodulated plants such as NIMl-overexpressors. Furthermore, 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.

C. Conventional Microbicides In Conjunction With Plant Activating Microbicides For even greater disease resistance, both a conventional microbicide and a plant activating nicrobicide 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.

Disease Resistance Evaluation Disease resistance evaluation is performed by methods known in the art. See, Uknes et al, (1993) Molecular Plant Microbe Interactions 6: 680-685; Gorlach et al., (1996) Plant Cell 8:629-643; Alexander et al., Proc. Natl. Acad. Sci. USA 90:7327-7331 (1993). For example, several representative disease resistance assays are described below.

WO 98/29537 PCT/EP97/07253 -38 A. Phytophthora parasitica (Black shank) Resistance Assay 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 0 C night temperature. The wilt index used for the assay is as follows: 0=no symptoms; 1=no symptoms; 1=some sign of wilting, with reduced turgidity; 2=clear wilting symptoms, but no rotting or stunting; 3=clear wilting symptoms with stunting, but no apparent stem rot; 4=severe wilting, with visible stem rot and some damage to root system; for 4, but plants near death or dead, and with severe reduction of root system. All assays are scored blind on plants arrayed in a random design.

B. Pseudomonas syringae Resistance Assay Pseudomonas syringae pv. tabaci strain #551 is injected into the two lower leaves of several 6-7-week-old plants at a concentration of 106 or 3 x 106 per ml in H 2 0. Six individual plants are evaluated at each time point. Pseudomonas tabaciinfected plants are rated on a point disease severity scale, 5=100% dead tissue, 0=no symptoms. 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.

C. Cercospora nicotianae Resistance Assay A spore suspension of Cercospora nicotianae (ATCC #18366) (100,000-150,000 spores per ml) is sprayed to imminent run-off onto the surface of the leaves. The plants are maintained in 100% humidity for five days. Thereafter the plants are misted with water 5-10 times per day. Six individual plants are evaluated at each time point. Cercospora nicotianae is rated on a leaf area showing disease symptoms basis. A T-test (LSD) is conducted on the evaluations for each day and the groupings are indicated after the Mean disease rating value. Values followed by the same letter on that day of evaluation are not statistically significantly different.

WO 98/29537 PCT/EP97/07253 -39 D. Peronospora parasitica Resistance Assay 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 170 C in a growth chamber with a 14-hr day/10-hr night cycle. Plants are examined at 3-14 days, preferably 7-12 days, after inoculation for the presence of conidiophores. In addition, several plants from each treatment are randomly selected and stained with lactophenol-trypan blue (Keogh et al., Trans. Br. Mycol. Soc. 74: 329-333 (1980)) for microscopic examination.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 is a sequence alignment of the NIM1 protein sequence with IBa from mouse, rat, and pig. Vertical bars above the sequences indicate amino acid identity between NIM1 and the IhBa sequences (matrix score equals double dots above the sequences indicate a similarity score single dots above the sequences indicate a similarity score <0.5 but and a score <0.0 indicates no similarity and has no indicia above the sequences (see Examples). Locations of the mammalian IBa 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 IhBa proteins. The five ankyrin repeats in IKB0X 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 sequences every 10 amino acids.

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 NIMl-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. and "7C" are individual NIMI-overexpressing plant lines produced according to Example 21. "0 BTH" is water treatment; "10 BTH" is 10 pM BTH treatment; "100 WO 98/29537 PCT/EP97/07253 BTH" is 100 gpM BTH treatment. is day zero control samples; and are samples at days 1, 3, and BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING SEQ ID NO:1 is a 5655-bp genomic sequence comprising the coding region of the wild-type Arabidopsis thaliana NIM1 gene.

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 IKBa amino acid sequence from Figure 1.

SEQ ID NO:4 is the rat IxBa amino acid sequence from Figure 1.

SEQ ID NO:5 is the pig licBa 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 Cterminal 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 NIM1.

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.

WO 98/29537 PCT/EP9/07253 -41 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.

DEFINITIONS

The following definitions will assist in the understanding of the present invention: Plant Cell: the structural and physiological unit of plants, consisting of a protoplast and the cell wall. The term "plant cell" refers to any cell which is either part of or derived from a plant. Some examples of cells include differentiated cells that are part of a living plant; differentiated cells in culture; undifferentiated cells in culture; the cells of undifferentiated tissue such as callus or tumors; differentiated cells of seeds, embryos, propagules and pollen.

Plant Tissue: a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.

Protoplast: a plant cell without a cell wall.

Descendant Plant: a sexually or asexually derived future generation plant which includes, but is not limited to, progeny plants.

Transgenic Plant: a plant having stably incorporated recombinant DNA in its genome.

Recombinant DNA: Any DNA molecule formed by joining DNA segments from different sources and produced using recombinant DNA technology.

Recombinant DNA Technology: Technology which produces recombinant DNA in vitro and transfers the recombinant DNA into cells where it can be expressed or propagated (See, Concise Dictionary of Biomedicine and Molecular Biology, Ed. Juo, CRC Press, Boca Raton (1996)), for example, transfer of DNA into a protoplast(s) or cell(s) in various forms, including, for example, naked DNA in circular, linear or supercoiled forms, DNA contained in nucleosomes or chromosomes or nuclei or parts thereof, DNA complexed or associated with other molecules, DNA enclosed in W OR/Q5317 PrT/E.P07/7253 -42liposomes, spheroplasts, cells or protoplasts or 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 et al., WO 94/12650 and Treco et al., WO 95/31560 which can be applied to increasing peroxidase activity in a monocot. Specifically, regulatory regions (ex. promoters) can be introduced into the plant genome to increase the expression of the endogenous peroxidase.

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 Ro 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, 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 Polymorphism avrRpt2: avirulence gene Rpt2, isolated from Pseudomonas syringae WO 98/29537 FCUEP97/0725 -43- BAC: Bacterial Artificial Chromosome BTH: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester CIM: Constitutive IMmunity phenotype (SAR is constitutively activated) cim: constitutive immunity mutant plant cM: centimorgans cprl: constitutive expresser of PR genes mutant plant Col-O: Arabidopsis ecotype Columbia ECs: Enzyme combinations Emwa: Peronospora parasitica isolate compatible in the Ws-O ecotype of Arabidopsis EMS: ethyl methane sulfonate INA: 2,6-dichloroisonicotinic acid Ler: Arabidopsis ecotype Landsberg erecta Isd. lesions simulating disease mutant plant nahG: salicylate hydroxylase Pseudomonas putida that converts salicylic acid to catechol 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 Noco: Peronospora parasitica isolate compatible in the Col-O ecotype of Arabidopsis ORF: open reading frame PCs: Primer combinations PR: Pathogenesis Related SA: salicylic acid SAR: Systemic Acquired Resistance SAR-on: Immunomodulated plants in which SAR is activated, typically exhibiting greaterthan-wild-type SAR gene expression and having a disease resistant phenotype SSLP: Simple Sequence Length Polymorphism UDS: Universal Disease Susceptible phenotype Wela: Peronospora parasitica isolate compatible in the Weiningen ecotype of Arabidopsis WO 98/29537 PCT/EP97/07253 -44- Ws-O: Arabidopsis ecotype Issilewskija WT: wild type YAC: Yeast Artificial Chromosome

EXAMPLES

The invention is illustrated in further detail by the following detailed procedures, preparations, and examples. The examples are for illustration only, and are not to be construed as limiting the scope of the present invention.

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, et al., Molecular Cloning, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and by T.J. Silhavy, M.L.

Berman, and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and by Ausubel, F.M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-lnterscience (1987).

I. Synergistic Disease Resistance Effects Achieved By Coordinate Application To Plants Of A Chemical Inducer Of Systemic Acquired Resistance With A Conventional Microbicide In this set of examples, SAR was induced in plants by application of a chemical inducer of SAR such as a benzothiadiazole. In addition, 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.greater-than-additive, synergistic, disease resistance. This was determined as the synergy factor the ratio of observed effect to expected effect.

The expected effect for a given combination of active ingredients can be described by the so-called Colby formula and can be calculated as follows (Colby, "Calculating synergistic and antagonistic responses of herbicide combination". Weeds, Vol. 15, pages 20-22 (1967)): ppm milligrams of active ingredient per liter of spray mixture, X action caused by active ingredient I at a rate of application of p ppm of active ingredient, Y action caused by active ingredient II at a rate of application of q ppm active ingredient, WO 98/29537 PCT/EP97/07253 E expected effect of active ingredients I II at a rate of application of p q ppm of active ingredient (additive action).

Colby's formula reads E X Y- XxY.

100 Example 1: Action Against Erysiphe graminis On Barley Residual-protective action: Barley plants about 8 cm in height were sprayed to drip point with an aqueous spray mixture (max. 0.02 active ingredient) and were dusted 3 to 4 days later with conidia of the fungus. The infected plants were stood in a greenhouse at 22". Fungus infestation was generally evaluated 10 days after infection.

Systemic action: Barley plants about 8 cm in height were watered with an aqueous spray mixture (max. 0.002 active ingredient, based on the volume of the soil). Care was taken that the spray mixture did not come into contact with parts of the plants above the soil. The plants were dusted with conidia of the fungus 3 to 4 days later. The infected plants were stood in a greenhouse at 22". Fungus infestation was generally evaluated 10 days after infection.

Table 1 Action against Erysiphe graminis on barley component I: benzothiadiazole-7-carboxylic acid component II: metconazole Test no. mg a.i. per litre (ppm) I:11 action SF comp. I comp. II O (observed) E (expected) O/E 1 0.6 0 2 2 3 6 89 4 0.6 2 6 6 51 7 20 8 0.6 0.6 1:1 37 10 3.7 9 0.6 2 1:3 59 40 0.6 6 1:10 81 51 1.6 11 0.6 20 1:30 78 65 1.2 12 2 6 1:3 78 71 1.1 13 2 20 1:10 98 79 1.2 WO 98/29537 WO 9829537PCT/EP97/07253 -46- Table 2 Action against Erysiphe graminis on barley component 1: benzothiadiazole-7-carboxyic acid component 11: tetraconazole Test- no. mg ai. per litre (ppm) 1:11 action SF comp. I comp. 1I 0 (observed) E (expected) OlE 1 0.6 14 2 2 27 3 0.6 4 2 63 0.6 0.6 1:1 70 53 1.3 6 0.6 2 1: 2681.

7 2 0.6 3:1 79 601.

Table 3 Action against Etysiphe graminis on barley component 1: benzoll ,2,3]thiadiazole-7-carbothioic acid-S-methyl ester component II: metconazole Test no. mg a.i. per litre (ppm) 1:11 action SF comp. I comp. II 0 (observed) E (expected) O/E 1 0.6 0 2 2 33 3 6 17 4 20 33 60 6 0.6 6 1:10 33 17 1.9 7 0.6 20 1:30 50 33 8 0.6 60 1:100 83 50 1.7 WO 98/29537 PCT/EP97/07253 -47- Example 2: Action Against Colletotrichum lagenarium On Cucumis sativus L.

After a cultivation period of 10 to 14 days, 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 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 to 23"C. Evaluation of protective action was made 7 to days after infection and was based on fungus infestation.

After a cultivation period of 10 to 14 days, 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 5 spores/ml) of the fungus and incubated for 30 hours at high humidity and a temperature of 23 0 C. Incubation was then continued at normal humidity and 22 0 C. Evaluation of protective action was made 7 to 10 days after infection and was based on fungus infestation.

Table 4 Action Against Colletotrichum lagenarium On Cucumis sativus L. Foliar Application component I: benzothiadiazole-7-carboxylic acid component II: azoxystrobin Test no. mg a.i. per litre (ppm) 1:ll action SF comp. I comp. II O (observed) E (expected) O/E 1 0.06 0 2 0.2 3 2 22 4 0.06 0.2 9 6 0.6 12 7 6 17 8 0.06 0.06 1:1 16 5 3.2 9 2 0.2 10:1 65 29 2.2 2 0.6 3:1 49 31 1.6 11 2 6 1:3 44 35 1.3 WO 98129537 PCT/EP97/07253 48 Table Action Against Colletotrichum lagenanum On Cucumis sativus L. Soil Application component 1: benzothiadiazole-7-carboxylic acid component 11: azoxystrobin Test no. mg a.i. per litre (ppm) 1:11 action SF comp. I comp. 11 0 (observed) E (expected) OlE 1 0.00F 0 2 0.02 3 0.06 49 4 0.2 91 0.2 0 6 0.6 9 7 2 28 8 6 66 9 0.006 0.2 1:30 11 0 0.6 1:100 30 9 3.3 11 2 1:300 83 28 12 0.02 6 1:300 97 80 1.2 13 0.06 6 1:100 100 82 1.2 synergy factor SF cannot be calculated Table 6 Action Against Colletotrichum lagenarium On Cucumis sat ivus Foliar Application component 1: benzothiadiazole-7-carboxylic acid component 1I: kresoxime, methyl Test no. mg a.i. per litre (ppm) 1:11 action SF comp. I comp. 11 0 (observed) E (expected) O/E 1 0.2 3 2 0.6 51 3 2 0 4 20 41 0.2 2 1:10 15 3 6 0.2 20 1 :100 61 43 1.4 WO 98/29537 PCT/EP97/07253 -49 Table 7 Action Against Colletotrichum lagenanium On Cucumis sativus L. Foliar Application component 1: benzo[1 ,2,3]thiadiazole-7-carbothioic acid-S-methyl ester component 11: azoxystrobin Test no. mg a.i. per litre, (ppm) 1:11 action SF comp. I comp. 1I 0 (observed) E (expected) OlE 1 0.06 16 2 0.2 22 3 6 4 2 18 6 6 0.06 2 1:30 43 31 1.4 7 0.2 2 1:10 57 36 1.6 Table 8 Action Against Colletotrichum lagenarium On Cucumis sativus L. /Soil Application component 1: benzo[1 ,2,3]thiadiazole-7-carbothioic acid-S-methyl ester component 11: azoxystrobin Test no. mg a.i. per litre (ppm) 1:11 action SF comp. I comp. II 0 (observed) E (expected) OlE 1 0.006 0 2 0.02 6 3 0.06 23 4 0.2 36 0.02 1 6 0.06 7 0.6 27 8 2 61 9 6 93 0.006 0.006 0.006 0.02 0.6 2 1:3 1:100 1:300 44 84 WO 98/29537 PCT/EP97/07253 Example 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.

Table 9 Action Against Cercospora nicotianae On Tobacco Plants component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester component II: tebuconazole Test no. mg a.i. per litre (ppm) 1:11 action SF comp. I comp. II O (observed) E (expected) O/E 1 0.2 0 2 2 17 3 6 4 20 78 2 O 6 6 O 7 0.2 2 1:10 87 0 8 0.2 6 1:30 97 0 9 2 2 1:1 87 17 5.1 2 6 1:3 94 17 11 6 2 3:1 87 55 1.6 12 6 6 1:1 90 55 1.6 13 20 2 10:1 97 78 1.2 14 20 6 3:1 97 78 1.2 WO 98/29537 PCT/EP97/07253 -51- Table Action Against Cercospora nicotianae On Tobacco Plants component 1: benzo[1 ,2,3]thiadiazole-7-carbothioic acid-S-methyl ester component 11: cyproconazole Test no. mg a.i. per litre, (ppmn) 1:11 action SF comp. I comp. 11 0 (observed) E (expected) O/E 1 0.2 0 2 2 17 3 6 4 20 78 2 0 6 6 0 7 0.2 2 1:10 78 0 8 0.2 6 1:30 84 0 9 2 2 1:1 90 17 5.3 2 6 1:3 94 17 11 6 2 3:1 87 55 1.6 12 6 6 1:1 93 55 1.7 13 20 2 10:1 100 78 1.3 14 20 6 3:1 100 781.3 Table 11 Action Against Cercospora nicotianae On Tobacco Plants component 1: benzothiadiazole-7-carboxylic acid component 11: fenpropimorph Test no. kg of a.i. per ha 1:11 action SF comp. I comp. II 0 (observed) E (expected) OlE 0 0 (control) 1 0.2 0 2 0.6 3 3 2 69 4 6 79 WO 98/29537 PCT/EP97/07253 -52- 8 0.2 2 1:10 52 13 4 9 0.2 6 1:30 61 23 2.7 0.6 2 1:3 71 16 4.4 11 6 6 1:1 100 83 1.2 Table 12 Action Against Cercospora nicotianae On Tobacco Plants Component I: benzothiadiazole-7-carboxylic acid Component II: difenoconazole Test no. kg of a.i. per ha 1:1I action SF comp. I comp. II O (observed) E (expected) O/E 0 0 (control) 1 2 69 2 6 79 3 20 100 4 0.6 3 2 23 6 6 32 7 2 0.6 3:1 90 70 1.3 8 6 0.6 10:1 100 80 1.3 Example 4: Action Against Pyricularia oryzae On Rice Plants 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 0

C.

WO 98/29537 PCT/EP97/07253 -53- Table 13 Action Against Pyricularia oryzae On Rice Plants component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester component II: KTU 3616 Test no. mg a.i. per litre (ppm) I:11 action SF comp. I comp. II O (observed) E (expected) O/E 1 6 2 0.02 0 3 0.06 28 4 0.2 47 0.6 79 6 2 83 7 6 91 8 6 0.02 300:1 42 15 2.8 9 6 0.06 100:1 76 39 1.9 6 0.2 30:1 98 55 1.8 11 6 0.6 10:1 98 82 1.2 12 6 2 3:1 100 86 1.2 13 6 6 1:1 98 92 1.1 On a 12m 2 plot, rice plants were sprayed with a spray mixture prepared with a wettable powder of the active ingredient. Infection was naturally. For evaluation, the leaf area infested with the fungus was measured 44 days post-application. The following results were obtained: Table 14 Action Against Pyricularia oryzae On Rice Plants in the open Component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component II: pyroquilon Test no. kg of a.i. per ha 1:1I action SF comp. I comp. II O (observed) E (expected) O/E 0 (control) WO 98/29537 PCT/EP97/07253 -54- 1 0.25 22 2 0.5 3 0.75 46 4 1.5 82 0.25 0.75 1:3 80 58 1.4 6 0.5 0.75 1:1.5 85 73 1.2 Rice plants about 2 weeks old were placed together with the soil around the roots in a container filled with spray mixture. Fungus infestation was evaluated 36 days later. Infestation of the untreated plants corresponded to 0 action.

Table Action Against Pyricularia oryzae On Rice Plants Component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component II: tricyclazole Test No. mg a.i. per litre (ppm) 1:11 action SF comp. I comp. II 0 (observed) E (expected) O/E 1 0.5 2 0.25 39 3 0.1 18 4 0.05 1 74 6 0.5 71 7 0.25 48 8 0.1 32 9 0.25 0.25 1:1 75 68 1.1 0.1 0.25 1:2.5 69 57 1.2 11 0.1 0.1 1:1 61 44 1.4 12 0.05 1 1:20 80 75 1.1 13 0.05 0.25 1:5 58 50 1.2 WO 98/29537 PCT/EP97/07253 Example 5: Action Against Colletotrichum sp. (Anthracnose) and Cercospora sp. (Leaf Spot) On Chili Effects on crop yield: In a plot of land about 10 m 2 (test location: Cikampek, Java, Indonesia), chili plants were sprayed a total of 7 times at intervals of about 7 days with 500-700 litres of spray mixture per hectare. Three days after the first spraying, the plants were infected artificially with the fungus.

Table 16 Action Against Colletotrichum: Evaluation was made by assessing infestation on the chili fruits after the fifth spraying.

component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester component II: mancozeb Test no. mg a.i. per litre (ppm) 1:11 action SF comp. I comp. II O (observed) E (expected) O/E 1 5 2 100 12 3 5 100 1:20 77 59 1.3 Table 17 Action Against Cercospora: Evaluation was made by assessing infestation on the leaves after the sixth spraying.

Component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component II: mancozeb Test no. mg a.i. per litre (ppm) 1:1 action SF comp. I comp. II O (observed) E (expected) O/E 1 5 76 2 100 8 3 5 100 1:20 87 78 1.1 WO 98/29537 PCT/EP97/07253 -56- Table 18 Action On Crop Yield: The chilis were harvested after the sixth spraying.

Component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component II: mancozeb Test no. mg a.i. per litre (ppm) 1:1 Crop yield in kg per hectare SF comp. I comp. II 0 (observed) E (expected) O/E 1 5 459 2 100 8 3 5 100 1:20 1400 ca 460 ca 3 Example 6: Action Against Puccinia recondita In Wheat 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 postinfection, the fungus infestation was assessed.

Table 19 Action Against Puccinia recondita In Wheat Component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component II: propiconazole Test no. mg of a.i. per litre I:11 action SF comp. I comp. II O (observed) E (expected) O/E 0 (control) 1 100 51 2 5 3 100 5 20:1 79 56 1.4 WO 98/29537 PCV/EP97/072 3 -57- Table Action Against Puccinia recondita In Wheat Component 1: benzothiadiazole-7-carboxylic acid Component II: fenpropidine Test no. kg of a.i. per ha 1:11 action SF comp. I comp. II O (observed) E (expected) O/E 0 (control) 1 6 2 20 3 20 4 60 6 20 1:3 73 52 1.4 6 6 20 1:10 75 68 1.1 Example 7: Action Against Erysiphe graminis In Wheat In field trials (10m2), winter wheat in the growth phase was sprayed with a spray mixture prepared with a wettable powder of the active ingredient. Infection was naturally. days post-infection, the fungus infestation was assessed. The following results were obtained: Table 21 Action Against Erysiphe graminis In Wheat Component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component II: propiconazole Test no. g of a.i. per ha I:11 action SF comp. I comp. II O (observed) E (expected) O/E (control) 1 5 29 2 50 2 3 100 31 4 5 50 1:10 49 32 5 100 1:20 59 51 1.2 WO 98/29537 PCT/EP97/07253 -58- Table 22 Action Against Erysiphe graminis In Wheat Component 1: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component II: cyprodinil Test no. g of a.i. per ha 1:I action SF comp. I comp. II O (observed) E (expected) O/E 0 (control) 1 5 29 2 50 2 3 100 31 4 5 50 1:10 49 32 5 100 1:20 59 51 1.2 Example 8: Action Against Mycosphaerella fijiensis In Bananas banana plants in a 300m 2 plot were sprayed at 17-19 day intervals with a spray mixture prepared with the wettable powder of the active ingredient; in total 6 times. Infection was naturally. For evaluation, the leaf infested with the fungus was measured. The following results were obtained: Table 23 Action Against Mycosphaerella fijiensis In Bananas Component 1: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component II: propiconazole Test no. g of a.i. per ha 1:11 action SF comp. I comp. II O (observed) E (expected) O/E 0 (control) 1 50 19 2 50 26 3 50 50 1:1 46 40 1.15 WO 98/29537 PCT/EP97/07253 -59- Example 9: Action Against Altemaria solaniln Tomatoes 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: Table 24 Action Against Altemaria solani In Tomatoes in the open Component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component II: cyprodinil Test no. g of a.i. per ha 1:1I action SF comp. I comp. II O (observed) E (expected) O/E -0 (control) 1 2.5 32 2 12.5 3 25 51 4 2.5 12.5 1:5 79 53 2.5 25 1:10 80 67 1.2 Example 10: Action Against Phytophthora infestans In Tomatoes 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 18-20C and a relative atmospheric humidity of 90-100%. 5 days post-infection, the fungus infestation was assessed. The following results were obtained: WO 98/29537 PCT/EP97/07253 Table Action Against Phytophthora infestans In Tomatoes Component 1: benzo[1 ,2,3]thiadiazote-7-carbothioic acid-S-methyl ester Component II: metalaxyl Test no. mg of a.i. per litre 1:11 action SF comp. I comp. 11 0 (observed) E (expected) OlE 0 (control) 1 5 14 2 25 36 3 100 61 4 500 72 0.1 13 6 1 23 7 10 6 50 68 9 5 0.1 50:1 50 25 5 1 5:1 62 34 1.8 11 5 10 1:2 87 44 12 5 50 1:10 84 73 1.2 13 25 50 1:2 92 80 1.2 14 100 10 10:1 85 75 1.1 100 50 2:1 95 88 1.1 16 500 10 50:1 97 82 1.2 Table 26 Action Against Phytophthora infestans In Tomatoes Component 1: benzothiadiazole-7-carboxylic acid Component 11: metalaxyl Test no. mg of a.i. per litre 1:11 action SF camp. I comp. I1 0 (observed) E (expected) O/E 100(control)I WO 98/29537 PCT/EP97/07253 -61- 1 13 6 10 33 7 50 63 8 100 83 9 0.1 1 1:10 36 13 2.8 0.5 1 1:2 29 21 1.4 11 1 1 1:1 57 32 1.8 12 1 10 1:10 79 48 1.6 13 5 1 5:1 61 52 1.2 Example 11: Action Against Pseudoperonospora cubensis In Cucumbers 16-19-day-old cucumber plants ("Wisconsin") were sprayed to drip point with a spray mixture prepared with the formulated active ingredient, or combination of active ingredient, or combination of active ingredients. After 4 days, the treated plants were infected with sporangia of Pseudoperonospora cubenswas (strain 365, Ciba; max. 5000 per ml), and the treated plants were subsequently incubated for 1-2 days at 18-20 C and a relative atmospheric humidity of 70-90%. 10 days post-infection, the fungus infestation was assessed and compared with the infestation on untreated plants. The following results were obtained: Table 27 Action Against Pseudoperonospora cubensis In Cucumbers Component I: benzothiadiazole-7-carboxylic acid Component II: metalaxyl Test no. mg of a.i. per litre 1:il action SF comp. I comp. II O (observed) E (expected) O/E 0 (control) 1 0.05 0 WO 98/29537 PCT/EP97/07253 -62- 4 0.5 31 5 66 6 50 91 7 0.05 0.5 1:10 66 31 2.1 8 0.05 5 1:100 83 66 1.3 9 0.5 0.5 1:1 83 35 2.4 0.5 5 1:10 83 68 1.2 Example 12: Action Against Peronospora tabacina On Tobacco Plants 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.

Table 28 Action Against Peronospora tabacina On Tobacco Plants Component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component II: dimethomorph Test No. mg a.i. per litre (ppm) 1:ll action SF comp. I comp. II O (observed) E (expected) O/E 1 0.03 14 2 0.1 34 3 0.3 88 4 0.3 52 1 52 6 0.03 1 1:33 74 59 1.3 7 0.1 0.3 1:3 92 68 1.4 8 0.1 1 1:10 95 68 1.4 WO 98/29537 PCT/EP97/07253 -63- Example 13: Action Against Peronospora parasitica In Arabidopsis thaliana The fungicides metalaxyl, fosetyl, and copper hydroxide, and the SAR activator benzo(1,2,3)-thiadiazole-7-carbothioc acid S-methyl ester (BTH), formulated as 25%, and 25% active ingredient (ai) respectively, with a wettable powder carrier, were applied as fine mist to leaves of three week-old plants. The wettable powder alone was applied as a control. Three days later, plants were inoculated with a Peronospora parasitica conidial suspension as described in Delaney et al. (1995). Ws plants were inoculated with the compatible P. parasitica isolate Emwa (1-2 x 10s spores/mi); Col plants were inoculated with the compatible P. parasitica isolate Noco2 (0.5-1 x 10 s spores/ml). 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.

Fungal infection progression was followed for 12 days by viewing under a dissecting microscope to score development of conidiophores (Delaney, et al. (1994); Dietrich, et al.

(1994)). Lactophenoltrypan blue staining of individual leaves was carried out to observe fungal growth within leaf tissue. Fungal growth was quantified using a rRNA fungal probe that was obtained by PCR according to White et al. (1990; PCR Protocols: A guide to Methods and Application, 315-322) using primers NS1 and NS2 and P. parasitica EmWa DNA as templates.

RNA was purified from frozen tissue by phenol/chloroform extraction following lithium chloride precipitation (Lagrimini et al, 1987: PNAS, 84: 7542-7546). Samples (7.5 ug) were separated by electrophoresis through formaldehyde agarose gels and blotted to nylon membranes (Hybond- Amersham) as described by Ausbel et al. (1987). Hybridizations and washing were according to Church and Gilbert (1984, PNAS, 81: 1991-1995). Relative amounts of the transcript were determined using a Phosphor Imager Molecular Dynamics, Sunnyvale, CA) following manufacturers instructions. Sample loading was normalized by probing stripped filter blots with the constitutively expressed b-tubulin Arabidopsis cDNA. The infestation of the untreated plants corresponded to 0 fungal growth inhibition. The following results were obtained: WO 98/29537 PCVEP97/072 3 -64- Table 29 Action Against Peronospora parasiica NoCo2 In Arabidopsis thaliana (001-0) Component 1: benzo[1 ,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component 11: metalaxyl_______ Test no. Components Fungal Growth Inhibition Synergy Factor BTH metalaxyl 0 (observed) E (expected) OlE control -0 1 0.01 mM- 0 2 0.1 mg/I 0 3 0.01 mM 0.1 mg/i 40.7 0 c Table Action Against Peronospora parasitica Emwa In Arabidopsis thaliana (Ws) Component 1: benzo 1 ,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component 11: metalaxyl Test no. Components Fungal Growth Inhibition Synergy Factor BTH metalaxyl 0 (observed) E (expected) OlE control 0 1 0.01 mM 2 0.003 mM 0 3 2.5 mg/I 4 0.5 mg/l 0. 1 mg/I 6 0.01 mM 2.5 mg/I 100 90 1.1 7 0.01 mM 0.5 mg/I 95 70 1.4 8 0.01 mM 0. 1 mg/I 88 70 1.3 9 0.003 mM 2.5 mg/i 100 75 1.3 Table 31 Action Against Peronospora parasitica Emwa In Arabidopsis thaliana (Ws) WO 98/29537 PCT/EP97/07253 Component 1: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component II: fosetyl Test no. Components Fungal Growth Inhibition Synergy Factor BTH fosetyl O (observed) E (expected) O/E control 0 1 0.01 mM 2 1.0 g/I 3 0.2 g/I 4 0.04 g/ 0 0.01 mM 1.0 g/l 100 70 1.4 6 0.01 mM 0.2 g/l 100 40 7 0.01 mM 0.04 g/l 95 30 3.2 Table 32 Action Against Peronospora parasitica Emwa In Arabidopsis thaliana (Ws) Component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component II: copper hydroxide Test no. Components Fungal Growth Inhibition Synergy Factor BTH Cu(OH) 2 O (observed) E (expected) O/E control 0 1 0.01 mM 2 0.01 g/ 0 3 0.01 mM 0.01 g/l 85 30 2.8 As can be seen in Table 29, synergistic disease-resistant effects were demonstrated in the wild-type Arabidopsis Col-0 plants. No fungal growth inhibition was observed by separately applying either 0.01 mM BTH or 0.0001 g/L metalaxyl to the plants, because these concentrations are normally insufficient for efficacy. However, by applying both of these compounds to the plants at these normally insufficient concentrations, 40.7% fungal growth inhibition was observed, which is clearly a synergistic effect. Tables 30-32 show WO 98/29537 PCT/EP97/07253 -66 synergistic disease-resistant effects in wild-type Arabidopsis Ws plants. Only 20-30% fungal growth inhibition was observed by applying 0.01 mM BTH to the Ws plants.

However, by simultaneously applying BTH and either metalaxyl, fosetyl, or copper hydroxide to the plants, synergistic disease resistance was observed. These combined antifungal effects, which result in a decrease in the effective concentration of the fungicide and BTH required for pathogen control, allow the reduction of the chemical dose needed to stop fungal growth and therefore mitigate the incidence of foliar damage due to chemical tolerance.

II. Synergistic Disease Resistance Effects Achieved By Application Of Conventional Microbicides and/or Chemical Inducers of Systemic Acquired Resistance To Constitutive Immunity (CIM) Mutant Plants In this set of examples, 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 et al. (1994); and Weymann et a. (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.

Two classes of SAR signal transduction mutants have been isolated using this screen.

One class has been designated as Isd mutants (Isd lesion 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 incorporated 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).

The second class, called cim (cim constitutive immunity), is described below and has all the characteristics of the Isd mutants except spontaneous lesions. 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.

WO 98/29537 PCT/EP97/07253 -67- Example 14: Isolation and Characterization of cim Mutants With Constitutive SAR Gene Expression 1100 individual M2 mutagenized (EMS) 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 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 0 C. When the plants were three- to fourweeks-old, one or two leaves, weighing 50 to 100 mg, were harvested and total 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 nontreated A. thaliana Col-0 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.

Back crosses to Columbia utilized the recessive glabrous trait as a marker for identification of F1 descendants. Col-gll flower buds were emasculated prior to pollen shed, and pollen from the mutants was applied immediately and the following day. F1 plants were grown in soil and the out crossed plants were identified by the presence of trichomes.

Following crosses of cim2 and cim3 to ecotype Col-0 or La-er, a large proportion of F1 plants were identified with high SAR gene expression, suggesting these traits were dominant. In the case of cim2, some, but not all, F1 plants had constitutive SAR gene expression. Such a result would be expected if the cim2 mutant were dominant and carried as a heterozygote in the wn o8/29537 PCT/EP907253 -68parent. Further genetic testing of cim2 showed continued variable segregation in the F2 generation, consistent with incomplete penetrance.

cim3 demonstrated a 1:1 segregation in the F1 generation whereupon two individual F1 plants expressing a high level of PR-1 mRNA were selfed to form an F2 population. F2 segregation, obtained by scoring PR-1 mRNA accumulation, showed 93 F2 plants with high PR- 1 mRNA and 25 F2 plants without significant PR-1 mRNA accumulation giving a 3.7:1 ratio (c 2 1.77; 0.5 P which is consistent with the hypothesis that cim3 is a dominant, single gene mutation. Subsequent outcrosses confirmed that cim3 was inherited as a dominant mutation.

For cim3, the original M2 plant identified in the screen and the M3 population appeared normal. However, as the cim3 plants were selfed some of the best expressing lines had low fertility. Following the back cross to Col-gll, plants with normal appearance and fertility and strong PR-1 expression were obtained.

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.

Example 15: SAR Gene Expression In addition to PR-1, two other SAR genes, PR-2 and PR-5, are also highly expressed in cim3. Levels of 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.

Example 16: Salicylic Acid Analysis Endogenous concentrations of SA have been shown to increase following pathogeninduced necrosis in Arabidopsis (Uknes et al., 1993, supra). Salicylic acid and its glucose conjugate were analyzed as described in Uknes et al., 1993. Leaf tissue was harvested from cim3 and 10 control, 4 week-old plants. Leaves from individual plants were harvested and analyzed for PR-1 gene expression. SA levels were measured from plants expressing PR-1. The WO 98/29537 PCT/EP97/07253 -69 concentration of free SA in cim3 was 3.4-fold higher than in non-infected wild-type Arabidopsis (233±35 vs. 69±8 ng/g fresh weight, respectively). The glucose conjugate of SA (SAG) 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.

Example 17: Disease Resistance 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. Mycol. Soc. 74, 329-333, and in Koch and Slusarenko (1990) Plant Cell2, 437-445. Wild-type (Col-0) 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. This trailing necrosis is similar to that found in wild-type plants treated with low doses of SA or INA (Uknes et al., 1992, supra; Uknes et al., 1993, supra). However, sporulation was never observed on cim3 plants while all control plants showed sporulation. No spontaneous lesions were observed on uninoculated cim3 leaves when stained with trypan blue.

In addition to resistance to the fungal pathogen P. parasitica, cim3 was also resistant to infection with the bacterial pathogen Pseudomonas syringae DC3000. Six-week-old wild-type INA treatment), and cim3 plants were inoculated with a suspension of P. syringae DC3000 and the progress of the disease was followed by monitoring the growth of the bacteria extracted from infected leaves over time. The difference in bacterial titers between Col-O, Col-O INA and cim3 at either day 0 or day 2 was not statistically significant. However, by day four, there was a 31-fold decrease in bacterial growth between wild-type and cim3 plants 0.003; Sokal and Rohlf, 1981). The plants were also visually inspected for disease symptoms. Leaves from wild- WO 98/29537 PCT/EP97/07253 type plants were severely chlorotic with disease symptoms spreading well beyond the initial zone of injection. In contrast, either wild-type plants pretreated with INA or cim3 plants were nearly devoid of disease symptoms.

For this example, cultures of Pseudomonas syringae pv. tomato strain DC3000 were grown on King's B media (agar plates or liquid) plus rifampicin (50 pg/ml) at 28*C (Walen et al.

(1991) Plant Cell3, 49-59). An ovemight 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 pl of the diluted bacterial solution with a 1 cc syringe. At various time points, random samples consisting of 3 random leaf punches from a #1 cork borer were taken from plants from each treatment. The 3 leaf punches were placed in an eppendorf tube with 300 pl of 10 mM MgCI 2 and ground with a pestle. The resulting bacterial suspension was appropriately diluted and plated on King's B media plus rifampicin (50 pg/ml) and grown for 4 days at 28 0 C. Bacterial colonies were counted and the data were subjected to Student's t statistical analysis (Sokal and Rohlf (1981), Biometry, 2 d ed. New York: W.H. Freeman and Company).

Also for this example, 2,6-Dichloroisonicotinic acid (INA) was suspended in sterile, distilled water as a 25% active ingredient formulated in a wetable powder (0.25 mg/ml, 325 pM; Kessmann et al. (1994) Annu. Rev. Phytopathol. 32,439-59). All plants were sprayed with water or INA solutions to the point of imminent runoff.

Example 18: The Role of SA in SAR Gene Expression and Disease Resistance To investigate the relationship between SA, SAR gene expression and resistance in cim3, crosses were carried out with Arabidopsis plants expressing the salicylate hydroxylase (nahG) gene (Delaney et al., 1994). These "NahG plants" were made by transformation of the driven nahG gene into Arabidopsis using Agrobactenum-mediated transformation. See, Huang, H. Ma, H. (1992) Plant Mol. Biol. Rep. 10, 372-383, herein incorporated by reference; Gaffney, et al. (1993) Science 261,754-756, herein incorporated by reference; and Delaney, et al. (1994) Science 266, 1247-1250, herein incorporated by reference. Col-nahG Arabidopsis carries a dominant kanamycin resistance gene in addition to the dominant nahG gene, so ColnahG 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 WO 98/29537 PCT/EP97/07253 -71sterile water. Seeds were plated onto germination media (GM, Murashige and Skoog medium containing 10g/L sucrose buffered with 0.5 g/L 2-(N-morpholino) ethanesulfonic acid, pH 5.7 with KOH) containing 25 mg/ml kanamycin to select for F 1 plants. See Valvekens et al. (1988) Proc.

Natl. Acad. Sci, USA 85, 5536-5540. Kanamycin resistant F, 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.

Because both the cim3 mutant and nahG phenotypes are dominant, epistasis between the two genes could be analyzed in F1 plants. Seventy F1 plants from a dm3 X nahG cross were analyzed for PR-1 and nahG gene expression. In Northern blot analysis of mRNA expression, the presence of the nahG gene correlated with suppressed SAR gene expression.

The presence of cim3 in each F1 was confirmed by assessing PR-1 mRNA in the resulting F2 segregants.

To determine if the cim3 mutation was epistatic to nahG with respect to disease resistance, 5 F1 plants from the cim3 X nahG cross, which had been confirmed for the presence of nahG and absence of PR-1 mRNA, were seffed and 20-30 F2 seed were planted.

Expression of nahG and PR-1 mRNA was analyzed in individuals from this F2 population, which were then challanged with P. parasitica (NoCo2) to assess their disease susceptibility. Disease resistance conferred by cim3 was eliminated by the presence of the nahG gene, demonstrating that nahG is epistatic to cim3 for the SAR gene expression and disease resistance phenotypes.

Example 19: Synergistic Disease-Resistance Attained by Applying Microbicide and/or BTH to cim Mutants Three days before pathogen inoculation, the chemical inducer of systemic acquired resistance BTH benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester) formulated as active ingredient (ai) with a wettable powder carrier (Metraux et al., 1991) and/or the microbicide metalaxyl (CGA 48988) formulated as 25% ai, or the wettable powder alone was applied as a fine mist to leaves of 4 week-old plants. Plants were inoculated with a conidial suspension (1.8 x 10 5 spores/ml) of the compatible pathogen Peronospora parasitica NoCo2.

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.

WO 98/29537 PCT/EP97/07253 -72- Fungal growth was determined using a rRNA fungal probe that was obtained by PCR according to White et al. (1990; PCR Protocols: A guide to Methods and Application, 315-322) using primers NS1 and NS2 and P. parasftica EmWa DNA as templates. RNA was purified from S frozen tissue by phenol/chloroform extraction following lithium chloride precipitation (Lagrimini et al, 1987: PNAS, 84: 7542-7546). Samples (7.5 ug) were separated by electrophoresis through formaldehyde agarose gels and blotted to nylon membranes (Hybond-N+, Amersham) as described by Ausbel et al. (1987). Hybridizations and washing were according to Church and Gilbert (1984, PNAS, 81: 1991-1995). Relative amounts of the transcript were determined using a Phosphor Imager Molecular Dynamics, Sunnyvale, CA) following manufacturers instructions.

Sample loading was normalized by probing stripped filter blots with the constitutively expressed b-tubulin ArabidopsiscDNA. The infestation of the untreated plants corresponded to 0 fungal growth inhibition.

Application of metalaxyl alone, the "plant activator" BTH alone, or both metalaxyl and BTH to the cim3 mutants described above produced a greater-than-additive, synergistic, disease-resistant effect. This effect was determined as the synergy factor which is the ratio of observed effect to expected effect. The following results were obtained: Table 33 Action Against Peronospora parasitica In Arabidopsis Component I: cim3 mutation Component II: metalaxyl Test no. Components Fungal Growth Inhibition Synergy Factor cim3 metalaxyl 0 (observed) E (expected) O/E control wt 0 1 cim3 12.5 2 wt 12.5 mg/ 52.7 3 wt 2.5 mg/I 0 4 wt 0.1 mg/I 0 wt 0.02 mg/I ND 6 cim3 12.5 mg/I ND ND ND 7 cim3 2.5 mg/I 82.2 12.5 6.6 8 cim3 0.1 mg/I 57.8 12.5 4.6 WO 98/29537 WO 9829537PCT/EP97/07253 .73 9 dim3 0.02 Am/ 5 5.6 ND ND ild-type Col-O ND not determined Table 34 Action Against Peronospora parasitica In Arabidopsis Component 1: dim3 mutation Component II: BTH Test no. Components Fungal Growth Inhibition Synergy Factor cim3 BTH 0 (observed) E (expected) OlE control wt -0 1 cim3 -12.5 2 wt 0.1 mM 85.7 3 wt 0.03 mM 20.8 4 wt 0.01 mM 0 cim3 0.1 mM ND 98.2 ND 6 cim3 0.03 mM 73.1 33.3 2.2 7 cim3 0.01 mM 16.6 12.5 1.3 wt wild-type Col-0 ND not determined Table Action Against Peronospora parasitica In Arabidopsis Component 1: cim3 mutation Component II: BTH and metalaxyl Test no. Components Fungal Growth Inhibition Synergy Factor cim3 BTH+M 0 (observed) E (expected) O/E control wt -0 1cim3 12.5 2 wt BTH 0.01 mM 100 M0.5 WO 98/29537 PCT/EP97/07253 -74- 3 wt BTH 0.01 mM 40.7 M 0.1 mg/I 4 wt BTH 0.01 mM ND M 0.02 mg/I cim3 BTH 0.01 mM ND 100 ND M 0.5 mg/I 6 cim3 BTH 0.01 mM 100 53.2 1.9 M 0.1 mg/l 7 cim3 BTH 0.01 mM 77.7 ND ND M 0.02 mg/l wt wild-type Col-0 ND not determined As can be seen from the above tables, synergistic disease-resistant effects were demonstrated in the cim3 plants by application of metalaxyl alone, by application of BTH alone, and by application of metalaxyl and BTH in combination. For example, in the untreated cim3 plant, 12.5% fungal growth inhibition was seen relative to the untreated wildtype plant; this demonstrates that the constitutive SAR gene expression in the cim3 mutant correlates with disease resistance. As shown in Table 30, however, by applying metalaxyl at 0.0001 g/l (a concentration normally insufficient for efficacy) to the immunomodulated (SAR-on) cim3 plant, the observed level of fungal growth inhibition increased to 57.8%.

The synergy factor of 4.6 calculated from these data clearly demonstrates the synergistic effect achieved by applying a microbicide to an immunomodulated plant.

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. For example, in wild-type plants, a 0.03 mM concentration of BTH is normally insufficient to confer effective disease resistance, providing only 20.8% fungal growth inhibition. However, in cim3 plants, 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.

WO 98/29537 PCT/EP97/07253 The effects on disease resistance were even more dramatic when both BTH and metalaxyl were applied to the cim3 plant. As set forth above in Example 13 (Table 29), in wild-type plants, no fungal growth inhibition is achieved by separately applying either 0.01 mM BTH or 0.0001 g/I metalaxyl, because these concentrations are normally insufficient for efficacy. However, by applying both of these compounds to the plants at these normally insufficient concentrations, 40.7% fungal growth inhibition was observed, which is a synergistic effect with respect to the wild-type plants. In the cim3 plants, the simultaneous application of 0.01 mM BTH and 0.0001 g/I metalaxyl resulted in 100% fungal growth inhibition, clearly demonstrating even further synergistic activity.

Thus, the combined use of of immunomodulated cim plants with low, normally ineffective concentrations of chemicals to achieve disease resistance provide advantages that should be apparent to those skilled in the agricultural arts. Normally toxic or otherwise undesirable concentrations of chemicals can be avoided by taking advantage of the synergies demonstrated herein. In addition, economic gains can be realized as a result of the decreased quantity of chemicals required to provide a given level of protection to plants.

II. Synergistic Disease Resistance Effects Achieved By Application Of Conventional Microbicides and/or Chemical Inducers of Systemic Acquired Resistance To Transgenic Plants Containing Forms of the NIM1 Gene The NIM1 gene is a key component of the systemic acquired resistance (SAR) pathway in plants (Ryals et a.,1996). 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 nim 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 a., 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 WO 98/29537 PCT/EP97/07253 -76 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, incorporated herein by reference.

SFurthermore, the NIM1 gene product has been shown to be a structural homologue of the mammalian signal transduction factor IKB subclass a (Ryals et al., 1997). Mutations of IKB have been described that act as super-repressors or dominant-negatives of the NF- KB/IKB 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" incorporated herein by reference.

Example 20: Transformation of Plants with Cosmid Clones Containing the Wild-Type NIM1 Gene 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 El 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 El 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. These cosmids were then used to transform a kanamycin-sensitive nim I mutant Arabidopsis line using vacuum infiltration (Mindrinos et al., 1994, Cell 78, 1089-1099). Seed from the infiltrated plants was harvested and allowed to germinate on GM agar plates containing 50 mg/ml kanamycin as a selection agent. Seedlings that survived the selection were transferred to soil approximately two weeks after plating.

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 WO 98/29537 PCT/EP97/07253 -77grown under high humidity conditions in a growing chamber with 19°C day/17 0 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 nim1 S plants were treated in the same way to serve as controls for each experiment.

Total RNA was extracted from the collected tissue using a LiCI/phenol extraction buffer (Verwoerd et al., 1989, Nuc Acid Res, 2362). RNA samples were run on a formaldehyde agarose gel and blotted to GeneScreen Plus (DuPont) membranes. Blots were hybridized with a 32P-labeled PR-1 cDNA probe. The resulting blots were exposed to film to determine which transformants were able to induce PR-1 expression after INA treatment.

To see if any of the D7 and El transformants overexpressed NIM1 due to insertion site (position) effect, primary transformants containing the D7 or El cosmids were selfed and the T2 seed collected. Seeds from one El line and 95 D7 lines were sown on soil and grown as described above. When the T2 plants had obtained at least four true leaves, a single leaf was harvested separately for each plant. RNA was extracted from this tissue and analyzed for PR-1 and NIM1 expression. Plants were then inoculated with P. parasitica (EmWa) and analyzed for fungal growth at 10 days following infection. A number of transformants showed less than normal fungal growth and four of them, namely, lines D7-2, D7-74, D7-89 and E1-1, showed no visible fungal growth at all. Plants showing higher than normal NIM1 and PR-1 expression and displaying fungal resistance demonstrate that overexpression of NIM1 confers disease resistance.

Example 21: NIM1 Overexpression Under Its Native Promoter 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 pSGCGO1 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 pg/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 WO 98/29537 PCCUEP97/0725 -78several 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 S 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.

RNA was prepared from all of the collected samples and analyzed as previously described (Delaney et al, 1995). The blot was hybridized to the Arabidopsis gene probe PR-1 (Uknes et al, 1992). Five of the 13 transgenic lines analyzed showed early induction of PR1 gene expression. For these lines, PR-1 mRNA was evident by 24 or 48 hours following fungal treatment. These five lines also had no visible fungal growth. Leaves were stained with lactophenol blue as described (Dietrich et al., 1994) to verify the absence of fungal hyphae in the leaves. PR-1 gene expression was not induced in the other eight lines by 48 hours and these plants did not show resistance to Emwa.

A subset of the resistant lines were also tested for increased resistance to the bacterial pathogen Pseudomonas syringae DC3000 to evaluate the spectrum of resistance evident as described by Uknes et al. (1993). Experiments were done essentially as described by Lawton et al. (1996). Bacterial growth was slower in those lines that also demonstrated constitutive resistance to Emwa. This shows that plants overexpressing the NIM1 gene under its native promoter have constitutive immunity against pathogens.

To assess additional characteristics of the CIM phenotype in these lines, unifected plants are evaluated for free and glucose-conjugated salicylic acid and leaves are stained with lactophenol blue to evaluate for the presence of microscopic lesions. Resistance plants are sexually crossed with SAR mutants such as NahG 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 salicylic acid-dependent feedback loop.

Example 22: 35S Driven Overexpression of NIM1 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 Xbalfragment containing an enhanced CaMV 35S promoter, the NIM1 cDNA in the correct orientation for transcription, and a tml 3' terminator was obtained. This fragment WO 98/29537 PCT/EP9707253 -79 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 pg/ml kanamycin. Surviving plantlets were transferred to soil and tested as described above. Selected plants were selfed and selected for two subsequent generations to generate homozygous lines. Nine of the 58 lines tested demonstrated resistance when they were treated with Emwa without prior chemical treatment. Thus, overexpression of the NIM1 cDNA also results in disease-resistant plants.

Example 23: NIM1 Is A Homolog Of licBa A multiple sequence alignment between the protein gene products of NIM1 and IkB was performed by which it was determined that the NIM1 gene product is a homolog of IKBa (Figure 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, WI). The sequences used in the alignment were NIM1 (SEQ ID NO:2), mouse IkBoa (SEQ ID NO:3, GenBank Accession 1022734), rat liBa (SEQ ID NO:4, GenBank accession Nos. 57674 and X63594; Tewari et al., Nucleic Acids Res. 20, 607 (1992)), and pig IBa (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)). Parameters used in the Clustal analysis were gap penalty of 10 and gap length penalty of 10. Evolutionary divergence distances were calculated using the PAM250 weight table (Dayhoff et al., "A model of evolutionary change in proteins. Matrices for detecting distant relationships." In Atlas of Protein Sequence and Structure, Vol. 5, Suppl. 3, Dayhoff, ed (National Biomedical Research Foundation, Washington, pp. 345-358 (1978)). Residue similarity was calculated using a modified Dayhoff table (Schwartz and Dayhoff, "A model of evolutionary change in proteins." In Atlas of Protein Sequence and Structure, M.O. Dayhoff, ed (National Biomedical Research Foundation, Washington, pp. 353-358 (1979); Gribskov and Burgess, Nucleic Acids Res. 14, 6745-6763 (1986)).

Homology searches indicate similarity of NIM1 to ankyrin domains of several proteins including: ankyrin, NF-KB and IKB. The best overall homology is to IKB and related molecules (Figure NIM1 contains 2 serines at amino acid positions 55 and 59; the WO 98/29537 PCT/EP97/07253 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 IKBa's have these N-terminal serines and they are required for inactivation of IKB and subsequent release of NF-KB. NIM1 has ankyrin domains (amino acids 262-290 and 323-371). Ankyrin domains are believed to be involved in protein-protein interactions and are a ubiquitous feature for kB and NF-KB molecules. The C-termini of IKB's can be dissimilar. NIM1 has some homology to a QL-rich region (amino acids 491-499) found in the C-termini of some IBs.

Example 24: Generation Of Altered Forms Of NIM1 Changes Of Serine Residues 55 and 59 To Alanine Residues Phosphorylation of serine residues in human IBac is required for stimulus-activated degradation of IKBa thereby activating NF-KB. Mutagenesis of the serine residues (S32- S36) in human IKBa to alanine residues inhibits stimulus-induced phosphorylation thus blocking IcBa proteosome-mediated degradation Britta-Mareen Traenckner et al., EMBO J. 14: 2876-2883 (1995); Brown et al., Science 267:1485-1488 (1996); Brockman et al., Molecular and Cellular Biology 15: 2809-2818 (1995); Wang et al., Science 274:784-787 (1996)).

This altered form of IKBa functions as a dominant negative form by retaining NF-KB in the cytoplasm, thereby blocking downstream signaling events. Based on sequence comparisons between NIM1 and IKB, serines 55 (S55) and 59 (S59) of NIM1 are homologous to S32 and S36 in human I Ba. To construct dominant-negative forms of NIM1, 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).

Using a full length NIM1 cDNA (SEQ ID NO:6) including 42 bp of 5' untranslated sequence (UTR) and 187 bp of 3' UTR, the mutagenized construct can be made per the manufacturer's instructions using the following primers (SEQ ID NO:6, positions 192-226): CAA CAG CTT CGA AGC CGT CTT TGA CGC GCC GGA TG-3' (SEQ ID NO:25) and 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 WO 98/29537 PCT/EP97/07253 -81 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.colistrain 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 tm 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.

Example 25: Generation Of Altered Forms Of NIM1 N-terminal Deletion Deletion of amino acids 1-36 (Brockman et al.; Sun et al.) or 1-72 (Sun et al.) of human IkBa, which includes K21, K22, S32 and S36, results in a dominant-negative hlBa phenotype in transfected human cell cultures. An N-terminal deletion of approximately the first 125 amino acids of the encoded product of the NIM1 cDNA removes eight lysine residues that may serve as potential ubiquitination sites and also removes putative phosphorylation sites at S55 and S59 (see Example This altered gene construct may be produced by any means known to those skilled in the art. For example, using the method of Ho et al., Gene 77:51-59 (1989), a NIM1 form may be generated in which DNA encoding approximately the first 125 amino acids is deleted. The following primers produce a 1612bp PCR product (SEQ ID NO:6: 418 to 2011): 5'-gg aat tca-ATG GAT TCG 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 KCV2 mM MgC 2 /0.001% gelatin/0.25 mM each dNTP/0.2 mM of each primer and 1 unit rTth DNA polymerase in a final volume of WO 98/29537 PCTIEP97/07253 -82mL 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 S released by restriction endonuclease digestion using EcoRI and ligated into the EcoRI site of dephosphorylated pCGN1761, under the transcriptional regulation of the double 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 tmlterminator 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 NIM1 gene having an N-terminal amino acid deletion.

Example 26: Generation Of Altered Forms Of NIM1 C-terminal Deletion The deletion of amino acids 261-317 of human IBa 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 NIM1. 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. (1989), the C-terminal coding region and 3' UTR of the NIM1 cDNA (SEQ ID NO:6:1606-2011) is deleted by PCR, generating a 1623 bp fragment using the following primers: 5'-cggaattcGATCTCTTTAATTTGTGAATTT C-3' (SEQ ID NO:29) and 5'-ggaattcTCAACAGTT CATAATCTGGTCG-3' (SEQ ID NO:30) in which a synthetic stop codon is underlined (TGA on complementary strand) and EcoRI linker sequences are in lower case. PCR reaction components are as previously described and cycling parameters are as follows: 94°C 3 min: 35x (940C 30 sec: 520C 30 sec: 72°C 2 min); 720C 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 WO 98/29537 PCT/EP97/07253 -83terminator is released from pCGN1761 by partial restriction digestion with Xbaland ligated into the Xbalsite 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.

Example 27: Generation Of Altered Forms Of NIM1 N-terminalC-terminal Deletion Chimera An N-terminal and C-terminal deletion form of NIM1 is generated using a unique Kpnl restriction site at position 819 (SEQ ID NO:6). The N-terminal deletion form (Example is restriction endonuclease digested with EcoRI/Kpnl and the 415 bp fragment corresponding to the modified N-terminus is recovered by gel electrophoresis. Likewise, 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/Cterminal deletion form of NIM1 is under the transcriptional regulation of the double promoter. Similarly, 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 promoter, NIM1 chimera and tm/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 Cterminal amino acid deletions.

Example 28: Generation Of Altered Forms Of NIM1 Ankyrin Domains NIM1 exhibits homology to ankyrin motifs at approximately amino acids 103-362.

Using the method of Ho et al. (1989), the DNA sequence encoding the putative ankyrin domains (SEQ ID NO:1: 3093-3951) is PCR amplified (conditions: 94C 3 min:35x (94 0 C WO 98/29537 PCT/EP97/07253 -84sec: 62 0 C 30 sec: 72 0 C 2 min): 72 0 C 10 min) from the NIM1 cDNA (SEQ ID NO:6: 349- 1128) using the following primers: 5'-ggaattcaATGGACTCCAACAACACCGCCGC-3'

(SEQ

ID NO:31) and 5'-ggaattcTCAACCTTCCAAAGTTGCTTCTGATG-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 tmlterminator is released from pCGN1761 by partial restriction digestion with Xbaland ligated into the Xbalsite 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 NIM1.

Example 29: Construction Of Chimeric Genes To increase the likelihood of appropriate spatial and temporal expression of altered NIM1 forms, 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. Although 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. Therefore, 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. Likewise, the altered NIM1 forms may be expressed under the regulation of the pathogen-responsive promoter PR-1 Pat. No. 5,614,395). Such expression permits strong expression of the altered NIM1 forms only under pathogen attack or other SARactivating conditions. Furthermore, disease resistance may be evident in the transformants expressing altered NIM1 forms under PR-1 promoter regulation when treated with concentrations of SAR activator compounds BTH or INA) which normally do not activate SAR, thereby activating a feedback loop (Weymann et al., (1995) Plant Cell 7: 2013-2022).

WO 98/29537 I PCT/EP97/07253 Example 30: Transformation Of Altered Forms Of The NIM1 Into Arabidopsis thaliana The constructs generated (Examples 24-29) are moved into Agrobacterium tumefaciens by electroporation into strain GV3101. These constructs are used to transform Arabidopsis ecotypes Col-0 and Ws-0 by vacuum infiltration (Mindrinos et al., Cell78, 1089- 1099 (1994)) or by standard root transformation. Seed from these plants is harvested and allowed to germinate on agar plates with kanamycin (or another appropriate antibiotic) as selection agent. Only plantlets that are transformed 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 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 5-10x104 spores/ml from two compatible P. parasitica isolates, Emwa and Noco these fungal strains cause disease on wildtype Ws-O and Col-0 plants, respectively), are prepared, and transformants are sprayed with the appropriate isolate depending on the ecotype of the transformant. Inoculated plants are incubated under high humidity for 7 days. Plants are disease rated at day 7 and a single leaf is harvested for RNA blot analysis utilizing a probe which provides a means to measure fungal infection.

Transformants that exhibit a CIM phenotype are taken to the T1 generation and homozygous plants are identified. Transformants are subjected to a battery of disease resistance tests as described below. Fungal infection with Noco and Emwa is repeated and leaves are stained with lactophenol blue to identify the presence of fungal hyphae as described in Dietrich et al., (1994). Transformants are infected with the bacterial pathogen 4 WO 98/29537 PCT/EP97/07253 -86 Pseudomonas syringae DC3000 to evaluate the spectrum of resistance evident as described in Uknes et al. (1993). Uninfected plants are evaluated for both free and glucose-conjugated SA and leaves are stained with lactophenol blue to evaluate for the presence of microscopic lesions. Resistant plants are sexually crossed with SAR mutants such as NahG 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.

Example 32: Isolation Of NIM1 Homologs 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 Tm, 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 0 C below the calculated melting temperature Tm, preferably in the range of about 12-15°C below the calculated melting temperature and, in the case of oligonucleotides, in the range of about 5-10°C below the melting temperature Tm.

Using the NIM1 cDNA (SEQ ID NO:6) as a probe, homologs of Arabidopsis NIM1 are identified through screening genomic or cDNA libraries from different crops such as, but not limited to those listed below in Example 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.

WO 98/29537 PCT/EP97/07253 -87- 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. Following UV-crosslinking to affix the DNA, the membrane was hybridized under low stringency conditions [(1%BSA; 520mM NaPO 4 pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride) at 0 C for 18-24h] with 3P-radiolabelled Arabidopsis thaliana NIM1 cDNA. Following hybridization the blots were washed under low stringency conditions [6XSSC for 15 min.

(X3) 3XSSC for 15 min. (X1) at 55°C; 1XSSC is 0.15M NaCI, 15mM Na-citrate (pH7.0)] and exposed to X-ray film to visualize bands that correspond to NIM1.

In addition, expressed sequence tags (EST) identified with similarity to the NIM1 gene can be used to isolate homologues. For example, several rice expressed sequence tags (ESTs) have been identified with similarity to the NIM1 gene. A multiple sequence alignment was constructed using Clustal V (Higgins, Desmond G. and Paul M. Sharp (1989), Fast and sensitive multiple sequence alignments on a microcomputer,

CABIOS

5:151-153) as part of the DNA* (1228 South Park Street, Madison Wisconsin, 53715) Lasergene Biocomputing Software package for the Macintosh (1994). 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 NIM1 sequences in a GenBank BLAST search. Comparisons of the regions of homology in 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 ESTs 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 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 d codon position. This diversity of substitution in the third position may be constrained depending on the species that is being targeted. For example, because maize WO 98/29537 PCT/EP97/07253 -88is GC rich, primers are designed that utilize a G or a C in the 3" position, if possible. The PCR reaction is performed from cDNA or genomic DNA under a variety of standard conditions. When a band is apparent, it is cloned and/or sequenced to determine if it is a NIM1 homologue.

Example 33: Expression of a Form of NIM1 In Crop Plants Those constructs conferring a CIM phenotype in Col-0 or Ws-0 are transformed into crop plants for evaluation. Alternatively, altered native NIMI genes isolated from crops in the preceding example are put back into the respective crops. Although the NIM1 gene can be inserted into any plant cell falling within these broad classes, it is particularly useful in crop plant cells, such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane. Transformants are evaluated for enhanced disease resistance. In a preferred embodiment of the invention, 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 active ingredient respectively, with a wettable powder carrier, were applied as fine mist to leaves of three week-old transgenic Ws plants constitutively expressing the NIM1 gene.

The wettable powder alone was applied as a control. Three days later, plants were inoculated with a Peronospora parasitica isolate Emwa conidial suspension (1-2 x 10 spores/ml), as described in Delaney et aL (1995). Following inoculation, plants were covered to WO 98/29537 PCT/EP97/07253 -89maintain high humidity and were placed in a Percival growth chamber at 17 0 C with a 14-hr night cycle (Uknes et al., 1993). Tissue was harvested 8 days after inoculation.

Fungal infection progression was followed for 12 days by viewing under a dissecting microscope to score development of conidiophores (Delaney, et al. (1994); Dietrich, et al.

(1994)). Lactophenoltrypan blue staining of individual leaves was carred out to observe fungal growth within leaf tissue. Fungal growth was quantified using a rRNA fungal probe obtained by PCR according to White et al. (1990; PCR Protocols: A guide to Methods and Application, 315- 322) using primers NS1 and NS2 and P. parasitica EmWa DNA as templates. RNA was purified from frozen tissue by phenol/chloroform extraction following lithium chloride precipitation (Lagrimini et al, 1987: PNAS, 84: 7542-7546). Samples (7.5 pg) were separated by electrophoresis through formaldehyde agarose gels and blotted to nylon membranes (Hybond- Amersham) as described by Ausbel et al. (1987). Hybridizations and washing were according to Church and Gilbert (1984, PNAS, 81:1991-1995). Relative amounts of the transcript were determined using a Phosphor Imager Molecular Dynamics, Sunnyvale, CA) following manufacturers instructions. Sample loading was normalized by probing stripped filter blots with the constitutively expressed b-tubulin Arabidopsis cDNA. The infestation of the untreated plants corresponded to 0 fungal growth inhibition.

Application of metalaxyl, fosetyl, or copper hydroxide to plant lines overexpressing NIM1 produced a greater-than-additive, synergistic, disease-resistant effect. This effect was determined as the synergy factor which is the ratio of observed effect to expected (E) effect. The following results were obtained: Table 36 Action Against Peronospora parasitica In Arabidopsis Component I: NIM1 overexpression (line 6E) Component II: metalaxyl Test no. Components Fungal Growth Inhibition Synergy Factor NIM1 metalaxyl O (observed) E (expected) O/E control wt 0 1 NIM1 2 wt 0.0125 g/ 59 3 wt 0.0012 g/i 27 4 NIM1 0.0125 g/ 76 69 1.1 WO 98/29537 WO 9829537PCT/EP97/07253 -9o- 1 N/Mi 0.0012 g 56 371.5 wt wild-type Ws Table 37 Action Against Peronospora parastica In Arabidopsis Component I: NIMi overexpression (line 6E) Component II: tosetyl Test no. Components Fungal Growth Inhibition Synergy Factor N/M1 fosetyl 0 (observed) E (expected) O/E control wt -0 1 N/Mi 2 wt 5.0Og/I 7 3 wt 0.5 g 2 4 wt 0.05 gf 0 NIMi 5.0Ogl 93 17 6 NIMi 0.5 g/I 83 12 6.9 7 N/Mi 0.05 gI 42 10 4.2 wt wild-type Ws Table 38 Action Against Peronospora parasitica In Arabidopsis Component 1: N/Mi overexpression (line 7C) Component II: fosetyl Test no. Components Fungal Growth Inhibition Synergy Factor N/Mi fosetyl 0 (observed) E (expected) O/E control wt -0 1 N/Mi 14 2 wt 5.0Og/l 7 3 wt 0.5 g/l 2 4 N/Mi 5.0 g/l 80 21 3.8 0.5 gI 56 16 WO 98/29537 WO 9829537PCT/EP97/07253 -91- NIMi 1 wt wild-type Ws Table 39 Action Against Peronospora parasiica In Arabidopsis Component 1: N/Mi overexpression (line 6E) Component 11: copper hydroxide_______ Test no. Components Fungal Growth Inhibition Synergy Factor N/Mi Cu(OH) 2 0 (observed) E (expected) OlE control wt -0 1 N/Mi 2 wt 2.0Og/l 0 3 wt 0.2 g/I 0 4 wt 0.02 g/ 0 N/Mi 2.0 gI 66 10 6.6 6 N/Mi 0.2 g/l 14 10 1.4 7 N/Mi 0.02 g/l 20 10 wt wild-type Ws Table Action Against Peronospora parasitica In Arabidopsis Component 1: N/Mi overexpression (line 7C) Component II: copper hydroxide Test no. Components Fungal Growth Inhibition Synergy Factor N/Mi Cu(OH) 2 0 (observed) E (expected) O/E control WI 0 1 NIMi 14 2 WI 2.0 g/I 0 3 wt 0.2 g 0 4 wt 0.02 g/I 0 WO 98/29537 PCT/EP97/07253 -92- NIM1 2.0 g/l 77 14 6 NIM1 0.2 g/ 51 14 3.6 7 NIM1 0.02 g/i 55 14 3.9 wt wild-type Ws As can be seen from the above tables, synergistic disease-resistant effects were demonstrated in plants overexpressing NIM1 by application of metalaxyl, fosetyl, and copper hydroxide. For example, in the untreated NIM1 plant (line 6E), 10% fungal growth inhibition was seen relative to the untreated wild-type plant; this demonstrates that the constitutive SAR gene expression in this NIM1 overexpressor correlates with disease resistance. As shown above in Table 37, however, by applying fosetyl at 5.0 g/I (a concentration normally insufficient for efficacy) to the immunomodulated (SAR-on) NIM1 overexpressing plant, the observed level of fungal growth inhibition increased to 93%. 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. In another example, in the untreated NIM1 plant (line 7C), 14% fungal growth inhibition was seen relative to the untreated wild-type plant, demonstrating that the constitutive SAR gene expression in this NIM1 overexpressor correlates with disease resistance. As shown above in Table 40, however, by applying copper hydroxide at 2.0 g/I (a concentration normally insufficient for efficacy) to the immunomodulated (SAR-on) NIM1 overexpressing plant, the observed level of fungal growth inhibition increased to 77%. The synergy factor of calculated from these data further demonstrates the synergistic effect achieved by applying a microbicide to an immunomodulated (SAR-on) plant.

Thus, the combined use of of immunomodulated plants overexpressing NIM1 with low, normally ineffective concentrations of microbicides to achieve disease resistance provides advantages that should be apparent to those skilled in the agricultural arts.

Normally toxic or otherwise undesirable concentrations of microbicides can be avoided by taking advantage of the synergies demonstrated herein. In addition, economic gains can be realized as a result of the decreased quantity of microbicides required to provide a given level of protection to plants.

Example 35: Synergistic Disease Resistance Attained by Applying A Chemical Inducer Of SAR to Transgenic Plants Overexpressing NIM1 WO 98/29537 PCT/EP97/07253 -93- 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 H20, 10pM BTH, or 100pM 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.

The results of Northern analysis for Ws and four of the NIM-overexpressing lines (3A, 6E, and 7C) are shown in Figure 3. PR-1 gene expression in the wild-type Ws line was barely detectable after the low level 10pM BTH treatment (a BTH concentration of 100-300 gM is normally required for efficacy). Ws plants from this treatment were also still susceptible to the fungal pathogen P. parasitica (Emwa). In all of the NIMl-overexpressing lines, however, there was a much stronger response for PR-1 gene expression following the low-level BTH treatment. In addition, all of the NIMl-overexpressing lines treated with BTH showed complete or almost complete resistance to P. parasitica. Leaves stained with lactophenol blue to identify the presence of fungal hyphae (Dietrich et al.

(1994)) confirmed the absence of fungal growth in the NIMi-overexpressing lines. PR-1 gene expression in leaf tissue following the 100pM BTH treatment was also much stronger and quicker in the NIM1-overexpressing lines relative to wild-type. Thus, immunomodulated plants are able to respond much faster and to much lower doses of BTH, as shown by PR-1 gene expression and resistance to P. parasitica, than wild-type plants. This data demonstrates that synergistic disease resistance is achieved by applying a chemical inducer of systemic acquired resistance such as BTH to an immunomodulated (SAR-on) plant such as a NIMI-overexpressing plant.

Thus, the combined use of immunomodulated plants overexpressing NIM1 with low, normally ineffective concentrations of SAR-inducing chemicals such as BTH to achieve disease resistance provides advantages that should be apparent to those skilled in the agricultural arts. Normally toxic or otherwise undesirable concentrations of SAR-inducing chemicals can be avoided by taking advantage of the synergies demonstrated herein. In -94addition, economic gains can be realized as a result of the decreased quantity of SARinducing chemicals required to provide a given level of protection to plants.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

9@ S* WO 98/29537 PCTIEP97/07253 SEQU= LIS=~l~ GENRAL INONAIC

APPLICANT:

NAM: Novartis AG STREr: Schwmarzwaldaliee 215 CI=: Easel Cott='R: Switzerland pogW]AL CODE (ZIP): 4002 TmEpH=: +41 61 69 11 11 TmLEFAX: 41 61 696 79 76 TELEx: 962 991 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentln Release Version #1.30 (ii) TITLE OF INVENTION: METHOD FOR PROTECTING PLANTS (iii) NUMBER OF SEQUENCES: 32 INFORMATION FOR SEQ ID NO:1: Wi SEQUENCE CHARACTERISTICS: LENGTH: 5655 base pairs TYPE: nucleic acid STRA2NDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO WO 98/29537 PCT/EP97/07253 96- (ix) FEATURE: NAME/KEY: exon LOCATION: 2787. .3347 OTHER INFORMATION: /product= w1st. exon of NIMI" (ix) FEATURE: NAME/KEY: exon LOCATION: 3427. .4162 OTHER INFORMATION: /product= w2nd exon of (ix) FEATURE: NAME/KEY: exon LOCATION: 4271. .4474 OTHER INFORMATION: /product= 63rd exon of NIMla (ix) FEATURE: NAME/KEY: exon LOCATION: 4586. .4866 OTHER INFORMATION: /product= "4th exon of NIMI" (ix) FEATURE: NAME/KEY: CDS LOCATION: join(2787. .3347, 3427. .4162, 4271. .4474, 4586. .4866) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: TGTGATGCAA GTCATGGGAT ATTGCTTTGT GTTAAGTATA CAAAACCATC ACGTGGATAC ATAGTCTTCA AACCAACCAC TAAACAGTAT CAGGTCATAC CAAAGCCAGA AGTGAAGGGT 120 TGGGATATGT CATTGGGTTT AGCGGTAATC .GGATTGAACC CTTTCCGGTA TAAAATACAA 180 AGGCTTTCGC AGTCTCGGCG TATGTGTATG TCTCGGGGTA TCTACCATTT GAATCACAGA 240 ACTTTTATGT GCGAAGTTTT CGATTCTGAT TCGTTTACCT GGAAGAGATT AGAAAATTTG 300 CGTCTACCAA AAACAGACAG ATTAATTTTT TCCAACCCGA TACAAGTTTC GGGGTTCTTG 360 CATTGGATAT CACGGAACAA CAATGTGATC CGGTTTTGTC TCAAAACCGA AACTTGGTCC 420 WO 98/29537 WO 9829537PCT/EP97/07253 97-

TTCTTCCATA

GGTGCTATTC

AAGAGCAGTT

CTCCGAACTC

GTCAGTGGAC

TTGAAAAGTC

TGATGTTTTC

AAACAAAGAT

GTGGGTTAAA

TTGATTACGT GGACTCCAAG CAACGACGTT

CTCTACAACA

TGTTCTTTCG

AGCAACGGGC

GTTGACAAGG

ATCTAGTGAT

GGTTTATGTA

TATTTTATTA

TGTCCCAATT

ATATACATAA

TTACATATAT

ATATATGTAC

AAATTTATTA

CTATAATAGA

TATATCATTT

TAAACGCAGA

TTTGTTTTCC

CGACACTTTA

ATCTCAAGTC

GTTTAATTGT

ATAATACCAA

TATCAAGGGT

TTCTCTCTTA

TATCTAAAGC

TTATAGCTTA

GGTATGCTGT

AATATTTGGC

TGGTAGAAGA

TAAAAAATT)

GAAGTTGAAT

GTTTTGTTCT

AAAAAAAAAT

TCAAGTCTCA

TTTTTATAAG

ACATTGTTTT

TCCTGTTTAT

AATAATATAT

AACACATATT

CCAATATAAC

CCACGTATAT

AATTGGGTGT

,TAAAAAAAT

T

I

LATTAAAAGAPI

TCAGGATTAG

CAAGAAGATG

GTGAAAGATA

GTATTGTTTC

TTAGTTTATG

GATTACGAGA

AAAAAAAATG

ATTGGCTCGC

GTAAAAAGGA

ATGAATATTT

AGTTGAAAAC

TAGTTAATAA

TAGACACAAC

CCGTATCTAT

ATATTCTCCA

TTATCTAAAG

ATATCAGATT

AACTATTTCA

TTGAGTTATA

TCAGATACGA

TTCACGAGTTI

TTAAAAGCAT'

GTAGTAGTGA

CAAAAAAAGA

GGGTTGATCT4

GGCCGACAAA

TCATTGTGGG

ATATTGAATT

AATCTGATTT

AGTTACTGTA

AAGATATTTT

ACGTAATATC

GTTTTATAAG

AAAAAAACGC

TTTATCACAA

GATTCAATTA

TAAAATTGTT

LGAGAGTTATT

GGGAAGCTA

kTGGGTTTTA rGGAGTAGAT

E'CGTGGTTGC

GGGATCTGAT

GAACGGAAGA

TGCAAACGTA

GCATAAATAT

TTGTTTCTTA

TTTGGCTAGT

TAGAAAATAG

AATATATTAG

TTACTATTGT

CTTTTATACA

ATGGTACACA

TATTTATCAA

AATTTTATAA

CAAAAGATAA

GTAAATTTAC

AAAAGTAAAA

480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 TTAGTAAAAT TAATTAAATA TGTGATGCTP TTAAAATCAT ACAAATCTTA TCCTAATTTA ACTTATCATT TAAGAAATAC AACGCGGAAA GCAATAATTT ATTTACCTTA TTATAACTCC TATATAAAGT ACTCTGTA WO 98/29537 PCT/EP97/07253 -98- TTCAACATAA TCTTACGTTG TTGTATTCAT AGGCATCTTT AACCTATCTT TTCATTTTCT 1740 GATCTCGATC GTTTTCGATC CAACAAAATG AGTCTACCGG TGAGGAACCA

ATGCAGATTC

GTGAAGGATG

CTTCTTCTTC

AGCCAAATTT

TCAGTTTCCA

GTTTAGACGT

TGAAAAAGCT GATTTATCGC ATGATTCAGA

GTCTTTTATA

ATTATGACTT

AGAGCGTTTT

TAATTATAGT

ATATACATTA

TTTTACTTCA

TTAAAAAATA

ATATATTTAT

TTCTCATATA

CCCGA.ACCGG

TTCCTGGAAA

ATCTCACCAC

TTTAACCAAA

GAATTTCAAT

TGTATACAAT

TCATGTTTTT

CATTTGCTAT

GTAAACATGC

CAAA.ACTTAT

AAGAAXATAA

ATATTTATAT

ATCATCTCCA

CAAAAATTAG

TTTAGCTTCC

TTTACCGGT'I

CACTCTCGT'I

TCCAGTTGA1

TCATCGGAAC

AATTGTTTTT

AATGTAATTT

AGAACAAGGA

TGAACACTGA

GTGAATAAAG

ACAGAAATGT

ATTTATATGA

AATCTAGTT'I

CAACACAAAP

TGTTATATCVI

'TTGGTGAAA]

GACTTGACT9

AAGGTCTT'

CTGTTG AT( Mel

GCAACATCGA

GTTATGAATT

ACGAGAAGTT

AA.ATCAAATC

ATTCCTATAT

GAATAGTTCC

AAATTACTTT

CATGAAACTT

AACTTTCACA

AAATAACGAA

GGTTCAGGGG

TGTCTCCGGT

TTTTAAAAAA

GTAAACCGTG

GTCCGGAAAA

TGCTTTTACG

GAAGGCAAAT

CTAATTAAAA

CTATAATGAT

AGGAANTATT

TTCAATAA.AC

AATATACGTT

TGTAAATCTA

CCGGATGAAA

CTTACCGAAC

ATAAATACTA

GATCTCTGAC

A~GAGGTGATT

CACCAATCAA

TCGTAGTTAT

AACTAAAGAA

AAATATATTC

TTTGTTGTGA

CGACTTGATT

GAAUAAATATA

CCCTTTATCA

ATTCTTAAAT

AATAAATTTT

CGGATTGAAC

ACATTTATAA

AAAGATTCCT

1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2813 GGACGAGGAT GCTTCTTCAT GGCTCTGCTC GTCAATGGTT ATCTTCGATC CGTTGATTAG CAGAGATCTC TTTAATTTGT GAC ACC ACC ATT GAT QGA TTC GCC Asp Thr Thr Ile Asp Gly Phe Ala GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC GTC OCT ACC GAT AAC ACC Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val Ala Thr Asp Asn Thr 15 20 2861 WO 98/29537 WO 9829537PCT/EP97/07253 99-

GAC

Asp TCC TCT ATT GTT TAT Ser Ser Ile Val Tyr CTG GCC GCC GAA CAA GTA CTC ACC GGA CCT Leu Ala Ala Giu Gin Val Leu Thr Gly Pro 2909 GAT GTA TCT Asp Val Ser GAC TCG CCG Asp Ser Pro

GOT

Ala CTG CAA TTG CTC Leu Gin Leu Leu

TCC

Ser 50 AAC AGC TTC Asn Ser Phe GAT GAT TTC TAC Asp Asp Phe Tyr

AGO

Ser 65 GAO GOT AAG CTT Asp Ala Lys Leu GAA TCC GTC TTT Glu Ser Val Phe OTT CTC TCC GAC Val Leu Ser Asp GCG AGA AGC TOT Ala Arg Ser Ser 2957 3005 000 CG Gly Arg GAA OTT TCT TTC Giu Val Ser Phe

CAC

His 80 CGO TGC GTT TTG TCA Arg Cys Val Leu Ser

TTC

Phe

AAC

Asn TTC AAG AGC GCT Phe Lys Ser Ala ACC GCC GCC OTG Thr Ala Ala Val 110 TTA GCC Leu Ala 95 GCC GCT AAG Ala Ala Lys

AAG

Lys 100 GAG AAA GAO TCC Giu Lys Asp Ser

AAC

Asn 105 3053 3101 3149 AAG CTC GAG OTT Lys Leu Giu Leu

AAG

Lys 115 GAG ATT GCC AAG Glu le Ala Lys OAT TAC Asp Tyr 120 GAA GTC GOT Glu Val Oly

TTC

Phe 125

GAT

Asp TCG GTT GTG Ser Val Val

ACT

Thr 130 GTT TTG OCT TAT Val Leu Ala Tyr AGC AGA Ser Arg AAT TGC Asn Cys 155

GTG

Val 140 AGA COG CCG COT Arg Pro Pro Pro

AAA

Lys 145

GGA

Gly OTT TCT GMA Val Ser Oiu

TOO

150 GTT TAO AOC Val Tyr Ser 135 GCA GAC GAO Ala Asp Glu ATO TTG GAG Met Leu Glu 3245 3293 3197 TOO CAC GTO GOT TOO Cys His Val Ala Cys 160 COG CCG GCG OTG Arg Pro Ala Val OAT TTC Asp Phe 165

GTT

Val 170 OTO TAT Leu Tyr TTG OCT TTC ATC TTC MAG ATO CCT Leu Ala Phe Ile Phe Lys Ile Pro 175 180 GMA TTA ATT ACT Glu Leu Ile Thr

OTC

Leu 185 3341 TAT CAG GTAAAMCACC ATCTGCATTA AGCTATGGTT ACACATTCAT GAATATGTTC Tyr Gin 3397 WO 98/29537 WO 9829537PCT/EP97/07253 -100- TTACTTGAGT ACTTGTATTT GTATTTCAG AGO CAC TTA TTG GAC GTT Arg His Leu Leu Asp Val 190 OTA GAC Val Asp 195 AAT ATA Asn Ile 210 3450 AAA OTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT Leu Lys Leu Ala 3498 Lys Val Val Ile TGT GGT AAA OCT Cys Gly Lys Ala 215 GTC AAG TCT AAT Val Lys Ser Asn 230 Glu 200 Asp Thr Leu Val le 205 TOT ATG AAG CTA Cys Met Lys Leu TTG GAT AGA TGT Leu Asp Arg Cys 220 AGT CTT OAA ANO Ser Leu Glu Lys MAA GAG ATT ATT Lys (flu Ile Il.e 225 3546 3594 GTA GAT ATG Val Asp Met

GTT

Val 235 TTG CCG GAA Leu Pro Giu GAG CTT Giu Leu 245 OTT AAA GAG ATA Val Lys Giu Ile

ATT

Ile 250 GAT AGA CGT AAA Asp Arg Arg Lys GAG CTT GOT TTG GAG Glu Leu Gly Leu Glu 255 CAT AAO OCA CTT GAC His Lys Ala Leu Asp 275

GTA

Val 260 CCT AMA OTA AAO Pro Lys Val Lys

AMA

Lys 265 CAT OTC TCG AAT His Val Ser Asn

GTA

Val 270 3642 3690 3738 TCG OAT GAT Ser Asp Asp ATT GAG Ile Oiu 280 TTA OTC AAG TTG Leu Val Lys Leu TTG AAA GAG OAT Leu Lys Giu Asp CAC ACC His Thr 290 AAT CTA OAT Asn Leu Asp

OAT

ASP

295 OCG TOT GCT CTT Ala CYS Ala Leu TTC OCT OTT GCA Phe Ala Val Ala TAT TGC AAT Tyr Cys Asn 305 3786 GTG AAG Val1 Lys CAT AGO His Arg 325

ACC

Thr 310 OCA ACA OAT CTT Ala Thr Asp Leu TTA AMA Leu Lys 315 CTT OAT CTT Leu Asp Leu GCC OAT GTC AAC Ala Asp Val Asn 320 OCT C ATO COO Ala Ala Met Arg 3834 3882 AAT CCG AGO GGA Asn Pro Arg Gly

TAT

Tyr 330 ACO GTG CTT CAT Thr Val Leu His AAO GAG CCA CAA TTG ATA CTA TCT CTA TTG GA AAA GOT GCA ACT OCA 33 3930 WO 98/29537 WO 9829537PCT/EP97/07253 -101- Lys Glu Pro Gin Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser -4A n 345 350 Ala 355 TCA GAN GCA ACT Ser Glu Ala Thr

TTG

Leu 360 GAA GGT AGA ACC Giu Gly Arg Thr

GCA

Ala 365 CTC ATG ATC OCA AAA CAA Leu Met Ile Ala Lys Gin 370 3978 4026 GCC ACT ATG Ala Thr Met GTT GAP. TGT AAT Val Giu Cys Asn

AAT

Asn 380 ATC CCG GAG CA.

Ile Pro Glu Gin AP.G CAT LYS His TCT CTC APA Ser Leu Lys 390 GGC CGA CTA TGT Giy Arg Leu Cys

GTA

Val 395 GAP. ATA CTA GAG Giu Ile LeU GlU

CAP.

Gin 400 GA. GAC AAAP Giu Asp Lys 4074 CGA GAA Arg Giu 405 CAA. ATT CCT AGA Gin Ile Pro Arg GAT GTT Asp Val 410 CCT CCC TCT Pro Pro Ser TTT GCA GTG GCG GCC Phe Ala Val Ala Ala 4122 GAT GAP. TTG AAG ATG Asp Giu Leu Lys Met 420 ACG CTG CTC GAT CTT GAA AAT AGA G 4162 Thr Leu Leu Asp Leu Glu Asn Arg 425 430 GTATCTATCA AGTCTTATTT CTTATATGTT TGAATTAAAT TTATGTCCTC TCTATTAGGA AACTGAGTGA ACTAATGATA ACTATTCTTT GTGTCGTCCA CTGTTTAG TT GCA CTT GCT CAA.

Ala Gin CGT CTT TTT Arg Leu Phe 440 GAA ATG AAG Giu Met Lys GGA ACA Gly Thr 455 GGT ACG G ly Thr CCA ACO GAP. GCA CAA GCT Pro Thr Giu Ala Gin Ala 445 TGT GAG TTC ATA GTG ACT Cys Giu Phe Ile Val Thr 460 AP.G AGA ACA TCA CCG GGT Lys Arg Thr Ser Pro Gly 475 GCA ATG Ala Met AGC CTC Ser Leu GTA AAG Val Lys 480 Val Ala Leu 435 GAG ATC GCC Giu Ile Ala 450 GAG CCT GAC Giu Pro Asp 465 ATA GCA CCT Ile Ala Pro 4222 4278 4326 4374 4422 CGT CTC Arg Leu

ACT

Thr 470 TTC AGA ATC Phe Arg Ile CTA GA. GAG CAT CA. AGT AGA CTA AAA. GCG CTT TCT AAA.

Leu Giu Glu His Gin Ser Arg Leu Lys Ala Leu Ser Lys 4470 WO 98/29537 WO 9829537PCT/EP97/07253 -102- 485 490 495 ACC G GTATGGATTC TCACCCACTT CATCGGACTC CTTATCACAA AAAACAAAAC Thr 500 TAAATGATCT TTAAACATGG TTTTGTTACT TGCTGTC!TGA CCTTGTTTTT TTTATCATCA G TG GAA CTC GGG AAM CGA TTC TTC CCG CGC TGT TCG GCA GTG CTC Val Giu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu 505 510 515 4524 4584 4629 GAC CAG ATT ATG Asp Gin Ile Met GAC GAC ACT GCT Asp Asp Thr Ala 535 ATA CAA GAG ACA Ile Gin Glu Thr

AAC

Asn 520

GAG

Giu TGT GAG GAC TTG Cys Glu Asp Leu AAA CGA CTA CAM Lys Arg Leu Gin 540 AAG AAG GCC TTT Lys Lys Ala Phe ACT CAA Thr Gin 525 MAG MAG LYS LYS CTG GCT TGC GGA GAA Leu Ala Cys Giy Giu 530 CAA AGG TAC ATG GAA Gin Arg Tyr Met Giu 545 4677 4725 4773 4821

CTA

Leu AGT GAG GAC Ser Glu Asp 550 GGA AAT TCG Gly Asn Ser 565 GGT GGA AAG Giy Gly Lys 580 555

TCG

Ser TCC CTG ACA Ser Leu Thr AGG TCT AAC Arg Ser Asn

GAT

Asp 570

CGT

Arg ACT TCT TCC Thr Ser Ser

ACA

Thr 575

CGT

Arg MAT TTG GMA TTA Asn Leu Giu Leu 560 TCG AMA TCA ACC Her Lys Ser Thr CGT CGG TGA Arg Arg AMA CTC TCT CAT Lys Leu Ser His 590 4866

GACTCTTGCC

TAACTGTTTA

ATTATTGCTC

ATTTGTAATA

GAATCAMAGT

TCTTAGTGTA

TGTCTATCGT

CAGGTGTGCT

TATATTTATG

GTGAAATAAT

ATTTTTGCTG

TGGCGTCATA

TCAAACAAAT

TACATCAACA

GTCAAATTGT

TACCATATAA

TAGTTTCGCT

GTTGTAACAA

ATAACCCATG

TCATCTGTTG

TTCTGTTTTC

CTTCGTTTTG

TTTGMACCAA

ATGGTGTTAC

GATATTTTCC

ATGATGACTG

CATCCTGTGT

TGGTATACAG

AGAGTTGCTA

ACCAAGAACC

4926 4986 5046 5106 5166 5226 AAAGAATAT TCAAGTTCCC TGAACTTCTG GCMACATTCA TGTTIATATGT ATCTTCCTAA WO 98/29537 WO 9829537PCTIEP97/07253 -103-

TTCTTCCTTT

AAGAGAACAC

ATTTGTGAAT

TTCTTCGATT

ACTGAAAGCT

TCCGACCACT

CGAGCTTCTG

CTTGTGGAT

AACCTTTTGT

TGAGTGGGCG

GACACAAGTT

GAAACTTCCC

TTCACAAATT

GGTCATGAGC

AGTCCTTCTT

AACTCGAATT ACACAGCAAG TGTAAGGTGC ATTCTCCTAG AACAATCCTT TGCACCATTT ACATGTGCAG GTGCGTTCGC GCCCTCAAAT CTTCTGTTTC CAGAGCCCAC TGATTTTGAG TTTGATGTCC TTTATGTAGG

TTAGTTTCAG

TCAGCTCCAT

CTGGGTGCAT

TGTCACTGAT

TATCGTCATG

GGAATTGGGC

AATCAAATTC

GTCTAGAGAT

TGCATCCAA&C

ACATGGAAAC

AGACCAAGAG

ACTCCATATC

TAACCATTTC

TTCCTTCTGA

5286 5346 5406 5466 5526 5586 5646 5655 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 594 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Asp Thr Thr Ile Asp Gly Phe Ala Asp Ser Tyr 1 5 10 Thr Ser Phe Val Ala Thr Asp Asn Thr Asp Ser Ser 25 Ala Ala Glu Gin Val Leu Thr Gly Pro Asp Val Ser Glu Ile Ser Ser Ile Val Tyr Leu Ala Leu Gin Leu Val Leu Ser Asn Ser Phe Glu Phe ASP Ser Pro Glu ASP Asp Phe Tyr Asp Ala Lys Leu Val Ser Asp Gly Arg 75 Val Ser Phe His WO 98/29537 WO 9829537PCTIEP97/07253 -104-- Arg Cys Val Leu Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala Ala Ala Lys Glu Leu Lys 115 Lys 100 Glu Lys Asp Ser Asn 105 Asn Thr Ala Ala Val Lys Leu 110 Asp Ser Val Glu Ile Ala Lys Asp 120 Tyr Glu Val Gly Val Thr 130 Val Leu Ala Tyr Val 135 Tyr Ser Ser Arg Val 140 Arg Pro Pro Pro Lys 145 Gly Val Ser Glu Cys 150 Ala Asp Glu Asn Cys 155 Cys His Val Ala Cys 160 Arg Pro Ala Val Asp 165 Phe Met Leu Glu Val 170 Leu Tyr Leu Ala Phe Ile 175 Phe Lys Ile Val Val Asp 195 Giu Leu Ile Thr Tyr Gin Arg His Leu Leu Asp 190 Leu Lys Leu Lys Val Val Ile Glu 200 Asp Thr Leu Val Ala Asn 210 Ile Cys Gly Lys Cys Met Lys Leu Leu 220 Asp.Arg Cys Lys Giu 225 Ile Ile Val Lys Asn Val Asp Met Val 235 Ser Leu Glu Lys Ser 240 Leu Pro Glu Glu Leu 245 Val Lys Glu Ile le 250 Asp Arg Arg Lys Glu Leu 255 Gly Leu Glu Val 260 Pro Lys Val Lys His Val Ser Asn Val His Lys 270 Leu Lys Glu Ala Leu Asp Ser 275 Asp Asp Ile Glu 280 Leu Val Lys Leu Cys Ala Leu His 300 Asp His 290 Thr Asn Leu Asp Asp Ala 295 Phe Ala Val Ala WO 98/29537 WO 9829537PCT/EP97/07253 105 Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala 305 310 315 320 Asp Val Asn His Arg 325 Asn Pro Arg Gly Tyr 330 Thr Val Leu His Val Ala 335 Ala Met Arg Ala Ser Ala 355 Lys 340 Giu Pro Gln Leu Leu Ser Leu Leu Giu Lys Gly 350 Leu Met Ile Ser Giu Ala Thr Leu 360 Giu Gly Arg Thr Ala Lys 370 Gin Ala Thr Met Ala 375 Val Giu Cys Asn Asn 380 Ile Pro Giu Gin Cys 385 Lys His Ser Leu Lys 390 Gly Arg Leu Cys Val 395 Glu Ile Leu Glu Gin 400 Giu Asp Lys Arg Giu 405 Gin Ile Pro Arg Asp 410 Val Pro Pro Ser Phe Aia 415 Val Ala Aia Val Ala Leu 435 Asp 420 Giu Leu Lys Met Thr 425 Leu Leu Asp Leu Giu Asn Arg 430 Ala Ala Met Ala Gin Arg Leu Phe 440 Pro Thr Giu Ala Gin 445 Giu Ile 450 Ala Giu Met Lys Giy 455 Thr Cys Glu Phe Ile Val Thr Ser 460 Ser Pro Giy Val Leu Giu 465 Pro Asp Arg Leu Thr 470 Giy Thr Lys Arg Lys 480 Ile Ala Pro Phe Arg 485 Ile Leu Giu Glu His 490 Gin Ser Arg Leu Lys Ala 495q Leu Ser Lys Thr Val Giu 500 Leu Gly Lys 505 Arg Phe Phe Pro Arg Cys Ser 510 Gin Leu Ala Ala Val Leu 515 Asp Gin Ile Met Asn Cys Glu Asp 520 Leu Thr 525 Cys Gly Giu Asp Asp Thr Ala Giu Lys Arg Leu Gin Lys Lys Gin Arg WO 98/29537 WO 9829537PCTIEP97/07253 -106- 530 Tyr 545 Met Glu Ile Giln Giu 550 Thr Leu Lys Lys Ala 555 Phe Ser Giu Asp Asn 560 Thr Ser 575 Leu Glu Leu Gly Asn 565 Ser Ser Leu Th~r Asp 570 Ser Thr Ser Ser Lys Ser Thr Gly Gly Lys Arg Ser Asn 580 5859 Arg Lys Leu Ser His Arg Arg 590 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 314 amino acids TYPE: amino acid STP.ANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Phe Gin Pro Ala Gly His Gly Gin Asp Trp, Ala Met Glu Gly Pro Arg Asp Gly Gly Leu Asp Leu Lys Lys Glu Arg Leu 25 Val Asp Asp Arg His Asp Ser Val Lys Glu Ser Met Lys Asp Giu 40 Glu Tyr Giu Gin Met Leu Arg Glu Ile Arg Leu Gin 55 Pro Gin Glu Ala Pro Leu Ala Ala Glu Pro Trp Lys Gin Gin Leu Thr Giu Asp Gly Asp Ser Phe Leu His Leu WO 98/29537 PCT/EP9707253 -107- Ala Gin Ile Ile His Glu Leu Glu Lys Pro Leu Thr Met Glu Val Ile Gly Phe Val Lys Gly Thr Pro Leu 115 Ala Leu Leu Ala Phe Leu Asn 105 Thr Gin Asn Asn Leu Ala Val lie 120 Asp Asn Gin Pro Gly 125 Asp Leu Gin Gin 110 Ile Ala Glu Phe Arg Gly Lys Ala Gly 130 Asn Thr Cys 135 Ala Pro Giu Leu Pro Leu His 145 Ala Leu 150 Thr Cys Glu Gin Gly 155 His Leu Ala Ser Val 160 Val Leu Thr Gin 165 Tyr Cys Thr Pro Gin 170 Cys Leu His Ser Val Leu 175 Gin Ala Thr His Gly Tyr 195 Val Asn Ala Asn 180 Leu Asn Giy His Thr 185 His Leu His Leu Ala Ile Val Giu 200 Asn Leu Val Thr Leu 205 Leu Ala Ser Thr 190 Gly Ala Asp His Leu Ala Gin Glu Pro 210 Asp Cys 215 Asp Gly Arg Thr Ala 220 Leu Val 225 Ala Leu Gln Asn Pro 230 Val Leu Val Ser Leu 235 Tyr Leu Lys Cys Gly 240 Asp Val Asn Arg 245 Thr Tyr Gin Gly 250 Gin Ser Pro Tyr Gin Leu 255 Thr Trp Gly Thr Leu Glu 275 Arg Pro 260 Asn Leu Ser Thr Arg Gin Met Leu 280 Ile 265 Pro Gin Gin Leu Gly Gin Leu 270 Glu Glu Ser Glu Ser Giu Asp Tyr Asp 290 Thr Glu Ser Glu Phe Thr 295 Glu Asp Giu Leu 300 Pro Tyr Asp Asp WO 98/29537 -108- Cys Val Phe Gly Gly Gin Arg Leu Thr Leu 305 310 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 314 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: PCT/EP97/07253 met 1.

Phe Gin Pro Ala Gly His Gly Gin Asp Trp Ala Met Giu Gly Pro Arg Asp Gly Leu Lys Lys Giu Arg Leu 25 Val Asp Asp Arg His Asp Ser Gly Leu Asp Ser Met Lys Asp Asp Tyr Giu Gin Met Val Lys Giu Leu Arg Giu Ile Arg Leu Gin 55 Pro Gin Giu Ala Pro Leu Ala Ala Giu Pro Trp Lys Gin Gin Ala Ile Ile His Giu Leu 70 Thr GiU Asp Gly ASP .75 Ser Phe Leu His Leu Giu Lys Thr Leu Thr 90 Met Giu Val Ile Giy Gin Val Lys Giy Asp 100 Leu Ala Phe Leu Asn Phe Gin Asn Asn 105 Leu Gin Gin 110 Thr Pro Leu His 115 Leu Ala Val Ile Thr Asn Gin Pro Gly Ile Ala Giu 120 125 WO 98/29537 PCTIEP97/07253 109- Ala Leu Leu Lys Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly Asfl 145 Ala Thr Pro Leu Val Leu Thr His Ala Leu 150 Thr Cys Glu Gin Gly 155 His Cys Leu Ala Ser Val 160 Gin 165 Cys Thr Pro Leu His Ser Val Leu 175 Gin Ala Thr His Gly Tyr 195 Val Asn Ala Asn 180 Leu Asn Gly His Thr 185 His Leu His Leu Gly Ile Val Giu 200 Asn Leu Val Thr Leu 205 Leu Ala Ser Ile 190 Gly Ala Asp His Leu Ala Gin Glu Pro 210 Val Asp Cys 215 Asp Gly Arg Thr Ala 220 Leu Leu Gin Asn 225 Ala Pro 230 Val Leu Val Ser Leu 235 Tyr Leu Lys Cys Gly 240 Asp Val Asn Arg 245 Pro Thr Tyr Gin Ser Pro Tyr Gin Leu 255 Thr TrP Gly Thr Leu Giu 275 Tyr Asp Thr 290 Arg 260 Asn Ser Thr Arg Ile 265 Pro Gin Gln Leu Leu Gin Thr Leu 280 Thr Glu Ser Glu Asp 285 Pro Gly Gin Leu 270 Glu Glu Ser Tyr Asp ASP Glu Ser Glu Phe 295 Giu Asp Glu Cys 305 Val Phe Gly Gly Gin 310 Arg Leu Thr Leu INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 314 amino acids TYPE: amino acid WO 98/29537 -110- STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein PCT/EP97/07253 (xi) SEQUENCE DESCRIPTION: SEQ ID 110:5: Met Phe Gin Pro Ala Glu Pro Gly Gin Glu Trp Ala Met Lys Arg Asp Ala Gly Leu Asp LeU Arg Glu Leu Lys Glu Arg Ser Met Lys Asp Glu 40 Pro Leu 25 Glu Gln Leu Asp Asp Arg Tyr Glu Gin Met Glu Ala Pro Arg Gly Asp Ser Phe Glu Gly Pro His Asp Ser Val Lys Glu Gly Ala Glu Ile Arg Leu Pro Trp Glu 55 Thr Lys Gin Gin Ala Leu 70 Glu Glu Asp 75 Met Leu His Leu Ile Ile His Giu Leu Lys Ala Leu Thr 90 Phe Glu Val Val Arg Gin Vai Lys Giy Thr Pro Leu 115 Ala Leu Leu Asp 100 His Ala Phe Leu Asn 105 Thr Gin Asn Asn Leu Ala Val Ile 120 Asp Asn Gin Pro Glu 125 Asp Leu Gin Gin 110 Ile Ala Glu Phe Arg Gly Giu Ala Giv 130 Asn Thr 145 Pro Leu His Leu 150 Cys 135 Ala Pro Glu Leu Arg 140 Cys Giu Gin Gly Cys Leu Ala Ser Val 155 160 Gly Val Leu Thr Gin Pro Arg Gly Thr Gin 165 170 His Leu His Ser Ile Leu 175 WO 98/29537 WO 9829537PCT/EP97/07253 -111 Gin Ala Thr Asn Tyr Asn Gly His 180 Tbr 185 Cys Leu His Leu Ala Ser Ile 190 His Gly Tyr 195 Leu Gly Ile Val Glu 200 Leu Leu Val Ser Leu 205 Gly Ala Asp Val Asn 210 Ala Gin Giu Pro Asn Gly Arg Tbr Leu His Leu Ala Asp Leu Gin Asn Pro 230 Asp Leu Val Ser Leu 235 Leu Leu Lys Cys Gly 240 Ala Asp Val Asn Arg 245 Val Thr Tyr Gin Gly 250 Tyr Ser Pro Tyr Gin Leu 255 Thr Trp Gly Thr Leu Glu 275 Arg 260 Pro Ser Thr Arg Gin Gin Gin Leu Gly Gin Leu 270 Giu Glu Ser Asn Leu Gin Met Leu 280 Pro Glu Ser Glu Asp 285 Tyr Asp 290 Thr Glu Ser Glu Phe 295 Thr Glu Asp Glu Pro Tyr Asp Asp Cys 305 Val Leu Gly Gly Gin 310 Arg Leu Thr Leu INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 2011 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Arabidopsis thaliana (ix) FEATURE: NAME/KEY: misc-feature WO 98/29537 WO 9829537PCTIEP97/07253 -112- LOCATION: 1.-.2011 OTHER INFORMATION: /note= "NIMI cDNA sequencem (ix) FEATURE: NAME/KEY: CDS LOCATION: 43. .1824 OTHER INFORMATION: /product= "NINI protein" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGOAACCTGT TO ATO GAC ACC ACC Met Asp Thr Thr 1 ATT GAT GGA TTC GCC GAT TCT TAT GAA ATC AGC AGC Ile Asp Gly Phe Ala Asp Ser Tyr Giu Ile Ser Ser ACT AGT TTC Thr Ser Phe 102 GCT ACC GAT AAC Ala Thr Asp Asn GAC TCC TCT ATT Asp Ser Ser Ile TAT CTG GCC CC Tyr Leu Ala Ala GAA CAA Olu Gin 150 GTA CTC ACC Val Leu Thr

GGA

Gly COT GAT GTA TOT GCT CTO CAA TTG CTC Pro Asp Val Ser Ala Leu Gin Leu Leu 45 TCO AAO AGC Ser Asn Ser GAC OCT AAG Asp Ala Lys 198 TTC OAA TOO Phe Glu Ser OTO TTT GAO TCG CCG Val Phe Asp Ser Pro 60 GAT OAT TTC TAC AGC Asp Asp Phe Tyr Ser

OTT

Leu CTC TCC GAC 000 Leu Ser Asp Gly

CG

Arg OAA OTT TCT TTC CAC COO TOO OTT TTG Olu Val Ser Phe His Arg Cys Val Leu

TCA

Ser 000 AGA AGO TCT TTC Ala Arg Ser Ser Phe 90 AAA GAC TCC AAC AAC Lys ASP Ser Asn Asn 105 TTC AAG AGC OCT Phe Lys Ser Ala TTA GCC Leu Ala 95 AAG CTC Lys Leu GCC OCT AAG AAG Ala Ala Lys Lys 100 GAG CTT AAO GAG Oiu Leu Lys Glu 115

GAG

Glu ACC GCC GC Thr Ala Ala

GTG

Val 110 390 WO 98/29537 PCT/EP97/07293 113- ATT GCC AAG GAT Ile Ala Lys Asp 120 TAC GAA GTC Tyr Glu Val GGT TTC Oly Phe 125 GAT TCG Asp Ser GTT GTG ACT OTT TTG Val Val Thr Val Leu 130 438 GCT TAT Ala Tyr GAA TGC Glu Cys 150

OTT

Val 135 TAC AGC AGC AGA Tyr Ser Ser Arg AGA CCG CCG CCT Arg Pro Pro Pro

AAA

Lys 145 GGA OTT TCT Gly Val Ser 486 OCA GAC GAG AAT Ala Asp Glu Asn

TGC

Cys 155 TGC CAC GTG GCT Cys His Val Ala

TGC

Cys 160 CGG CCG GCG GTG Arg Pro Ala Val 534

OAT

Asp 165 TTC ATG TTG GAG Phe Met Leu Glu

OTT

Val 170 CTC TAT TTG OCT TTC Leu Tyr Leu Ala Phe 175 ATC TTC AAG ATC Ile Phe Lys Ile

CCT

Pro 180

AAA

Lys 582 GAA TTA ATT ACT Glu Leu Ile Thr TAT CAG AGO CAC TTA TTG GAC GTT OTA Tyr Gin Arg His Leu Leu Asp Val Val 190

GAC

Asp 195 630 OTT OTT ATA Val Val Ile

GAG

Glu 200 GAC ACA TTG OTT Asp Thr Leu Val

ATA

Ile 205 CTC AAG CTT GCT AAT ATA TGT Leu Lys Leu Ala Asn Ile Cys 210 GGT AAA OCT Oly Lys Ala 215 TGT ATO AAG CTA Cys Met Lys Leu OAT AGA TOT AAA GAG Asp Arg Cys Lys Glu 225 ATT ATT GTC Ile Ile Val 726

AAG

Lys

TCT

Ser 230 AAT OTA GAT ATG Asn Vai Asp Met AGT CTT OAA AAG Ser Leu Glu Lys

TCA

Ser 240 TTG CCO GAA GAG Leu Pro Glu Glu 774

CTT

Leu 245 OTT AAA GAG ATA Val Lys Glu Ile

ATT

Ile 250 OAT AGA CGT Asp Arg Arg AAA GAO Lys Glu 255 CTT GGT Leu Gly TTO GAG OTA 822 Leu Glu Val 260 CCT AAA OTA AAG Pro Lys Val Lys

AAA

Lys 265 CAT GTC TCG AAT His Val Ser Asn CAT AAG OCA OTT His Lys Ala Leu GAC TCG Asp Ser 275 OAT GAT ATT Asp Asp Ile GAG TTA Glu Leu 280 GTC AAG TTO Val Lys Leu

CTT

Leu 285 TTO AAA Leu Lys GAG GAT Glu Asp CAC ACC AAT His Thr Asr 290 WO 98/29537 PCT/EP97/07253 -114- CTA GAT GAT GCG TOT OCT CTT CAT TTC GCT GTT GCA TAT TOC AAT GTG 966 Leu Asp Asp 295 Ala Cys Ala Leu His 300 Phe Ala Val Ala Tyr 305 Cys Asn Val AAG ACC Lys Thr 310 GCA ACA GAT CTT Ala Thr Asp Leu

TTA

Leu 315 AAA CTT GAT CTT Lys Leu Asp Leu 0CC Ala 320 GAT GTC AAC CAT Asp Val Asn His 1014 1062 AGG Arg 325 AAT CCG AGO GGA Asn Pro Arg Gly

TAT

Tyr 330 ACG GTG CTT CAT Thr Val Leu His

GTT

Val 335 OCT GCG ATG CGG Ala Ala Met Arg

AAG

Lys 340 GAG CCA CAA TTG ATA CTA TCT CTA TTG Glu Pro Gin Leu Ile Leu Ser Leu Leu 345

GAA

Glu 350 AAA GOT GCA AGT GCA TCA Lys Gly Ala Ser Ala Ser 355 1110 GAA GCA ACT Glu Ala Thr

TTG

Leu 360 GAA GOT AGA ACC Glu Gly Arg Thr

GCA

Ala 365 CTC ATG ATC GCA AAA CAA GCC Leu Met Ile Ala Lys Gin Ala 370 1158 ACT ATG GCG Thr Met Ala 375 GTT GAA TGT AAT Val Giu Cys Asn

AAT

Asn 380 ATC CCG GAG CMh Ile Pro Giu Gin TGC AAG CAT TCT Cys Lys His Ser 385 GAA GAC AAA CGA Glu Asp Lys Arg 1206 CTC AAA Leu Lys 390 GGC CGA CTA TGT Gly Arg Leu Cys

OTA

Val 395 GAA ATA CTA GAG Glu Ile Leu Glu

CAA

Gin 400

GAA

Glu 405 CAA ATT CCT AGA Gin Ile Pro Arg

OAT

Asp 410 OTT CCT CCC TCT Val Pro Pro Ser GCA GTG GCG 0CC Ala Val Ala Ala

OAT

Asp 420 1254 1302 1350 GAA TTG Glu Leu AAG ATO ACO Lys Met Thr 425 CTG CTC OAT CTT GAA AAT AGA OTT Leu Leu Asp Leu Glu Asn Arg Val 430 GCA CTT OCT Ala Leu Ala 435 ATC GCC GAA Ile Ala Glu 450 CAA COT CTT Gin Arg Leu ATG AAG GGA Met Lys Gly

TTT

Phe 440 CCA ACO GAA GCA Pro Thr Giu Ala

CAA

Gin 445 OCT GCA ATO GAG Ala Ala Met Glu 1398 ACA TGT Thr Cys GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC CGT Olu Phe Ile Val Thr Ser Leu Olu Pro Asp Arg 1446 WO 98/29537 WO 9829537PCTIEP97/07253 -115- 455 CTC ACT GGT ACG AAG AGA ACA 460 465 TCA CCG GGT GTA Ser Pro Gly Val.

ATA GCA CCT TTC Ile Ala Pro Phe Leu Thr 470 Gly Thr Lys Arg Thr 475

AGA

Arg 485 ATC CTA GAA GAG Ile Leu Glu Giu CAA AGT AGA CTA AAA Gin Ser Arg Leu Lys 495 GCG CTT TCT AAA ACC Ala Leu Ser Lys Thr 500 1494 1542 1590 1638 GTG GAA CTC GGG Val Giu Leu Gly

AAA

Lys 505 CGA TTC TTC CCG Arg Phe Phe Pro

CGC

Arg 510 TGT TCG GCA GTG Cys Ser Ala Val CTC GAC Leu Asp 515 GAA GAC Giu Asp CAG ATT ATG Gin Ile Met

A.AC

Asn 520 TGT GAG GAC TTG Cys Glu Asp Leu

ACT

Thr 525 CAA CTG GCT TGC Gin Leu Ala Cys

GGA

Gly 530 GAC ACT Asp Thr CAA GAG Gin Glu 550 GAG AAA CGA CTA CAA Giu Lys Arg Leu Gin 540 AAG AAG CAA AGG Lys Lys Gin Arg

TAC

Tyr 545 ATG GAA ATA Met Giu Ile 1686 1734 ACA CTA AAG AAG Thr Leu Lys Lys

GCC

Ala 555 TTT AGT GAG GAC Phe Ser Glu Asp

AAT

Asn 560 TTG GAA TTA GGA Leu Glu Leu Gly AhT Asn 565 TTG TCC CTG ACA GAT TCG ACT TCT TCC Leu Ser Leu Thr Asp Ser Thr Ser Ser 570

ACA

Thr 575 TCG AAA TCA ACC Ser Lys Ser Thr

GGT

Gly 580 1782 GGA AAG AGG TCT AAC Gly Lys Arg Ser Asn 585 CGT AAA CTC TCT CAT Arg Lys Leu Ser His 590 CGT CGT CGG TGA Arg Arg Arg 1824 GACTCTTGCC TCTTAGTGTA ATTTTTGCTG TACCATATAA TTCTGTTTTC ATGATGACTG TAACTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG 1884 1944 2004 2011 ATTTGTA WO 98/29537 PCT/EP97/07253 -116- INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 2011 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 43..1824 OTHER INFORMATION: /product= "altered form of NIM1" /note= "Serine residues at amino acid positions 55 and 59 in wild-type NIM1 gene product have been changed to Alanine residues." (ix) FEATURE: NAME/KEY: misc_feature LOCATION: 205..217 OTHER INFORMATION: /note= "nucleotides 205 and 217 changed from T's to G's compared to wild-type sequence." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC Met Asp Thr Thr ATT GAT GGA TTC GCC GAT TCT TAT GAA ATC AGC Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser 10 15 GCT ACC GAT AAC ACC GAC TCC TCT ATT GTT TAT Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr 30 GTA CTC ACC GGA CCT GAT GTA TCT GCT CTG CAA Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gln AGC ACT Ser Thr AGT TTC GTC Ser Phe Val GCC GAA CAA Ala Glu Gin CTG GCC Leu Ala TTG CTC Leu Leu AAC AGC Asn Ser 198 WO 98/29537 WO 9829537PCT/EP97/07253 -117- TTC GAA GC Phe Glu Ala GTC TTT GAC GCG Val Phe Asp Ala CCG OAT GAT TTC TAC AGC Pro Asp Asp Phe Tyr Ser 60 GAA GTT TCT TTC CAC CGG Glu Val Ser Phe His Arg GAC GCT AAG Asp Ala Lys TGC GTT TTG Cys Val Leu 246 CTT OTT Leu Val CTC TCC GAC C Leu Ser Asp Gly

COO

Arg 75 294

TCA

Ser GCG AGA AGC TCT Ala Arg Ser Ser TTC TTC AAG Phe Phe Lys 90 AAC ACC CC Asn Thr Ala AGC OCT TTA GCC Ser Ala Leu Ala 95 GCC GTG AAG CTC Ala Val Lys Leu 110 GCC OCT AAG Ala Ala Lys

AAG

LYS

100 342 390 GAG AAA GAC TCC Glu Lys Asp Ser GAG CTT AAG, GAG Gl~u Leu Lys Glu ATT CCC AAG Ile Ala Lys

GAT

Asp 120 TAC GAA GTC GOT Tyr Glu Val Oly

TTC

Phe 125 GAT TCG OTT GTG ACT GTT TTG Asp Ser Val Val Thr Val Leu 130 OCT TAT Ala Tyr

GTT

Val 135 TAC AGC AGC AGA Tyr Ser Ser Arg

GTG

Val 140 AGA CCG CCG CCT Arg Pro Pro Pro AAA GGA OTT TCT Lys Gly Val Ser 145 COG CCG OCO GTG Arg Pro Ala Val

GAA

Glu

GAT

Asp 165 TOC OCA CAC GAG Cys Ala Asp Giu 150 TTC ATG TTG GAG Phe Met Leu Giu

AAT

Asn

TGC

Cys 155 TGC CAC OTG GCT Cys His Val Ala

TGC

Cys 160 OTT CTC TAT TTG OCT TTC Val Leu Tyr Leu Ala Phe 170 175 TAT CAG AGG CAC TTA TTG Tyr Gin Arg His Leu Leu 190 ATC TTC AAG ATC Ile Phe Lys Ile

CCT

Pro 180 582 OAA TTA Glu Leu ATT ACT CTC Ile Thr Leu 185 GAC OTT GTA GAC AAA Asp Val Val Asp Lys 195 OTT OTT ATA GAG Val Val Ile Glii 200 GAC ACA TTO OTT ATA Asp Thr Leu Val Ile 205 CTC AAO CTT GCT AAT ATA TOT Leu Lys Leu Ala Asn Ile Cys 210 GOT AAA OCT Gly LYS Ala TGT ATO AAG Cys Met Lys CTA TTG OAT Leu Leu Asp AGA TOT Arg Cys AAA GAG ATT ATT GTC Lys Giu le Ile Val WO 98/29537 WO 9829537PCT/EP97/07253 .118- 215 220 225 AAG TCT Lys Ser 230 AAT GTA GAT ATO Asn Val Asp Met GTT ACT Val Ser 235 CTT GAA AAG TCA Leu Giu Lys Ser TTG CCC GAA GAG Leu Pro Glu Glu

CTT

Leu 245 GTT AAA GAG ATA Val Lys Giu Ile GAT AGA CGT AAA GAG Asp Arg Arg Lys Giu 255 CTT GGT TTG GAG GTA Leu Gly Leu Glu Val 260 CCT AAA GTA AAG Pro Lys Val Lys GAT GAT ATT GAG Asp Asp Ile Clu 280 CAT GTC TCG A4AT GTA His Val Ser Asn Val 270 CAT AAG GCA CTT His Lys Ala Leu GAC TCG Asp Ser 275 870 TTA GTC AAG TTG Leu Val. Lys Leu

CTT

Leu 285 TTG AAA GAG GAT Leu Lys Giu Asp CAC ACC AAT His Thr Asn 290 CTA GAT Leu Asp AAG ACC LYS Thr 310 C TGT GCT CTT Ala Cys Ala Leu TTC GCT GTT GCA TAT TGC AAT GTG Phe Ala Val Ala Tyr Cys Asn Val 305 GCA ACA CAT Ala Thr Asp CTT TTA Leu Leu 315 AAA CTT GAT CTT Lys Leu Asp Leu

GC

Ala 320 CAT GTC AAC CAT Asp Val Asn His

AGG

Arg 325 AAT CCG AGG CCA Asn Pro Arg Gly

TAT

Tyr 330 ACG GTC CTT CAT Thr Val Leu His GCT GCG ATG CCC Ala Ala Met Arg 1014 1062 1110 1158 GAG CCA CAA TTG Giu Pro Gin Leu

ATA

Ile 345 CTA TCT CTA TTG Leu Ser Leu Leu GAA AAA COT GCA ACT GCA TCA Clu Lys Gly Ala Ser Ala Ser 350 355 CTC ATG ATC GCA AAA CAA CC Leu Met Ile Ala Lys Gin Ala 370 CAA GCA ACT Ciu Ala Thr

TTG

Leu 360 GAA GCT AGA ACC Giu Gly Arg Thr

GCA

Ala 365

ACT

Thr ATC C Met Ala 375 CTT CAA TGT AAT AAT Val Giu Cys Asn Asn 380 ATC CCC GAG CAA TGC AAG CAT TCT Ile Pro Giu Gin Cys Lys His Ser 385 1206 CTC AAA CCC CGA CTA TGT CTA GAA ATA CTA GAG CAA GAh CAC AAA CGA 1254 WO 98/29537 WO 9829537PCT/EP97/07253 -119- Leu Lys 390 Gly Axg Leu Cys Val Glu Ile Leu Giu 395 Gin 400 GlU ASP LYS Arg

GAA

Giu 405 CAA ATT CCT AGA Gin Ile Pro Arg

GAT

Asp 410 GTT CCT CCC TCT Val Pro Pro Ser

TTT

Ph.

415 GCA GTG GCG GCC GAT Ala Val Ala Ala Asp 420 1302 1350 GAA TTG AAG ATG Giu Leu Lys Met

ACG

Thr 425 CTG CTC CAT CTT Leu Leu Asp Leu

GAA

Giu 430 AAT AGA GTT GCA Asn Arg Val Ala CTT GCT Leu Ala 435 CAA CGT CTT Gin Arg Leu ATG AAG GGA Met Lys Giy 455 CCA ACG GAA GCA Pro Thr Glu Ala

CAA

Gin 445 GCT GCA ATG GAG Ala Ala Met Glu ATC GCC GAA Ile Ala Giu 450 CCT GAC CGT Pro Asp Arg 1398 1446 ACA TGT GAG TTC ATA Thr Cys Giu Phe Ile 460 OTO ACT AGC CTC Val Thr Ser Leu

GAG

Giu 465 CTC ACT Leu Thr 470 GGT ACG AAG AGA Gly Thr Lys Arg

ACA

Thr 475 TCA CCG GGT GTA Ser Pro Gly Val

AAG

Lys 480 ATA GCA CCT TTC Ile Ala Pro Phe CTT TCT AAA ACC Leu Ser Lys Thr 500

AGA

Arg 485 ATC CTA GAA GAG Ile Leu Clu Glu

CAT

His 490 CAA ACT AGA CTA Gin Ser Arg Leu AAA GCG Lys Ala 495 1494 1542 1590 1638 GTG GAA CTC GGG Val Glu Leu Gly CAG ATT ATG AAC Gin Ile Met Asn 520 AAA CGA TTC TTC CCG Lys Arg Phe Phe Pro 505 TGT GAG GAC TTG ACT Cys Glu Asp Leu Thr 525 CCC TGT TCG OCA GTG CTC CAC Arg Cys Ser Ala Val Leu Asp 510 515 CAA CTG GCT TCC GGA GAA GAC Gin Leu Ala Cys Gly Giu Asp 530 AAG CAA AGG TAC ATG GAA ATA Lys Gin Arg Tyr Met Glu Ile 545 GAC ACT GCT GAG AAA CGA CTA Asp Thr Ala Giu Lys Arg Leu 535 CAA AAG Gin Lys 540 1686 CAA GAG Gin Giu 550 ACA CTA Thr Leu AAG AAG Lys Lys

GCC

Ala 555 TTT ACT GAG Ph. Ser Giu GAC AMT Asp Asn 560 TTG GAA TTA GGA Leu Glu Leu Gly 1734 WO 98/29537 WO 9829537PCT/EP97/07253 -120 AAT TTG TCC CTG ACA GAT TCG ACT TCT TCC ACA Asn Leu Ser Leu Thr Asp Ser Thr Ser Ser Thr 565 570 575 GGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT Gly Lys .Arg Ser Asn Arg Lys Leu Ser His Arg 585 590 GACTCTTGCC TCTTAGTGTA ATTTTTGCTG TACCATATAA TAACTGTTTA TGTCTATC!GT TGGCGTCATA TAGTTTCGCT ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA

ATTTGTA

TCG AAA TCA ACC GGT Ser Lys Ser Tbr Gly 580 CGT CGG TGA Arg Arg TTCTGTTTTC ATGATGACTG CTTCGTTTTG CATCCTGTGT TTTGAACCAA TGGTATACAG 1782 1824 1884 1944 2004 2011 INFORMATION FOR SEQ ID NO:8: Wi SEQUENCE CHARACTERISTICS: LENGTH: 594 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Asp Thr Thr Ile Asp Gly Phe Ala Asp 1 5 10 Thr Ser Phe Val Ala Thr Asp Asn Thr Asp N~O: 8: Ser Tyr Ser Ser GJlu le Ser Ser Ile Val Tyr Leu Gln Pro Ala Ala Glu Val Leu Tbx ASP Val Ser Ala Asp Leu Gin Leu Asp Phe Tyr Leu Ser Asp Asn Scr Phe Glu Ala Lys Leu Val 70 Ala Leu Phe Asp Ala Scr Ser Asp Gly Arg Val Ser Phe WO 98/29537 PCT/EP97/07253 -121- Arg Cys Val Leu Ser Ala Arg Ser Ser Phe 90 Phe Lys Ser Ala Leu Ala Ala Ala Lys Lys 100 Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu 105 110 Glu Leu Lys 115 Glu Ile Ala Lys Asp 120 Tyr Glu Val Gly Phe 125 Asp Ser Val Val Thr 130 Val Leu Ala Tyr Val 135 Tyr Ser Ser Arg Arg Pro Pro Pro Gly Val Ser Glu Cys 150 Ala Asp Glu Asn Cys 155 Cys His Val Ala Cys 160 Arg Pro Ala Val Asp 165 Phe Met Leu Glu Leu Tyr Leu Ala Phe Ile 175 Phe Lys Ile Val Val Asp 195 Pro 180 Glu Leu Ile Thr Leu 185 Tyr Gin Arg His Leu Leu Asp 190 Leu Lys Leu Lys Val Val Ile Glu 200 Asp Thr Leu Val Ile 205 Ala Asn 210 Ile Cys Gly Lys Ala 215 Cys Met Lys Leu Leu 220 Asp Arg Cys Lys Ile Ile Val Lys Ser 230 Asn Val Asp Met Val 235 Ser Leu Glu Lys Ser 240 Leu Pro Glu Glu Leu 245 Val Lys Glu Ile Ile 250 Asp Arg Arg Lys Glu Leu 255 Gly Leu Glu Ala Leu Asp 275 Val 260 Pro Lys Val Lys Lys 265 His Val Ser Asn Val His Lys 270 Leu Lys Glu Ser Asp Asp Ile Glu 280 Leu Val Lys Leu Leu 285 Asp His 290 Thr Asn Leu Asp Asp Ala Cys Ala Leu 295 His 300 Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala WO 98/29537 PCT/EP97/07253 -122- 305 310 320 Asp Val Asn His Arg 325 Asn Pro Arg Gly Tyr Thr Val LeU 330 Ala Met Arg Ala Ser Ala 355 Lys 340 Glu Pro Gin Leu Ile 345 Leu Ser Leu Leu His Val Ala 335 Glu Lys Gly 350 Leu Met lie Ser Giu Ala Thr Glu Gly Arg Thr Ala 365 Ala Lys 370 Gin Ala Thr Met Ala 375 Val Giu Cys Asn Asn 380 Ile Pro Giu Gin Cys 385 Lys His Ser Leu Lys 390 Gly Arg Leu Cys Vai 395 Glu Ile Leu Glu Gin 400 Glu Asp Lys Arg Glu 405 Gin Ile Pro Arg Asp 410 Val Pro Pro Ser Phe Ala 415 Val Ala Ala Val Ala Leu 435 Asp 420 Glu Leu Lys Met Thr 425 Leu Leu Asp Leu Glu Asn Arg 430 Ala Ala Met Ala Gin Arg Leu Phe 440 Pro Thr Glu Aia Gin 445 Glu Ile 450 Ala Glu Met Lys Gly 455 Thr Cys Giu Phe Val Thr Ser Leu Glu 465 Pro Asp Arg Leu Thr 470 Gly Thr Lys Arg Thr 475 Ser Pro Gly Val Lys 480 Ile Aia Pro Phe Arg 485 Ile Leu Glu Glu His 490 Gin Ser Arg Leu Lys Ala 495 Leu Ser Lys Ala Val Leu 515 Thr 500 Val Glu Leu Gly Arg Phe Phe Pro Arg Cys Ser 510 Gln Leu Ala Asp Gin Ile Met Cys Giu Asp Leu Thr 525 Cys Gly 530 Glu Asp Asp Thr Ala 535 Glu Lys Arg Leu Gin 540 Lys Lys Gin Arg WO 98/29537 WO 9829537PCT/EP97/07253 -123- Tyr Met 545 Giu Ile Gin Giu 550 Tbr Leu Lys Lys Ala 555 Phe Ser Glu Asp Asn 560 Leu Glu Leu Gly Asn 565 Leu Ser Leu, Thr Asp 570 Ser Thr Ser Ser Thr Ser 575 Lys Ser Thr Giy 580 Gly Lys Arg Ser Asn Arg 585 LYS Leu Ser His Arg Arg 590 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 1597 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1410 OTHER INFORMATION: /product= "Altered form of NIMI" /note= *N-terminal deletion compared to wild-type NIMI sequence.' (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: ATG GAT TCG GTT GTG Met Asp Ser Val Val 1 ACT OTT TTG GCT TAT GTT TAC AGC AGC AGA GTG Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val AGA CCG Arg Pro CCG CCT AAA Pro Pro Lys OGA OTT TCT GAA TGC Gly Val Ser Glu Cys 25 GCA GAC GAG Ala Asp Giu AAT TGC TOC Asn Cys Cys CAC OTG OCT TGC COG CCG GCG OTO GAT TTC ATG TTG GAG GTT CTC TAT WO 98/29537 WO 9829537PCTIEP97107253 -124- His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr TTG GCT Leu Ala TTC ATC TTC AAG ATC CCT GAA TTA ATT Phe Ile Phe Lys Ile Pro Glu Leu Ile 55

ACT

Thr CTC TAT Leu Tyr CAG AG Gin Arg 192

CAC

His TTA TTG GAC GTT Leu Leu Asp Val GAC AAA GTT OTT ATA GAG GAC ACA TTG Asp Lys Val Val Ile Glu Asp Thr Leu 75

GTT

Val 240 ATA CTC AAG CTT Ile Leu Lys Leu

GCT

Ala AAT ATA TOT GOT Asn le Cys Gly GCT TOT ATG AAO Ala Cys Met Lys CTA TTO Leu Leu 288

GAT

Asp AGA TGT Arg Cys

AAA

Lys 100 GAG ATT ATT OTC GiU Ile Ile Val

AAO

LYS

105 TCT AAT GTA GAT ATO GTT AGT Ser Asn Val Asp Met Val Ser 110 CTT GAA AAO Leu Giu Lys 115 TCA TTG CCC GAA Ser Leu Pro Ciu

GAO

Ciu 120 CTT GTT AAA GAG Leu Val Lys Giu

ATA

Ile 125 ATT OAT AGA Ile Asp Arg CGT AAA Arg Lys 130 GAG CTT GOT TTO Giu Leu Cly Leu

GAG

Glu 135 OTA CCT AAA OTA AAO AAA CAT GTC TCG Val Pro Lys Val Lys Lys His Vai Ser 140

AAT

Asfl 145

CTT

Leu GTA CAT AAG OCA CTT Val His Lys Ala Leu 150 TTG AAA GAG OAT CAC Leu Lys Oiu Asp His 165 GAC TCO OAT OAT ATT Asp Ser Asp Asp Ile 155 ACC AAT CTA OAT OAT Thr Asn Leu Asp Asp 170 GAG TTA GTC AAO Glu Leu Val Lys

TTG

Leu 160 480 GCC TOT GCT Ala Cys Ala CTT CAT Leu His 175 528 TTC OCT GTT OCA TAT TOC AAT OTO Phe Ala Vai Ala Tyr Cys Asn Vai 180 CTT GAT CTT GCC OAT GTC AAC CAT Leu Asp Leu Ala Asp Val Asri His 195 200

AAO

Lys 185 ACC OCA ACA OAT CTT Thr Ala Thr Asp Leu 190 TTA AMA 576 Leu Lys AGO AMT CCCG AGO Arg Asn Pro Arg GGA TAT ACO OTO Gly Tyr Thr Val 205 WO 98/29537 WO 9829537PCT/EP97107253 125 CTT CAT Leu His 210 GTT GCT GCG ATG Val Ala Ala Met AAG GAG CCA CAA Lys Giu Pro Gin

TTG

Leu 220 ATA CTA TCT CTA Ilie Leu Ser Leu 672 720 TTG GAA AAA GGT GCA Leu Giu Lys Gly Ala 225

AGT

Ser 230 GCA TCA GAA GCA Ala Ser Giu Ala

ACT

Thr 235 TTG GAA GGT AGA ACC Leu Glu Gly Arg Thr 240 GCA CTC ATG ATC Ala Leu Met Ile

OCA

Ala 245 AAA CAA GCC ACT ATG GCG Lys Gin Ala Thr Met Ala 250 AAG CAT TCT CTC AAA GGC Lys His Ser Leu Lys Gly 265 GTT GAA TGT Val Glu Cys

AAT

Asn 255

AAT

Asn 768

ATC

le CCG GAG CAA TOC Pro Giu Gin Cys 260 OGA CTA TGT GTA GAA Arg Leu Cys Val Giu 270 ATA CTA GAG Ile Leu Glu 275 CAA GAA GAC AAA Gin GiU Asp Lys

CGA

Arg 280 GAA CAA ATT CCT Glu Gin Ile Pro AGA GAT GTT CCT Arg Asp Val Pro 285 CCC TCT Pro Ser 290 TTT GCA GTG GCG Phe Ala Val Ala GAT GAA TTG AAG ATG ACG CTG CTC GAT Asp Giu Leu Lys Met Thr Leu Leu Asp 300

CTT

Leu 305 GAA AAT AGA OTT Glu Asn Arg Val

GCA

Ala 310 CTT OCT CAA COT Leu Ala Gin Arg

CTT

Leu 315 TTT CCA ACG GAA Phe Pro Thr Glu

GCA

Ala 320 960 CAA GCT GCA ATO Gin Ala Ala Met

GAG

Giu 325 ATC GCC GAA ATO Ile Ala Giu Met

AAG

Lys 330 GGA ACA TGT GAG Gly Thr Cys Glu TTC ATA Phe Ile 335 1008 GTO ACT AGC Val Thr Ser

CTC

Leu 340 GAG CCT GAC CGT CTC ACT Giu Pro Asp Arg Leu Thr 345 GOT ACG AAG Gly Thr Lys AGA ACA TCA Arg Thr Ser 350 CAT CAA AGT His Gin Ser 1056 CCG GGT Pro Gly

GTA

Val 355 AAG ATA GCA CCT Lys Ile Ala Pro

TTC

Phe 360 AGA ATC CTA GAA GAG Arg Ile Leu Giu Glu 365 1104 AGA CTA AAA Arg Leu Lys 370 C CTT TCT Ala Leu Ser AAA ACC Lys Thr 375 GTG GAA CTC Val Giu Leu 000 Oly 380 AAA CGA TTC TTC Lys Arg Phe Phe 1152 WO 98/29537 WO 9829537PCT/EP97/07253 -126-

CCG

Pro 385 CGC TOT TCG GCA GTG Arg Cys Ser Ala Val 390 CTC GAC Leu Asp CAG ATT ATG Gin Ile Met 395 AAC TGT GAG GAC Asn CyS Giu ASP 1200 1248 ACT CAA CTG GCT Thr Gin Leu Ala

TGC

Cys 405 GGA GAA GAC GAC Gly Giu Asp Asp ACT OCT GAG AAA CGA CTA CAA Thr Ala Glu Lys Arg Leu Gin 410 415 AAG AAG CAA AGO Lys Lys Gin Arg 420 TAC ATG GAA ATA CAA Tyr Met Giu Ile Gin 425 GAG ACA CTA ANO Olu Thr Leu Lys AAG 0CC TTT Lys Ala Phe 430 1296 AGT GAG GAC AAT TTG OAA TTA GGA A&T TTG, TCC CTG Ser Giu Asp Asn Leu Giu Leu Oly Asn Leu Ser Leu 435 440 ACA GAT TCG ACT Thr Asp Ser Thr 445 1344 TCT TCC Ser Ser 450 ACA TCG AAA Thr Ser Lys TCA ACC Ser Thr 455 GOT GGA AAG AGG TCT AAC COT AAA CTC Gly Gly Lys Arg Ser Asn Arg Lys Leu 460 1392 TCT CAT CGT COT COG TGA GACTCTTOCC TCTTAGTOTA ATTTTTGCTG Ser His Arg Arg Arg 465 470 TACCATATAA TTCTGTTTTC ATOATOACTG TAACTGTTTA TGTCTATCGT TGOCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT ATTATTOCTG CAGOGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG ATTTGTA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 470 amino acids TYPE: amino acid TOPOLOGY:-linear (ii) MOLECULE TYPE: protein 1440 1500 1560 1597 (xi) SEQUENCE DESCRIPTION: SEQ ID WO 98/29537 WO 9829537PCTIEP97/07253 -127- Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arq Pro Pro His Val Ala Lys Gly Val Ser Glu Cys Ala Asp Giu Asn Cys Cys Cys Arg Pro Ala Val 40 Asp Phe Met Leu Giu Vai Leu Tyr Leu Ala Phe Ile Phe Lys Ile 55 Pro Glu Leu Ile Thr Leu Tyr Gin Glu Asp Thr Leu Arg Val Leu Leu Asp Val Val Asp Lys Val Val Ilie Leu Lys Leu Ala Asn Ile Cys Gly Lys 90 Ala Cys Met Lys Leu Leu Asp Arg Cys Leu Giu Lys 115 Lys 100 Gu Ile Ile Val Lys 105 Ser Asn Val Asp Met Val Ser 110 Ilie Asp Arg Ser Leu Pro Glu Giu 120 Leu Val Lys Glu Ile 125 Arg Lys 130 Giu Leu Gly Leu Giu 135 Val Pro Lys Val Lys Lys His Vai 140 Glu Leu Val Lys Ser Leu 160 Vai His Lys Ala Asp Ser Asp Asp Ile 155 Leu Leu Lys Giu Asp 165 His Thr Asn Leu Asp Ala Cys Ala Leu His 175 Phe Ala Val Leu Asp Leu 195 Ala 180 Tyr Cys Asn Val Lys 185 Thr Ala Thr Asp Leu Leu Lys 190 Tyr Thr Val Ala Asp Val Asn His 200 Arg Asn Pro Arg Gly 205 Leu His 210 Val Ala Ala Met Arg 215 Lys Giu Pro Gin Leu 220 Ile Leu Ser Leu Leu Giu Lys Gly Ala Ser Ala Ser Giu Ala Thr Leu Glu Gly Arg Thr WO 98/29537 WO 9829537PCT/EP97/07253 -128- 230 235 240 Ala Leu Met Ile Ala Lys 245 Gin Ala Thr Met Ala Val Glu Cys Asn Asn 250 255 Ile Pro Giu Gin 260 Ile Leu Glu Gin 275 Cys Lys His Ser Leu 265 Lys Gly Arg Leu Cys Val Giu 270 Asp Val Pro Giu Asp Lys Glu Gin Ile Pro Arg 285 Pro Ser 290 Phe Ala Val Ala Ala 295 Asp Giu Leu Lys Met Thr Leu LeU 300 Phe Pro Thr Giu

ASP

Leu 305 Glu Asn Arg Val Ala 310 Leu Ala Gin Arg Leu 315 Ala 320 Gin Ala Ala Met Ile Ala Glu Met Gly Thr Cys Glu Phe Ile 335 Val Thr Ser Pro Gly Val 355 Leu 340 Glu Pro Asp Arg Leu 345 Thr Gly Thr Lys Arg Thr Ser 350 His Gin Ser Lys Ile Ala Pro Phe 360 Arg Ile Leu Glu Glu 365 Arg Leu 370 Lys Ala Leu Ser Lys 375 Thr Val Glu Leu Gly 380 Lys Arg Phe Phe Pro 385 Arg Cys Ser Ala Val 390 Leu Asp Gin Ile Met Asn Cys Giu Asp 395 Leu 400 Thr Gin Leu Ala Cys 405 Gly Glu Asp Asp Thr 410 Ala Glu Lys Arg Leu Gin 415 Lys Lys Gin Arg 420 Tyr Met Giu Ile Gin 425 Giu Thr Leu Lys Lys Ala Phe 430 Asp Ser Thr Ser Giu Asp Asn 435 Leu Giu Leu Gly 440 Asn Leu Ser Leu Thr 445 Ser Ser 450 Thr Ser Lys Ser Thr 455 Gly Gly Lys Arg Ser 460 Asn Arg Lys Leu WO 98/29537 WO 9829537PCT/EP97/07253 -129- Ser 465 His Arg Arg Arg 470 INFORMATION 7FOR SEQ ID NO:11: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1608 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 43. .1608 OTHER INFORMATION: /product= 'Altered form of NI~i" /note= "C-terminal deletion compared to wild-type NIXi." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC Met Asp Thr Thr 1 ATT GAT GGA TTC GCC GAT TCT TAT GAA ATC AGC AGC Ile Asp Gly Phe Ala Asp 10 Ser Tyr Glu Ile Ser Ser 15 ACT AGT TTC GTC Tbr Ser Phe Val 102 GCT ACC GAT AAC ACC Ala Thr Asp Asn Thr GAC TCC TCT ATT Asp Ser Ser Ile TAT CTG GCC GCC Tyr Leu Ala Ala GAA CAA Glu Gin GTA CTC ACC Val Leu Thr CCT GAT GTA TCT Pro Asp Val Ser CTG CAA TTG CTC Leu Gln Leu Leu TCC AAC AGC Ser Asn Ser TTC GAA TCC Phe Giu Ser GTC TTT Val Phe GAC TCG Asp Ser CCG GAT Pro Asp 60 GAT TTC TAC AGC GAC OCT AAG Asp Phe Tyr Ser Asp Ala Lys 246 WO W29537 WO 9829537PCT/EP97/07253 -130- CTT GTT Leu Val CTC TCC GAC GGC Leu Ser Asp Gly

CGG

Arg GAA GTT TCT TTC Giu Val Ser Phe CAC CGG TGC His Arg Cys GTT TTG Val Leu

TCA

Ser GCG AGA AGC TCT Ala Arg Ser Ser

TTC

Phe 90 TTC AAG AGC GCT Phe Lys Ser Ala

TTA

Leu 95 GCC GCC GCT AAG Ala Ala Ala Lys

AAG

Lys 100 GAG AAA GAC TCC AAC Glu Lys Asp Ser Asn 105 AAC ACC GCC GCC Asn Thr Ala Ala GTG AAG Val Lys 110 CTC GAG CTT AA~G GAG Leu Glu Leu Lys Glu 115 390 ATT GCC AAG Ile Ala Lys

GAT

Asp 120 TAC GAA GTC GGT Tyr Glu Val Gly

TTC

Phe 125 CAT TCG GTT Asp Ser Val GTG ACT GTT TTG Val Thr Val Leu 130 AAA GGA GTT TCT Lys Gly Val Ser 145 GCT TAT GTT TAC Ala Tyr Val Tyr 135 AGC AGC AGA Ser Ser Arg

GTG

Val 140 AGA CCG CCG CCT Arg Pro Pro Pro GAA TGC Glu Cys 150 GCA GAC GAG AAT Ala Asp Glu Asn

TGC

Cys 155 TGC CAC GTG GCT Cys His Val. Ala TGC CGG CCG GCG GTG Cys Arg Pro Ala Val 160 ATC TTC AAG ATC CCT Ile Phe Lys Ile Pro

GAT

Asp 165 TTC ATO TTG GAG Phe Met Leu Glu CTC TAT TTG GCT Leu Tyr Leu Ala

TTC

Phe 175 582 GAA TTA ATT ACT Glu Leu Ile Thr

CTC

Leu 185 TAT CAG AGG CAC Tyr Gln Arg His TTG GAC GTT GTA Leu Asp Val. Val GAC AAA Asp Lys 195 ATA TGT Ile Cys GTT OTT ATA Val Val Ilie

GAG

Glu 200 GAC ACA TTC GTT Asp Thr Leu Val.

ATA

Ile 205 CTC AAG CTT Leu Lys Leu GCT AAT Ala Asn 210 GGT AAA GCT Gly Lys Ala 215 TOT ATG AAG CTA Cys Met Lys Leu

TTG

Leu 220 GAT AGA TGT AAA Asp Arg Cys Lys

GAG

Giu 225 ATT ATT GTC 726 Ile Ile Val AAG TCT Lys Ser AAT GTA GAT Asn Val. Asp ATC GTT AGT Met Val Ser CTT GAA Leu Ciu AAG TCA TTG CCC LYS Ser Leu Pro CAA GAG Glu Giu 774 WO 98/29537 WO 9829537PCT/EP97/07253 -131- 230 CTT GTT AAA GAG ATA ATT Leu Val Lys Glu Ile Ile 245 250 235 GAT AGA CGT AAA GAG Asp Arg Arg Lys GlU 255 240 CTT GOT TTG GAG GTA Leu Gly Leu Glu Val 260 822 870 CCT AAA GTA AAG Pro Lys Val Lys GAT OAT ATT GAG Asp Asp Ile Giu 280

AAA

Lys 265 CAT GTC TCO AAT His Val Ser Asn

GTA

Val 270 CAT AAG OCA CTT His Lys Ala Leu GAC TCG Asp Ser 275 TTA GTC AAG TTG CTT Leu Val Lys Leu Leu 285 TTG AAA GAG OAT Leu Lys Glu Asp CAC ACC AAT His Thr Asn 290 918 CTA GAT Leu Asp GCG TOT GCT CTT Ala Cys Ala Leu

CAT

His 300 TTC GCT OTT GCA Phe Ala Val Ala TAT TGC AAT GTG Tyr Cys Asn Val 305 OAT GTC AAC CAT Asp Val Asn His 966 AAG ACC Lys Thr 310 OCA ACA OAT CTT Ala Thr Asp Leu TTA AAA Leu Lys 315 CTT OAT CTT Leu Asp Leu

GCC

Ala 320

AGG

Arg 325 AAT CCG AGO GGA Asn Pro Arg Gly

TAT

330 ACO OTO CTT CAT Thr Val Leu His

OTT

Val 335 GCT OCG ATO CG Ala Ala Met Arg

AAG

Lys 340 1014 1062 1110 GAG CCA CAA TTG Olu Pro Gin Leu

ATA

Ile 345 CTA TCT CTA TTG Leu Ser Leu Leu

GAA

Olu 350 AAA GOT Lys Gly OAA OCA ACT Oiu Ala Thr ACT ATG GCG Thr Met Ala 375

TTG

Leu 360 GAA GOT AGA ACC Glu Gly Arg Thr

OCA

Ala 365 CTC ATO ATC Leu Met Ile OCA AGT GCA TCA Ala Ser Ala Ser 355 OCA AAA CAA 0CC Ala Lys Oln Ala 370 TGC AAG CAT TCT Cys Lys His Ser 385 GAA GAC AAA CGA Oiu Asp Lys Arg 1158 1206 OTT GAA TOT AAT Val Olu Cys Asn ATC CCG GAO CAA Ile Pro Glu Gin CTC AAA Leu Lys 390 GGC COA CTA TOT OTA Gly Arg Leu Cys Val 395 GAA ATA Olu Ile CTA GAG CAA Leu Oiu Gin 400 1254 GAA CAA ATT CCT AGA OAT OTT CCT CCC TCT TTT GCA OTO GCG 0CC GAT 1302 WO 98/29537 WO 9829537PCT/EP97/07253 -132 Glu Gin Ile Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala Asp 410 415 420 GAA TTG AAG ATG Glu Leu Lys Met

ACG

Thr 425 CTG CTC GAT CTT Leu Lou Asp Leu

GAA

GlU 430 AAT AGA OTT GCA Asn Arg Val Ala CTT GCT Leu Ala 435 1350 CAA COT CTT TTT Gin Arg Lou Phe 440 CCA ACG GAA GCA Pro Thr Glu Ala

CAA

Gin 445 OCT OCA ATG GAG Ala Ala Met Glu ATC 0CC GAA le Ala Glu 450 CCT GAC COT Pro Asp Arg 1398 ATG AAG Met Lys CTC ACT Leu Thr 470

GGA

Gly 455 ACA TGT GAG TTC Thr Cys Glu Phe

ATA

le 460 GTG ACT AGC CTC Val Thr Ser Lou CCG GOT GTA AAG, Pro Gly Val. Lys 480

GAG

Glu 465 GOT ACO AAG AGA Oly Thr Lys Arg ACA TCA Thr Ser 475 ATA OCA CCT TTC Ile Ala Pro Ph.

1446 1494 1542 1590

AGA

Arg 485 ATC CTA GAA GAG Ile Lou Glu Glu

CAT

His 490 CAA ACT AGA CTA Gin Ser Arg Lou

AAA

Lys 495 GCO CTT TCT AAA Ala Lou Ser Lys

ACC

Thr 500 GTG GAA CTC G Val Glu Lou Gly

AAA

Lys 505 CGA TTC TTC CCC Arg Phe Phe Pro

CGC

Arg 510 TGT TCG GCA OTO Cys Ser Ala Val.

CTC GAC Lou Asp 515 CAG ATT ATG AAC TOT TGA Gin Ile Met Asn Cys 520 INFORMATION FOR SEQ ID NO:12: Wi SEQUENCE CHARACTERISTICS: LENGTH: 522 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 1608 WO 98/29537 PCT/EP97/07253 -133- Met Asp Thr Thr Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Ala Ala Glu Val Ala Thr Asp Asn Thr 25 Asp Ser Ser Ile Val Tyr Leu Leu Gin Leu Gin Val Leu Thr Gly 40 Pro Asp Val Ser Ala Leu Ser Asn Ser Phe Glu Ser 55 Val Phe Asp Ser Pro Asp Asp Phe Tyr Ser Asp Ala Lys Leu Arg Cys Val Leu Ser Leu Ser Asp Gly Arg Glu Val Ser Phe Ala Arg Ser Ser Phe 90 Phe Lys Ser Ala Leu Ala Ala Ala Lys Glu Leu Lys 115 Lys 100 Glu Lys Asp Ser Asn 105 Asn Thr Ala Ala Val Lys Leu 110 Asp Ser Val Glu Ile Ala Lys Tyr Glu Val Gly Phe 125 Val Thr 130 Val Leu Ala Tyr Val 135 Tyr Ser Ser Arg Val 140 Arg Pro Pro Pro Lys 145 Gly Val Ser Glu Ala Asp Glu Asn Cys 155 Cys His Val Ala Cys 160 Arg Pro Ala Val Asp 165 Phe Met Leu Glu Val 170 Leu Tyr Leu Ala Phe Ile 175 Phe Lys Ile Val Val Asp 195 Ala Asn Ile 210 Pro 180 Glu Leu Ile Thr Leu 185 Tyr Gin Arg Lys Val Val Ile Asp Thr Leu Val His Leu Leu Asp 190 Ile Leu Lys Leu 205 Asp Arg Cys Lys Cys Gly Lys Ala 215 Cys Met Lys Leu Leu 220 Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser WO 98/29537 WO 9829537PCTIEP97/07253 -134- 235 Asp Arg Arg Lys 240 Leu Pro Glu Giu Leu 245 Val Lys Giu Ile Ile 250 Giu Leu 255 Gly Leu Glu Ala Leu Asp 275 Val 260 Pro Lys Val Lys Lays 265 His Val Ser Asn Val His Lys 270 Leu Lys Glu Ser Asp Asp Ile Glu 280 Leu Val Lys Leu Leu 285 Asp His 290 Thr Asn Leu Asp Asp 295 Ala Cys Ala Leu His 300 Phe Ala Val Ala Tyr 305 Asp Cys Asn Val Lys Val Asn His Arg 325 Thr 310 Ala Thr Asp Leu Leu 315 Lys Leu Asp Leu Asn Pro Arg Gly Tyr 330 Thr Val Leu His Val Ala 335 Ala Met Arg Ala Ser Ala 355 Lys 340 Glu Pro Gin Leu Ile 345 Leu Ser Leu Leu Giu Lys Gly 350 Leu Met Ile Ser Giu Ala Thr Leu 360 Giu Gly Arg Thr Al a 365 Ala Lys 370 Gin Ala Thr Met Val Glu Cys Asn Asn Ile Pro Ciu Gin 380 Giu le Leu Giu Gin Cys 385 Lys His Ser Leu Lys 390 Gly Arg Leu Cys Val 395 Glu Asp Lys Arg Giu 405 Gin Ile Pro Arg Asp 410 Val Pro Pro Ser Phe Ala 415 Val Ala Ala Val Ala Leu 435 Asp 420 Giu Leu Lys Met Thr 425 Leu Leu Asp Ala Gin Arg Leu Phe 440 Pro Thr Giu Ala Leu Glu Asn Arg 430 Gin Ala Ala Met 445 Val Thr Ser Leu Glu Ile 450 Ala Giu Met Lys Gly Thr Cys Glu Phe Ile 460 WO 98/29537 WO 9829537PCTIEP97/07253 -135- Glu 465 Pro Asp Arg Leu Thr 470 Gly Thr Lys Arg Thr 475 Ser Pro Gly Val Lys 480 Ile Ala Pro Ph. Arg 485 Ile Leu Giu Glu His 490 Gin Ser Arg Leu Lys Ala 495 Leu Ser Lys Ala Val Leu 515 Val Glu Leu Gly LYS 505 Arg Phe Phe Pro Arg Cys Ser 510 Asp Gin Ile Met Asn Cys 520 INFORMATION FOR SEQ ID 110:13: SEQUENCE CHARACTERISTICS: LENGTH: 1194 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1.-1194 OTHER INFORMATION: /product= "Altered form of NIMi" /note= "N-terminal/C-terminal chimera." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: ATG GAT TCG GTT GTG ACT GTT TTG GCT TAT GTT TAC AGC AGC AGA GTG Met Asp Ser Val Val Tbr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val AGA CCG CCG CCT AAA GGA GTT TCT GAA TGC GCA GAC Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp 25 GAG AAT TGC TGC Glu Asn Cys Cys CAC GTG GCT TGC CGG CCG GCG GTG GAT TTC ATO TTG GAG GTT CTC TAT His Val Ala Cys Arg Pro Ala Val Asp Ph. Met Leu Glu Val Leu Tyr 144 WO 98/29537 WO 9829537PCT/EP97/07253 -136- TTG GCT Leu Ala CAC TTA His Lou TTC ATC TTC AAG Phe Ile Phe Lys TTG GAC GTT OTA Lou Asp Val Val 70

ATC

Ile 55 CCT GAA TTA ATT Pro Giu Leu Ile

ACT

Thr CTC TAT CAG AGO Leu Tyr Gin Arg 192 GAC AAA GTT GTT ATA Asp Lys Val Val Ile 75 GAG GAC ACA TTG Glu Asp Thr Lou

GTT

Val s0 240 ATA CTC AAG CTT Ile Lou Lys Leu

OCT

Ala AAT ATA TOT Asn Ile Cys GOT AAA Giy Lys 90 GCT TGT ATG AAG Ala Cys Met Lys CTA TTG Leu Lou GAT AGA TGT AAA GAG ATT ATT GTC AAO Asp Arg Cys Lys Olu Ile Ile Val Lys 100 105 TCT AAT OTA GAT ATG GTT AGT Ser Asn Val Asp Met Val Ser 110 CTT GAA AAG Lou Glu Lys 115 TCA TTG CCG GAA Ser Leu Pro Giu

GAG

Oiu 120 CTT GTT AAA GAG Lou Val Lys Olu

ATA

Ile 125 ATT GAT AGA le Asp Arg 384 COT AAA Arg Lys 130 GAG CTT GOT TTO Olu Lou Oly Leu

GAG

Giu 135 OTA CCT AAA OTA Val Pro Lys Val

AAO

Lys 140 AAA CAT OTC TCO Lys His Val Ser 432

AAT

Asn 145 OTA CAT AAG OCA Val His Lys Ala GAC TCO OAT OAT Asp Ser Asp Asp GAG TTA GTC AAO Olu Leu Val Lys

TTO

Leu 160 480 CTT TTG AAA GAO Lou Leu Lys Giu

GAT

Asp 165 CAC ACC AAT His Thr Asn CTA OAT OAT GCG TOT OCT Leu Asp Asp Ala Cys Ala 170 CTT CAT Lou His 175 TTA AAA Leu Lys 528 TTC OCT OTT Phe Ala Val

GCA

Ala TAT TOC AAT OTO AAG Tyr Cys Asn Val Lys 185 ACC OCA. ACA OAT Thr Ala Thr Asp

CTT

Leu 190 CTT OAT Leu Asp 0CC OAT GTC AAC Ala Asp Val Asn AGO AAT CCG AGO Arg Asn Pro Arg GGA TAT ACO GTO Oly Tyr Thr Val 205 CTT CAT OTT OCT OCO ATG COO AAO GAG CCA CAA TTO ATA CTA TCT CTA WO 98/29537 WO 9829537PCTIEP97/07253 -137- Leu His 210 Val Ala Ala Met Arg 215 Lys Glu Pro Gin Leu 220 Ile Leu Ser Leu

TTG

Leu 225 GAA AAA GGT GCA Giu Lys Gly Ala

AGT

Ser 230 GCA TCA GAA GCA Ala Ser Giu Ala

ACT

Thr 235 TTG GAA GGT AGA Leu Glu Giy Arg

ACC

Thr 240 GCA CTC ATG ATC Ala Leu Met Ile

GCA

Ala 245 AAA CAA GCC ACT ATG Lys Gin Ala Thr Met 250 GCG GTT QAA TGT Ala Val Giu Cys AAT AAT Asn Asn 255 ATC CCG GAG Ile Pro Giu

CAA

Gin 260 TGC AAG CAT TCT CTC Cys Lys His Ser Leu 265 AAA GGC CGA CTA TGT GTA GAA Lys Giy Arg Leu Cys Val Glu 270 ATA CTA GAG Ile Leu Giu 275 CAA GAA GAC AAA Gin Giu Asp Lys

CGA

Arg 280 GA. CAA ATT CCT Glu Gin Ile Pro AGA GAT GTT CCT Arg Asp Val Pro 285 ACG CTG CTC GAT Thr Leu Leu Asp CCC TCT Pro Ser 290 TTT GCA GTG GCG Phe Ala Val Ala

GCC

Ala 295 GAT GA. TTG AAG Asp Glu Leu Lys

ATG

Met 300

CTT

Leu 305 GAA AAT AGA GTT Giu Asn Arg Val

OCA

Ala 310 CTT GCT CA. CGT Leu Ala Gin Arg

CTT

Leu 315 TTT CCA ACG GAA GCA Phe Pro Thr Giu Ala 320 CAA GCT GCA ATG Gin Ala Aia Met

GAG

Giu 325 ATC GCC GAP. ATG AP.G GGA ACA TGT GAG Ile Ala Glu Met Lys Gly Thr Cys Giu 330 TTC ATA Phe Ile 335 1008 GTG ACT AGC CTC Val Thr Ser Leu 340 GAG CCT GAC CGT Giu Pro Asp Arg ACT GGT ACG AAG Thr Gly Thr Lys AGA ACA TCA Arg Thr Ser 350 1056 CCG GGT Pro Gly AGA CTA Arg Leu 370

GTA

Val 355 AP.G ATA GCA CCT Lys Ile Ala Pro

TTC

Phe 360 AGA ATC CTA GAA Arg Ile Leu Glu GAG CAT CAA AGT Giu His Gin Ser 365 AAA. CGA TTC TTC LYS Arg Phe Phe 1104 1152 AAA GCG CTT TCT Lys Ala Leu Ser

AAA

Lys 375 ACC GTG GAA CTC Thr Val Giu Leu

GGG

Gly 380 WO 98/29537 WO 9829537PCT/EP97/07253 -138-

CCG

Pro 385 CGC TGT TCG GCA GTG CTC GAC CAG ATT ATG AAC TGT TGA Arg Cys Ser Ala Val Leu Asp Gin Ile Met Asn Cys* 390 395 1194 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 398 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Met 1 Asp Ser Val Thr Val Leu Ala Val Tyr Ser Ser Arg Val Arg Pro Pro His Val. Ala Pro Lys Gly Val Ser Glu 25 Cys Ala Asp Glu Asn Cys Cys Val Leu Tyr Cys Arg Pro Ala Asp Phe Met Leu Glu Leu Ala Phe Ile Phe Lys Ile 55 Pro Giu Leu Ile Thr Leu Tyr Gin Arg His Leu Leu Asp Val Asp Lys Val. Val. Glu Asp Thr Leu Ile Leu Lys Leu Ala Asn Ile Cys Gly Ala Cys Met Lys Leu Leu Asp Arg Cys Leu Clu Lys 115 Glu Ile Ile Val Lys 105 Ser Asn Val Asp Met Val Ser 110 le Asp Arg Ser Leu Pro Glu Leu Val Lys Giu le 125 Arg Lys 130 Glu Leu Gly Leu Glu 135 Val Pro Lys Val. Lys His Val Ser WO 98/29537 WO 9829537PCT/EP97/07253 -139- Asn 145 Val His Lys Ala Leu Asp Ser Asp Asp 150 Ile 155 Glu Leu Val Lys Leu Leu Lys Glu Asp 165 His Thr Asn Leu Asp 170 Asp Ala Cys Ala Leu His 175 Phe Ala Val Leu Asp Leu 195 Tyr Cys Asn Val Lys 185 Thr Ala Thr Asp Leu Leu Lys 190 Tyr Thr Val Ala Asp Val Asn His 200 Arg Asn Pro Arg Gly 205 Leu His 210 Val Ala Ala met Arg 215 Lys Glu Pro Gin Ile Leu Ser Leu Leu 225 Giu Lys Gly Ala Ser 230 Ala Ser Glu Ala Thr 235 Leu Glu Gly Arg Ala Leu Met Ile Ala 245 Lys Gin Ala Thr Met 250 Ala Val Glu Cys Asn Asn 255 Ile Pro Giu Ile Leu Giu 275 Gin 260 Cys Lys His Ser Lys Giy Arg Leu Cys Val Giu 270 Asp Val Pro Gin Giu Asp Lys Arg 280 Giu Gin Ile Pro Arg 285 Pro Ser 290 Phe Ala Val Ala Ala 295 Asp Giu Leu Lys Met 300 Thr Leu Leu Asp Giu Asn Arg Val Ala 310 Leu Ala Gin Arg Leu 315 Phe Pro Thr Glu Ala 320 Gin Ala Ala Met Ile Ala Giu met Gly Thr Cys Glu Phe Ile 335 Val Thr Ser Leu 340 Giu Pro Asp Arg Leu 345 Thr Gly Thr Lys Arg Thr Ser 350 His Gin Ser Pro Gly Val Lys 355 Ile Ala Pro Phe 360 Arg Ile Leu Glu Glu 365 Arg Leu Lys Ala Leu Ser Lys Thr Val Giu Leu Gly Lys Arg Phe Phe WO 98/29537 WO 9829537PCTIEP97/07253 140 370 Arg Cys Ser Ala 375 Val Leu Asp Gin Ile Met Asn Cys 390 395 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 786 base pairs TYPE: nucleic acid STRANDEDNESS: singie TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .786 OTHER INFORMATION: /product= "Altered fonrm of NIM1' /note= "Ankyrin domains of NIMi.* (xi) SEQUENCE DESCRIPTION: SEQ ID ATG GAC TCC MAC AAC ACC GCC Met Asp Ser Asn Asn Thr Ala 0CC GTG AAG CTC GAG Ala Val Lys Leu Glu CTT MAG Leu Lys GAG ATT Oiu Ile

GCC

Ala MAG OAT Lys Asp TAC GMA Tyr Glu GTC GOT Val Oly TTC GAT Phe Asp 25 TOG OTT GTG ACT GTT TTG OCT Ser Val Val Thr Val LeU Ala TAT OTT TAC Tyr Val Tyr TGC GCA GAO Cys Ala ASP AGC AGC AGA OTO Ser Ser Arg Val GAG MAT TGC TOO Glu Asn Cys Cys

AGA

Arg 40 CCG CCG CCT A Pro Pro Pro Lys

OGA

Gly GTT TCT GMA Val Ser Glu CAC GTG OCT TGC His Val Ala Cys CCG GCO GTG OAT Pro Ala Val Asp TTO ATG TTG GAG OTT CTC TAT TTG OCT TTC ATC 'rTC MAG ATC CCT GMA Phe Met Leu Glu Val Leu, Tyr Leu Ala Phe Ile Phe Lys Ile Pro Glu WO 98/29537 PTE9175 PCT/EP97/072i3 -141- TTA ATT ACT CTC Leu Ile Thr Leu

TAT

Tyr CAG AGO Gin Arg CAC TTA TTG His Leu Leu 90 GAC OTT GTA GAC Asp Val. Val Asp AAA OTT Lys Val 288 GTT ATA GAG Val Ile Giu

GAC

Asp 100 ACA TTG GTT ATA Thr Leu Vai Ile CTC AAO CTT GCT AAT ATA TGT GGT Leu Lys Leu Ala Asn le Cys Gly 105 .110 336 AAA OCT TGT Lys Ala Cys 115 ATG AAG CTA TTG GAT Met Lys Leu Leu Asp 120 AGA TGT AAA GAG ATT Arg Cys Lys Giu Ile 125 ATT GTC AAG le Val Lys 384 TCT AAT Ser Asn 130 GTA OAT ATG GTT Val Asp Met Val

AGT

Ser 135 CTT GAA AAG TCA Leu GJlu Lys Ser

TTG

Leu 140 CCG GAA GAG CTT Pro Giu Glu Leu

GTT

Val1 145 AAA GAG ATA ATT Lys Glu Ile Ilie

GAT

Asp 150 AGA COT AAA GAG Arg Arg Lys Giu CTT GGT TTG GAG Leu Gly Leu Giu 155 AAG GCA CTT GAC Lys Ala Leu Asp GTA CCT Val Pro 160 TCG OAT Ser Asp 175 AAA OTA AAG AAA Lys Val Lys Lys GTC TCG AAT GTA Val Ser Asn Val

CAT

His 170 528 OAT ATT GAG Asp Ile Glu

TTA

Leu 180 GTC AAO TTG CTT Val Lys Leu Leu AAA GAG OAT CAC Lys Giu Asp His ACC AAT CTA Thr Asn Leu 190 AAT GTO AAG Asn Val Lys 576 OAT OAT Asp Asp TOT OCT CTT CAT Cys Ala Leu His

TTC

Phe 200 OCT OTT GCA TAT Ala Val Ala Tyr

TOC

Cys 205 624

ACC

Thr

AAT

Asn 225 ACA OAT CTT TTA Thr Asp Leu Leu CTT OAT CTT 0CC GAT Leu Asp Leu Ala Asp 220 GTC AAC CAT AGO Val. Asn His Arg CCC AGO OGA TAT Pro Arg Gly Tyr

ACG

Thr 230 OTO CTT CAT OTT Vai LeU His Val

OCT

Ala 235 OCG ATG COG MOG Ala Met Arg Lys

GAG

Olu 240 CCA CMA TTG ATA CTA TCT CTA TTG GMA AAA GOT GCA ACT OCA TCA GMA WO 98/29537 WO 9829537PCTAEP97/07253 .142 Pro Gin Leu Ile Leu 245 Ser Leu Leu Giu Lys Gly Ala Ser Ala Ser Glu 250 255 GCA ACT TTG GAR GGT TGA Ala Thr Leu Glu Gly 260 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 262 amino acids TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Met 1 Asp Ser Asn Asn Thr Ala Aia Val 5 Leu Glu Leu Lys Glu Ile Ala Lys Asp TIyr Val Tyr Tyr Giu Val Gly Phe Asp 25 Ser Val Val Thr Val Leu Ala Val Ser Giu Ser Ser Arg Val Arg 40 Pro Pro Pro Lys Gly Cys Ala Asp Glu Asn Cys His Val Ala Cys Pro Ala Val Asp Phe Met Leu Glu Val Tyr Leu Ala Phe Ile 75 Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gin Arg His Leu Leu 90 Asp Val Vai Asp Lys Val Val Ile Glu Lys Ala Cys 115 Asp 100 Thr Leu Val Ile LYS Leu Ala Asn Ile Cys Gly 110 Ile Val Lys Met Lys Leu Leu Asp 120 Arg Cys Lys Giu Ile 125 WO 98/29537 WO 9829537PCT/EP97/07253 -143- Ser Asn Val 130 Asp Met Val Ser 135 Leu Glu Lys Ser Leu 140 Pro Glu Glu Leu Val 145 Lys Glu Ile Ile Asp 150 Arg Arg Lys Glu Leu Gly Leu Glu 155 Lys Ala Leu Asp Val Pro 160 Lys Val Lys Lys His 165 Val Ser Asn Val Ser Asp 175 Asp Ile Glu Asp Asp Ala 195 Leu 180 Val Lys Leu Leu LeU 185 Lys Glu Asp His Thr Asn Leu 190 Asn Val Lys Cys Ala Leu His Phe 200 Ala Val Ala Tyr Cys 205 Thr Ala 210 Thr Asp Leu Leu Arg Gly Tyr Thr 230

LYS

215 Leu Asp Leu Ala Asp 220 Val Asn His Arg Asn 225 Pro Val Leu His Val Ala 235 Ala Met Arg Lys Glu 240 Pro Gin Leu Ile Ser Leu Leu Glu Gly Ala Ser Ala Ser Glu 255 Ala Thr Leu Giu Gly 260 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 41 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: WO 98/29537 PCT/EP97/07253 -144- Ile Arg Arg Met Arg Arg Ala 1 5 Leu Asp Ala Ala Asp Ile 10 Glu Leu Val Ala Leu Ala Lys Leu Met Val Met Gly Glu Gly Leu 25 Asp Leu Asp Asp Val His Tyr Ala Val Gin His Cys Asn INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 38 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Pro 1 Thr Gly Lys Thr Ala Leu His Leu 5 Ala Ala Glu Met 10 His Ala Asp Xaa Val Ser Pro Asn Phe Arg Asp Met Val Ser Val Leu Leu Asp His 25 Thr Xaa Asp Gly Val Thr INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 41 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide WO 98/29537 PCT/EP97/07253 -145- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Ile Arg Arg Met Arg Arg Ala Leu 1 5 Asp Ala Ala Asp Ile Glu Leu Val 10 Lys Leu Met Val Met Gly Glu Gly Leu 25 Asp Leu Asp Asp Ala Leu Ala Val His Tyr Ala Val Gin His Cys Asn INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 27 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Arg Arg Pro Asp Ser Lys Thr Ala Leu His Leu Ala Ala Glu Met Val 1 5 10 Ser Pro Asp Met Val Ser Val Leu INFORMATION FOR SEQ ID NO:21: Leu Asp Gin SEQUENCE CHARACTERISTICS: LENGTH: 41 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant WO 98/29537 PCT/EP97/07253 -146- (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Ile Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile Glu Leu Val 1 5 10 Lys Leu Met Val Met Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala 25 Val His Tyr Ala Val Gin His Cys Asn INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 27 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Arg Arg Pro Asp Ser Lys Thr Ala Leu His Leu Ala Ala Glu Met Val 1 5 10 Ser Pro Asp Met Val Ser Val Leu Leu Asp Gin INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 41 amino acids TYPE: amino acid STRANDEDNESS: not relevant WO 98/29537 PCT/EP97/07253 -147- TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Ile Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile Glu Leu Val 1 5 10 Lys Leu Met Val Met Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala 25 Val His Tyr Ala Val Gin His Cys Asn INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Pro Thr Gly Lys Thr Ala Leu His Leu Ala Ala Glu Met Val Ser Pro 1 5 10 Asp Met Val INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs WO 98/29537 PCVEP97/073 -148- TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID CAACAGCTTC GAAGCCGTCT TTGACGCGCC GGATG INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CATCCGGCGC GTCAAAGACG GCTTCGAAGC TGTTG INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide" WO 98/29537 PCT/EP97/07253 -149- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GGAATTCAAT GGATTCGGTT GTGACTGTTT TG 32 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: GGAATTCTAC AAATCTGTAT ACCATTGG 28 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: CGGAATTCGA TCTCTTTAAT TTGTGAATTT C 31 WO 98/29537 PCT/EP97/07253 -150- INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID GGAATTCTCA ACAGTTCATA ATCTGGTCG 29 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: GGAATTCAAT GGACTCCAAC AACACCGCCG C 31 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 98/29537 PCT/EP97/07253 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc 'oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: GGAATTCTCA ACCTTCCAAA GTTGCTTCTG ATG

Claims (13)

1. A method for protecting a plant from pathogen attack through synergistic disease resistance, comprising the steps of: obtaining an immunomodulated plant having a first level of disease resistance by applying to a plant INA or SA; or by selecting a plant based on constitutive expression of SAR genes and/or a disease-resistant phenotype; or by genetically engineering a plant by transforming it with one or more SAR genes; and applying to said immunomodulated plant at least one microbicide that confers a second level of disease resistance; whereby application of said microbicide to said immunomodulated plant confers a synergistically enhanced third level of disease resistance that is greater than the sum of the first and second levels of disease resistance.

2. A method according to claim 1, wherein said immunomodulated plant is a constitutive immunity (cim) mutant plant; a lesion mimic mutant plant; obtained by recombinant expression in a plant of an SAR gene; obtained by applying to a plant a chemical capable of inducing SAR.

3. A method according to claim 2, wherein said cim mutant plant is selected from a population of plants according to the following steps: 20 evaluating the expression of SAR genes in uninfected plants that are phenotypically normal in that said uninfected plants lack a lesion mimic phenotype; and selecting uninfected plants that constitutively express SAR genes in the absence of viral, bacterial, or fungal infection.

4. A method according to claim 2 wherein said lesion mimic mutant plant is selected from a population of plants according to the following steps: evaluating the expression of SAR genes in uninfected plants that have a lesion mimic phenotype; and cQ selecting uninfected plants that constitutively express SAR genes in the S asnce of viral, bacterial, or fungal infection. P:\OPER\MKR\58597-98.175 -2416/99 -153- A method according to claim 2 wherein said SAR gene is a functional form of a NIM1 gene encoding a NIM1 protein involved in the signal transduction cascade leading to systemic acquired resistance in plants.

6. A method according to claim 5, wherein said NIM1 protein comprises the amino acid sequence set forth in SEQ ID NO:2.

7. A method according to claim 5, wherein said NIM1 gene comprises the coding sequence set forth in SEQ ID NO:1.

8. A method according to claim 2 wherein said SAR gene encodes an altered form of a NIM1 protein that acts as a dominant-negative regulator of the SAR signal transduction pathway.

9. A method according to claim 8, wherein said altered form of the NIM1 protein has alanines instead of serines in amino acid positions corresponding to positions and 59 of SEQ ID NO:2. A method according to claim 9, wherein said altered form of the NIM1 protein 20 comprises the amino acid sequence shown in SEQ ID NO:8.

11. A method according to claim 9, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:7.

12. A method according to claim 8, wherein 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.

13. A method according to claim 12 wherein said altered form of the NIM1 protein o omprises the amino acid sequence shown in SEQ ID P:\OPER\MKR\58597-98.175 24/6/99

154- 14. A method according to claim 12 wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:9. A method according to claim 8, wherein said altered form of the NIM1 protein has a C-terminal truncation of amino acids corresponding approximately to amino acid positions 522-593 of SEQ ID NO:2. 16. A method according to claim 15, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown SEQ ID NO:12. a.. 17. A method according to claim 15, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:11. 18. A method according to claim 8, wherein 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. 19. A method according to claim 18, wherein said altered form of the NIM1 protein 20 comprises the amino acid sequence shown in SEQ ID NO:14. A method according to claim 18, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:13. 21. A method according to claim 8, wherein said altered form of the NIM1 protein consists essentially of ankyrin motifs corresponding approximately to amino acid positions 103-362 of SEQ ID NO:2. 22. A method according to claim 21, wherein said altered form of the NIM1 protein omprises the amino acid sequence shown in SEQ ID NO:16. WO 98/29537 PCT/EP97107293 155 23. A method according to claim 21, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID 24. A method according to any one of claims 7, 11, 14, 17,20 and 23, wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID Nos: 1, 7, 9, 11, 13 or 15: hybridization in 1 %BSA; 520mM NaPO 4 pH7.2; 7% lauryl sulfate, sodium salt; I1mM EDTA; 250 mM sodium chloride at 55 0 C for 1 8-24h, and wash in 6XSSC for 15 min. (M3) 3XSSC for 15 min (X1) at 55 0 C. A method according to any of claims 1 to 24, wherein said microbicide is a fungicide selected from the following group: 4-[3-(4-chlorophenyl)-3-(3,4-dimthoxyphenyl)acyloyl]morpholine ("dimethomorphu); ,2,4-triazolo[3,4-b][1 ,3]benzothiazole (otricyclazoleo); 3-allyloxy-1 ,2-benzothiazole-1 ,1 -dioxide (aprobonazole*); ca-2-(4-chlorophenyl)ethyl]--a-(1, 1-dimethylethyl)-1 H-i ,2,4-triazole-1 -ethanol, (utebuconazole*); 1 -[[3-(2-chlorophenyl)-2-(4-fluorophenyl)oxiran-2-yl]methyl-1 H-i ,2,4-triazole, ("epoxyconazole"); a-(4-chlorophenyl)--a-(1 -cyclopropylethyl)--1 H-i ,2,4-triazole-1 -ethanol, ("cyproconazole"); 5-(4-chlorobenzyl)--2,2-dimethyl-1 (1 H-I ,2,4-triazol-1 -ymethyl)-cyclopentanol, (nmetconazole*); 2-(2+4dichlorophenyl)--3-(1 H-i ,2,4-triazol-i -yl)-propyl-1,1 ,2,2-tetrafluoroethyl-ether, (utetraconazolel); methyl-(E)-2-(2-[6-(2-cyanophenoxy)pyrimidin--4-yloxylphenyl)-3-methoxyacrylate, ("ICI A 5504", "azoxystrobino); methyl-(E)--2-methoximino--2-[ct-(o-tolyloxy)--o-tolyl]acetate, (OBAS 490 FO, "cresoxime methyl"); 2-(2-phenoxyphenyl)-(E)-2-methoximrino--N-methylacetamide); [2-(2,5-dimethylphenoxymethyl)-phenyl]-(E)-2-methoximino-N-methylacetamide); (1 R,3S/1 S,3R)-2,2-dichloro--N-[(R)-1 -(4-chlorophenyl)ethyl]-1 -ethyl-3- methylcyclopropanecarboxamide, ('KTU 361 6); WO 98/29537 PCTIEP97/07253

156- manganese ethylenebis(dithiocarbamate)polymr-zilc complex, (mmancozebo); 1 -[2-(2,4-dichlorophenyl)-4-pOpyl-1 ,3-dioxolan-2-ylmethyl]-l H-i ,2,4-triazole, ("propiconazole*); 1 -(2-[2-chloro-4-(4-chlorophenoxy)phenfl]-4-methyl-1 ,3-dioxolan-2-ytmethy l)-1 H- 1 ,2,4--triazole, cInifenoconazole*); 1 -[2-(2,4-dichlorophenyl)pentyl--1 H-i 2,4-triazole, T upenconazoleo); cis-4-[3-(4-tert-butylphenyl)-2-methylpropyl]--2,6-dimethylmorpholifle, ("fenpropimorph'); 1 -[3-(4-tert-butylphenyl)--2-methylpropyl]-piperidifle, ('fenpropidin"); 4-cyclopropyl-6- methyl-i-j-phenyl-2-pyrimidinamine (Ocyprodinilm); (RS)-N-(2,6-dimethylphenyl-N-(methoxyacetyl)-alalife methyl ester (ametalaxyra); (R)-N-(2,6-dimethylphenyl--(methoxyacetyl)-alalife methyl ester (OR-metalaxyl m 1 ,2,5,6-tetrahydro-4H-pyrrolo[3,2,1 -ijlquinolin-4-one (Mpyroquilono); and ethyl hydrogen phosphonate ("tosetyl"). 26. A method according to claim 25, wherein said fungicide is metalaxyl. 27. A method according to any of claims 2 to 26, wherein said chemical capable of inducing SAR is either a benzothiadiazole compound, an isonicotinic acid compound, or a salicylic acid compound. 28. A method according to any of claims 1 to 26, wherein said microbicide is either a benzothiadiazole compound, an isonicotinic acid compound, or a salicylic acid compound. 29.. A method according to any of claims 1 to 28, wherein two microbicides are concurrently applied to said immunomodulated plant. A method according to claim 29, wherein one of said microbicides is a fungicide selected from the following group: 4-[3-(4-chlorophenyI)-3-(3,4-dimethoxyphelyl)acryloyljmlorpholine (Odimethomorpho); ,2,4-triazolo[3,4-b][1 ,3]benzothiazole (tricyclazole'); 3-allyloxy-i ,2-benzothiazole-i ,1 -dioxide ('probonazolea); WO 98/29537 PCT/EP97/07253 -157- a-[2-(4-chlorophenyl)ethyl]--cG-(1 -dimethylethyl)-1 H-i ,2,4-triazole-1 ethanol, (*tebuconazole*); 1 .[3-(2-chlorophenyl)-2(4fluorophlyl)oxiral-2-yl]methy1]-1 H-I ,2,4-triazole, (uepoxyconazolo); a(4-chlorophenyl)--ae-(1 .cyclopropylethyl)-1 H-i ,2,4-triazole-I -ethanol, ('cyproconazol*); 5-(4-chlorobenzyl)-2,2-dimlethyl-1 H-i ,2,4-triazol-1 -ymethyit)-cyclopentanol, ("metconazole"); 2-(2,4dichlorophely)--3-(1 H-i ,2,4-tniazol-1 -yl)-propyl-1 ,1 ,2,2-tetrafluoroethyl-ether, ("tetraconazole"); methyl-(E)-2-{2-[6-(2-cyanophOfloxy)pyflmidifl-4-yloxy]pheflyl)-3-methoxyacrylate, ("IC A 5504", "azoxystrobin*) methyl-(E)-2-methoximino-2-[-(o-tolyloxy)--o-tolyl]acetate, (*BAS 490 170, acresoxime methyl'); 2-(2-phenoxypheny)-(E)-2-mtho)imilo-N-methylacetamide); [2-(2,5-dimethylphenoxymethyl)-phenl]-(E)--2-methoximiflo-N-methylacetamide); (1 R,3S/1 S,3R)-2,2-dichoro-N-[(R)-1 -(4-chlorophenyl)ethyl]-1 -ethyl-3- methylcyclopropanecarboxamide, (nKTU 3616"); manganese ethylenebis(dithiocarbamate)polymer-zinc complex, ("mancozebo); 1 -[2-(2,4-dichlorophenyl)-4-propyl-1 ,3-dioxolan-2-ylmethyl]-1 H-i ,2,4-triazole, ("propiconazole"); 1 -f2-[2-chloro-4-(4-chlorophenoxy)phenyl]-4-methyl-1 ,3-dioxolan-2-ylmethy I)-i H- 1 ,2,4-triazole, ('ditenoconazole'); 1 -[2-(2,4-dichlorophenyl)pentyl--I H-i ,2,4-triazole, (upenconazole*); cis-4-[3(4-tert-butylphenyly-2-methylpropyl]--2,6-dimethylmorpholine, ('fenpropimorph'); 1 -[3-(4-tert-butylphenyl)-2-methylpropyl]-pipendifle, ("fenpropidin'); 4-cyclopropyl-6- methyI-N ~phenyl-2-pyrimidinamine ("cyprodinil'); (RS)-N-(2,6-dimethylphenyl--N-(methoxyacetyl)-alanine methyl ester (ametalaxylo); (R)-N-(2,6-dimethylphenyl--N-(methoxyacetyl)-alanifle methyl ester (NR-metalaxylo); 1 ,2,5,6-tetrahydro-4H-pyrrolo[3,2,1 -ij]quinolin-4-one (opyroquilono); and ethyl hydrogen phosphonate ("fosetyl"); -158 and the other microbicide is is either a benzothiadiazole compound, an isonicotinic acid compound, or a salicylic acid compound. 31. A method according to claim 30, wherein the fungicide is metalaxyl and the other microbicide is a benzothiadiazole compound. 32. A method according to claim 1 substantially as hereinbefore described. DATED this 28 th day of July 2000 Novartis AG by DAVIES COLLISON CAVE Patent Attorneys for the Applicants S.. S S *SSS