HU224480B1 - Method for protecting plants - Google Patents

Abstract

The present invention relates to a method for protecting plants against pathogen attack by inducing synergistic disease resistance. The method comprises (a) producing a chimeric gene transformed plant, wherein said gene comprises a plant-active promoter operably linked to: (i) a nucleotide sequence encoding a wild-type NIM1 protein that participates in systemic resistance to systemic acquired resistance (SAR) , or (ii) a nucleotide sequence encoding a modified form of the NIM1 protein, which modified protein acts as a dominant-negative regulator of the SAR signal transduction pathway, wherein the resulting plant has a first level of disease resistance; and (b) applying to said plant at least one microbicide which confers a second level of disease resistance to the plant; wherein the application of the microbicide imparts to said plant a synergistically increased, third level of disease resistance greater than the sum of the first and second levels of disease resistance.

Description

The present invention relates to a method of protecting plants against pathogen attack by synergistic disease resistance, which is achieved by applying a microbicide to an immuno-modified plant.

Plants are constantly exposed to a wide range of pathogenic organisms, including viruses, bacteria, fungi and nematodes. Crop plants are particularly vulnerable because they are usually grown in genetically uniform monocultures; if the disease hits its head, the losses can be severe. However, most plants have their own innate defense mechanisms against pathogenic organisms. A natural variant of resistance to plant pathogens has already been identified by plant growers and pathologists and introduced into a variety of crop plants by breeding. These natural disease resistance genes often confer a high level of resistance or immunity to pathogens.

Systemic acquired resistance (SAR) is a component of the complex system used by the plant to protect itself from pathogens [Hunt and Ryals, Crit. Port. in Plant Sci. 15: 583-606 (1996); Ryals et al .: Plant Cell. 8: 1809-1819 (1996). See also U.S. Patent No. 5,614,395. SAR is particularly important in response to plant pathogens because it is a pathogen-inducible systemic resistance to a broad spectrum of infectious agents including viruses, bacteria and fungi. When the SAR block the signal transduction pathway, the plants are more susceptible to the pathogens that normally cause disease and become more sensitive to certain infectious agents that do not normally cause disease (Gaffney et al., Science 261: 754-756 (1993); Delaney et al. (1994) Science 266: 1247-1250; Delaney et al., Proc. Natl. Acad. Sci. USA 92: 6602-6606 (1995); Delaney: Plant. Phys. 113: 5-12 (1997); Bi et al. (1995) Plant J. 8: 235-245; Mauch-Mani and Slusarenko: Plant Cell. 8: 203-212 (1996). These observations indicate that the SAR signal transduction pathway is critical to maintaining plant health.

Theoretically, the SAR response can be divided into two phases. During the initial phase, the plant recognizes the pathogen infection and releases a signal that is transmitted to the distant tissues through the phloem. This systemic signal is sensed by target cells, which respond by expressing both SAR genes and disease resistance. The maintenance phase of SAR, which can last from weeks to full plant life, refers to the time during which the plant is approximately dormant and disease resistance is maintained (Ryals et al., 1996).

Salicylic acid (SA) accumulation appears to be required for SAR signal transduction. Plants that are unable to accumulate SA due to treatment with specific inhibitors, epigenetic repression of phenylalanine ammonia lyase, or transgenic expression of salicylate hydroxylase that specifically degrades SA are unable to induce expression of the SAR gene , nor 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); Pallas et al .: Plant. J. 10: 281-293 (1996)]. Although SA was supposed to serve as a systemic signal itself, this is still controversial, and until now it is only known with certainty that if SA cannot accumulate, SAR signal transduction is blocked [Pallas et al., 1996; Shulaev et al .: Plant Cell. 7, 1691-1701 (1995); Vernooij et al .: Plant Cell. 6: 959-965 (1994).

Currently, Arabidopsis has been selected as a model system for studying SAR [Uknes et al .: Molar. Plant-Microbe Interact. 6: 692-698 (1993); Cameron et al. (1994) Plant J. 5: 715-725; Mauch-Mani and Slusarenko: Mole. Plant Microbe Interact. 7: 378-383 (1994); Dempsey and Klessing (1995) Bulletin de L'Institut Pasteur 93, 167-186. It has been found that SAR can be activated in Arabidopsis by pathogens and chemicals such as SA, 2,6-dichloroisonicotinic acid (INA), and S-methyl ester of benzo [1.2.3] thiadiazole-7-carbothioic acid ( BTH) [Uknes et al., 1992; Vernooij et al., Mol. Plant-Microbe Interact. 8: 228-234 (1995); Lawton et al. (1996) Plant J. 10: 71-82. Following INA or BTH treatment or pathogen infection, at least three pathogenesis-related (PR) protein genes, namely PR-1, PR-2 and PR-5, are induced in concert, which is associated with the onset of resistance (Uknes et al., 1992, 1993). Treatment with a pathogen or an immunizing compound induces the expression of at least nine gene sets in the tobacco plant, which is the most characterized species [Ward et al., Plant Cell. 3: 1085-1094 (1991). Transgenic disease-resistant plants have already been produced by transforming plants with various SAR genes (US-PS 5,614,395).

A large number of Arabidopsis mutants with modified SAR signal transduction have been isolated (Delaney, 1997). The first of these mutants were the so-called Isd f / esions simulating disease) mutants and the acd2 (accelerated cell death) mutants [Dietrich et al., Cell. 77: 565-577 (1994); Greenberg et al., Cell. 77: 551-563 (1994). All of these mutants showed some degree of spontaneous necrotic lesion formation on their leaves, elevated SA levels, increased mRNA accumulation on SAR genes, and significantly increased disease resistance. At least seven different Isd mutants have been isolated and characterized [Dietrich et al., 1994; Weymann et al .: Plant Cell. 7, 2013-2022 (1995)]. Another interesting class of mutants is the constitutive / mmunity mutants [Lawton et al., "The Molecular Biology of Systemic Acquired Resistance" in B. Fritig and M. Legrand, Publisher, Dordrecht, The Netherlands: Kluwer Academic Publishers), 422-432 p. (1993); see also PCT Publication No. WO 94/16077]. Like the Isd and acd2 mutants, the title mutants show increased SA and SAR gene expression and resistance but

Unlike the Isd and acd2 mutants, they do not induce detectable lesions on their leaves. Epri (constitutive expresser of PR genes) may be one of the types of the title mutant; however, since the presence of microscopic lesions on epri leaves could not be excluded, one type of epri may be the Isd mutant [Bowling et al., Plant Cell. 6: 1845-1857 (1994).

Mutants in which SAR signaling was inhibited were also isolated. Ndr1 (non-race-specific disease zesistance) is a mutant that permits the growth of normal avirulent isolates of both Pseudomonas syringae and Peronospora parasitica containing different avirulent genes [Century et al., Proc. Natl. Acad. Sci. USA 92: 6597-6601 (1995)]. Currently, this mutant also inhibits initial SAR signaling. Npr1 (nonexpresser of PR genes) is a mutant that is unable to induce expression of the SAR signaling pathway following INA treatment [Cao et al., Plant Cell. 6: 1583-1592 (1994)]. Enhanced disease susceptibility mutants were isolated on the basis of their ability to maintain bacterial infection after inoculation with low bacterial concentrations (Glazebrook et al., 1996, Genetics 143: 973-982; Parker et al .: Plant Cell. 8: 2033-2046 (1996)]. Some eds mutants are phenotypically very similar to npr1 mutants, and recently eds5 and eds53 have been shown to be alleles of npr1 (Glazebrook et al., 1996). Nim1 (noninducible / mmunity) is a mutant that maintains the growth of P. parasitica (i.e., the cause of cotton wool disease) following INA treatment (Delaney et al., 1995; WO 94/16077). Although nim1 can accumulate SA after pathogen infection, it cannot induce SAR gene expression or disease resistance, assuming that the mutant blocks the progression pathway of SA. Nim1 is also defective in its ability to respond to INA or BTH, assuming that inhibition is mediated by the action of these chemicals (Delaney et al., 1995; Lawton et al., 1996).

Recently, two alleles of the Arabidopsis gene have been isolated and characterized, mutants responsible for the nim1 and npr1 phenotypes [Ryals et al., Plant Cell. 9: 425-439 (1997); Cao et al., Cell. 88: 57-63 (1997). The wild-type NIM1 gene product is present in both the SAR and the gene-to-gene disease-resistance signal transduction cascade in Arabidopsis (Ryals et al., 1997). Ryals et al. Also reported the isolation of five other alleles of nim1, showing a range of phenotypes ranging from weakly inhibited chemically induced PR-1 gene expression to fungal resistance to very highly inhibited. The introduction of the wild-type NPR1 gene into npr1 mutants not only complemented these mutants, restoring SAR induction sensitivity to PR gene expression and disease resistance, but also rendered transgenic plants more resistant to P. syringae infection in the absence of SAR et al. ., 1997).

Disease resistance responses include NF-κΒ / ΙκΒ signal transduction pathways across a wide range of organisms from Drosophila to mammals. In mammals, NF-κΒ / ΙκΒ signal transduction can be induced by a variety of stimuli, including exposure to cells for lipopolysaccharides, tumor necrosis factor, interleukin 1 (IL-1) or viral infection [Baeuerle and Baltimore, Cell. 87: 13-20 (1996); Baldwin: Annu. Port. Immunol. 14: 649-681 (1996)]. The activated pathway leads to the synthesis of several factors involved in inflammatory and immune responses, such as IL-2, IL-6, IL-8 and granulocyte / macrophage colony stimulating factor [deMartin et al., Gene 152, 253-255 ( 1995)]. In transgenic mouse studies, switching off the NF-κΒ / ΙκΒ signaling pathway led to a protective immune response, which resulted in increased susceptibility to bacterial and viral pathogens (Bég and Baltimore: Science 274: 782-784 (1996); Van Antwerp et al., Science 274: 787-789 (1996); Wang et al., 1996, Science 274: 784-787; Baeyerle and Baltimore (1996)]. In Arabidopsis, SAR is functionally analogous to inflammation, in which normal resistance processes are amplified following SAR activation and thus lead to increased disease resistance (Bi et al., 1995; Cao et al., 1994; Delaney et al., 1995; Delaney et al., 1995). al., 1994; Gaffney et al., 1993; Mauch-Mani and Slusarenko 1996; Delaney, 1997). In addition, inactivation of the pathway leads to increased susceptibility to bacterial, viral and fungal pathogens. Interestingly, SA has been reported to inhibit NF-κB activation in mammalian cells (Kopp and Ghosh, Science 265: 956-959 (1994)), while SA activates signal transduction in Arabidopsis. Bacterial infection of Drosophila activates the signal transduction cascade leading to the synthesis of a large number of antifungal proteins such as cercropin B, defensin, diptericin and drosomycin [Ip et al., Cell. 75: 753-763 (1993); Lemaitre et al .: Cell. 86: 973-983 (1996). This induction depends on the two NF-κΒ homologs, dorsal and diphene products, and can be repressed by an IkB homolog, cacfus, during the process. Mutants that reduce the synthesis of antifungal and antibacterial proteins dramatically reduce resistance to infection.

All references cited herein are hereby incorporated by reference in their entirety.

Despite a great deal of research and sophisticated and intensive plant protection procedures, including genetic transformation of plants, disease-related losses have remained in the billions of dollars each year. Therefore, there remains a need to develop new crop protection methods based on the ever-evolving understanding of the genetic basis of disease resistance in plants.

Accordingly, the present invention is primarily directed to a novel method of protecting plants against pathogen attack by synergistic disease resistance, which is achieved by applying a microbicide to immunomodulated plants. Immunomodulated plants are those in which SAR is activated, typically showing higher SAR gene expression3

EN 224 480 Β1 as a wild type and therefore referred to as "SAR-on" plants. Immunomodulated plants for use in the method of the present invention can be obtained by at least three different routes: chemical inducers of SAR, such as BTH, INA or SA, are applied to the plants; through a selective breeding program in which plants are selected based on the constitutive expression of SAR genes and / or a disease resistant phenotype; or genetically engineered to transform plants with one or more SAR genes, such as a functional form of the NIM1 gene. The microbicide used for immunomodulated plants may be a standard microbicide, such as the fungicidal metalaxyl, or, if the immunomodulated plant is obtained by selective breeding or genetic engineering, it may be a chemical inducer of SAR, such as BTH, INA or SA.

Immunomodulation in a plant results in some degree of disease resistance. Similarly, the application of a microbicide to a plant provides a degree of disease resistance. When combining immunomodulation with the use of a microbicide, the expected result would be the level of additive control obtained from the sum of the disease resistance provided by each procedure. However, when a microbicide is continuously applied to an immunomodulated plant, disease resistance will unexpectedly increase synergistically; that is, the level of disease resistance is higher than the expected additive levels of disease resistance.

Accordingly, the invention also relates to the production of immunomodulated plants and to the use of an appropriate amount of a conventional microbicide. A particularly preferred embodiment of the present invention relates to plants genetically modified to contain and express a functional form of the NIM1 gene or a homologue or variant thereof.

The process of the present invention results in greater pathogen control (inhibition) than is achieved by immunomodulation alone or by the use of a microbicide. Immunomodulation in a plant results in some degree of disease resistance. Similarly, the application of a microbicide to a plant provides a degree of disease resistance. When combining immunomodulation with the use of a microbicide, the expected result would be the level of additive control obtained from the sum of the disease resistance provided by each procedure. However, when a microbicide is continuously applied to an immunomodulated plant, disease resistance will unexpectedly increase synergistically; that is, the level of disease resistance is greater than the expected sum of the levels of disease resistance.

In addition to the greater disease resistance, another advantage of the invention is that less microbicides are needed to achieve the level of disease resistance provided by the method of the invention than is required for conventional wild-type plants.

As a result, the economic costs of the microbicide are lower and the potential for adverse environmental consequences from the toxicity of certain microbicides is lower. Furthermore, the combination of the effects of immunomodulation and the use of a microbicide in the plant protection method of the present invention results in a longer duration of the antipathogenic effect and thus higher yields. Another advantage of this method is that, since the combined modes of controlling the pathogen are completely different, the threat of the development of resistance can be effectively prevented.

The present invention therefore relates to a method for protecting plants against pathogenic attack by synergistic disease resistance, comprising the steps of:

(a) providing an immunomodulated plant having a first level disease resistance; and (b) applying to said immunomodulated plant at least one microbicide conferring a second level of disease resistance;

(c) whereby applying said microbicide to said immunomodulated plant provides a synergistically increased level of disease resistance greater than the sum of the first and second levels of disease resistance.

The present invention thus relates to a method for protecting plants against pathogen attack, comprising the steps of:

(a) providing a plant transformed with a chimeric gene, said gene comprising a promoter active in plants operably linked to:

(i) a nucleotide sequence encoding a wild-type NIM1 protein that participates in a signal transduction cascade leading to systemic acquired resistance (SAR) in plants, or (ii) a nucleotide sequence encoding a modified form of the wild-type NIM1 protein that encodes a SAR signal. acting as a dominant-negative regulator of the pathway, wherein said plant has a first-degree disease resistance; and (b) applying to said plant at least one microbicide that imparts to the plant a second level of disease resistance;

wherein the application of the microbicide to said plant provides a synergistically increased level of disease resistance greater than the sum of the first and second levels of disease resistance.

Preferred is the method of the invention wherein the wild-type NIM1 protein comprises the amino acid sequence set forth in SEQ ID NO: 2.

Particularly preferred is the method of the invention wherein said NIM1 gene is in SEQ ID NO: 1

EN 224,480 Β1 hybridizes under the following conditions: hybridization in 1% BSA; 520 mM NaPO ₄ , pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours and wash in 6xSSC three times for 15 minutes, then 3x SSC for 15 minutes once at 55 ° C.

Particularly preferred is the method of the invention wherein the wild-type NIM1 gene is SEQ ID NO; 1, and all DNA molecules hybridize to it under moderately stringent conditions.

In a particularly preferred method of the invention, the nucleotide sequence encodes an altered form of the NIM1 protein which acts as a dominant-negative regulator of the SAR signal transduction pathway.

Even more preferred is the method wherein the altered form of the NIM1 protein comprises alanine at amino acid positions 55 and 59 of SEQ ID NO: 2 instead of serine.

In a particularly preferred method of the invention, the altered form of the NIM1 protein comprises the amino acid sequence set forth in SEQ ID NO: 8.

Preferred is the method of the invention wherein said DNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 7 and all DNA molecules hybridize to it under moderately stringent conditions.

Particularly preferred is the method of the invention wherein said DNA molecule hybridizes to the nucleotide sequence set forth in SEQ ID NO: 7 under the following conditions: Hybridization in 1% BSA; 520 mM NaPO ₄ , pH 7.2; 7% lauryl sulfate, sodium salt;

mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours and wash in 6 * SSC for 3 times for 15 minutes and then 3xSSC for 15 minutes at 55 ° C.

Even more preferred is the method wherein the altered form of the NIM1 protein is as set forth in SEQ ID NO.

about 1-125. has a truncated N-terminal amino acid sequence corresponding to its amino acid position.

In a particularly preferred method of the invention, the altered form of the NIM1 protein comprises the amino acid sequence set forth in SEQ ID NO: 10.

Particularly preferred is the method of the invention wherein said DNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 9 and all DNA molecules hybridize to it under moderately stringent conditions.

Particularly preferred is the method of the invention wherein said DNA molecule hybridizes to the nucleotide sequence set forth in SEQ ID NO: 9 under the following conditions: Hybridization in 1% BSA; 520 mM NaPO ₄ , pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours and wash in 6xSSC three times for 15 minutes and then in 3xSSC once for 15 minutes at 55 ° C.

Particularly preferred is the method wherein the altered form of the NIM1 protein is as set forth in SEQ ID NO:

522-593. has a truncated C-terminal amino acid sequence corresponding to its amino acid position.

In a particularly preferred method of the invention, the altered form of the NIM1 protein comprises the amino acid sequence set forth in SEQ ID NO: 12.

Particularly preferred is the method of the invention wherein said DNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 11, thereby hybridizing all DNA molecules under moderately stringent conditions.

Particularly preferred is the method of the invention wherein said DNA molecule hybridizes to the nucleotide sequence set forth in SEQ ID NO: 11 under the following conditions: hybridization in 1% BSA; 520 mM NaPO ₄ , pH 7.2; 7% lauryl sulfate, sodium salt;

mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours and wash in 6xSSC three times for 15 minutes and then in 3xSSC once for 15 minutes at 55 ° C.

Even more preferred is a method wherein the altered form of the NIM1 protein is as shown in SEQ ID NO:

about 1-125. with a truncated N-terminal amino acid sequence corresponding to its amino acid position and SEQ ID NO: 2 is about 522-593. has a truncated C-terminal amino acid sequence corresponding to its amino acid position.

In a particularly preferred method of the invention, the altered form of the NIM1 protein comprises the amino acid sequence set forth in SEQ ID NO: 14.

Particularly preferred is the method of the invention wherein said DNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 13, whereby all DNA molecules hybridize under moderately stringent conditions.

Particularly preferred is the method of the invention wherein said DNA molecule hybridizes to the nucleotide sequence set forth in SEQ ID NO: 13 under the following conditions: hybridization in 1% BSA; 520 mM NaPO ₄ , pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM NaCl at 55 ° C for 18-24 hours, and washing 6 ^x SSC for 15 minutes three times, followed by 3 ^X SSC, once for 15 minutes at 55 ° C.

Even more preferred is a method wherein the altered form of the NIM1 protein consists essentially of ankyrin motifs which correspond to about 103-362 of SEQ ID NO: 2. of amino acid positions.

In a particularly preferred method of the invention, the altered form of the NIM1 protein comprises the amino acid sequence set forth in SEQ ID NO: 16.

Particularly preferred is the method of the invention wherein said DNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 15 and all DNA molecules hybridize to it under moderately stringent conditions.

Particularly preferred is the method of the present invention wherein said DNA molecule has the nucleotide sequence set forth in SEQ ID NO: 15 at about

HU 224,480 Β1 hybridization: hybridization in 1% BSA; 520 mM NaPO ₄ , pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours and wash in 6xSSC three times for 15 minutes and then in 3xSSC once for 15 minutes at 55 ° C.

Examples of crops used in the indication areas listed herein include, but are not limited to, cereals (corn, wheat, barley, rye, oats, rice, millet, and related plants); beets (sugar beet and fodder beet); apples, stone fruits and berries (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and blackberries); legumes (beans, lentils, peas, soy); oil crops (rape, mustard, poppy, olive, sunflower, coconut, castor, cocoa, peanut); cucumbers (pumpkin, cucumber, melon); fiber plants (cotton, flax, hemp, jute); citrus fruits (orange, lemon, grapefruit, tangerine); vegetables (spinach, lettuce, cabbage, carrots, onions, tomatoes, potatoes, peppers); laurels (avocado, cinnamon, camphor); or other plants such as tobacco, hazelnuts, coffee, sugar cane, tea, wine grapes, hops, bananas and natural rubber, and ornamental plants (flowers, shrubs, deciduous trees and evergreens such as conifers). This list is not a limitation.

The method of the invention can be used to confer resistance to a wide variety of plant pathogens, including, but not limited to, viruses or viroids such as tobacco or cucumber mosaic virus, blemish virus or necrosis virus, nutmeg leaf shrink virus. , purple clover blotch virus, tomato broom stunt virus and similar viruses; Ascomycetes fungi such as Venturia, Podosphaera, Erysiphe, Monolinia, Mycosphaerella and Uncinula; Basidiomycetes fungi such as Hemileia, Rhizoctonia and Puccinia; Fungi imperfecti such as the genera Botrytis, Helmínthosporium, Rhynchosporium, Fusarium (i.e. F. monoliforme), Septoria, Cercospora, Alternaria, Pyricularia and Pseudocercosporella (i.e. P. herpotrichoides); Oomycetes fungi such as Phytophthora (i.e. P. parasitica), Peronospora (i.e. P. tabacina), Bremia, Pythium and Plasmopara; and other fungi such as Sclerophthora macrospora, Sclerophthora rayissiae, Sclerospora graminicola, Peronospora , Peronosclerospora philippinensis, Peronosclerospora sacchari and Peronosclerospora maydis, Physopella zeae, Cercospora zeae-maydis, Colletotrichum graminicola, Gibberella zeae, Exserohilum turcicum, Kabatiellu zeae and Bipolar may; bacteria such as Pseudomonas syringae, Pseudomonas tabaci and Erwinia stewartii; insects such as aphids such as Myzus persicae; and scaly butterflies such as Heliothus spp .; and nematodes such as Meloidogyne incognita.

Production of immunomodulated plants

All three general pathways for the production of immunomodulated plants are related to those that fit into the SAR signal transduction pathway model introduced by Ryals et al. (1996). Upon activation of the SAR signal transduction pathway leading to disease resistance, the same sequence of SAR genes is activated ("on") and results in disease resistance, regardless of which of the three pathways selected below. The only difference between the three paths is at which point in the reaction path the mud begins to operate; the end result is the same for all three roads. Therefore, the immunomodulated plant assays and observed results obtained by one route can be extrapolated and applied to immuno-modified plants obtained by different routes.

A) Application of the chemical inducer of systemic acquired resistance

The first way to produce immuno-modified plants involves the use of an inducer that is chemically capable of inducing SAR. Particularly potent chemical inducers of SAR are benzothiazide diazoles, such as S-methyl ester of benzo [1.2.3] thiadiazole-7-carbothioic acid (BTH). Benzothiadiazole derivatives which are further useful as regulators are described in U.S. Patent Nos. 5,523,311 and 5,614,395, which are incorporated herein by reference. BTH-induced SAR, which provides protection in the open ground against a broad spectrum of diseases in various crop plants, has been described in detail by Freidrich et al., Plant Journal 10 (1), 61-70 (1996); Lawton et al. (1996) Plant Journal 10 (1): 71-82; and Gorlach et al., Plant Cell. 8, 629-643 (1996), each of which is incorporated herein by reference. Other chemical inducers of SAR can also be used in the process of the present invention to produce an immuno-modified plant, such as 2,6-dichloroisonicotinic acid (INA) and its lower carbon esters and salicylic acid (SA). Suitable INA and SA compounds are described in U.S. Patent No. 5,614,395.

B) Breeding of Constitutive Immunity (TITLE) Mutant Plants

A second pathway for the production of immuno-modified plants is a selective breeding program based on constitutive expression of SAR genes and / or a disease resistant phenotype. Comparative data show little correlation between SAR gene expression 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. (PNAS USA 90, 7321-7331 (1993), incorporated herein by reference), well-characterized examples of SAR genes in Arabidopsis were expressed by PR-1, PR-2 and PR-5, PR-1. at the highest level with the lowest background.

To identify and select plants that constitutively express SAR genes,

HU 224 480 Β1

Northern analysis was performed to detect expression of SAR genes. Known SAR DNA sequences can be used in cross-hybridization experiments as described by Uknes et al. (1992). Methods for hybridizing and cloning nucleic acid sequences are well known in the art. See, for example, Molecular Cloning, A Laboratory Manual, 2nd Edition, 1-3. , Sambrook et al. (eds.) Cold Spring Harbor Laboratory Press (1989) and references therein. At least two classes of signal transduction mutants that constitutively express SAR genes have been isolated. One class is designated as / sd mutants (lsd = / esion stimulating disease), hereinafter referred to as "cim class I" mutants (WO 94/16077). Isd (class I class) mutants form spontaneous lesions in leaves, accumulate at elevated concentrations in SA, have 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). The other class is referred to as "cim mutant (cim = constitutive immunity), hereinafter referred to as" cim class II "mutants (see WO 94/16077). Cim mutants have all the features of Isd mutants except spontaneous lesions. That is, cim mutants are visually phenotypically normal.

Once plants that have constitutively expressed SAR genes have been selected, they can be used in a breeding program to introduce constitutive expression of SAR genes and resistance to pathogens into plant cell lines. Progeny are selected for further crossing on the basis of expression of SAR genes and disease resistance, as well as other traits that are relevant to the production and quality of the plant breeding according to methods well known to those skilled in the art. For example, since Isd mutants cause lesion formation and necrosis, cim mutants with their normal phenotype are preferred in such breeding programs and methods of the invention, although Isd mutants can be used if desired.

C) Transforming plants with SAR genes

A third way to obtain immunomodulated plants is to transform plants with a 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 the NIM1 gene (SEQ ID NO: 1) induces a disease-resistant phenotype of transgenic plants. See U.S. Patent No. 6,091,004, which is incorporated herein by reference. Elevated levels of active NIM1 protein result in the same disease resistance effect as chemical induction with inducing compounds such as BTH, INA and SA. Preferably, the expression level of the NIM1 gene is at least twice that of the NIM1 gene in wild-type plants, and more preferably is at least ten times higher than that of wild-type expression. The "Recombinant DNA Technology" section below describes a protocol that can be used to recombinantly express the wild-type NIM1 gene in transgenic plants at "higher than wild-type" levels. Alternatively, the plants may be transformed with the wild-type NPR1 gene to produce disease-resistant plants as described by Cao et al. (1997).

2. Recombinant expression of an altered form of the NIM1 gene

Immunomodulated plants for use in the method of the invention may also be produced by recombinant expression of a modified form of the NIM1 gene, while modification of the NIM1 gene takes advantage of the recognition that SARs in plants exhibit functional parallels with NF-κΒ / ΙκΒ and regulatory systems. the discovery that a structural homologue of the NIM1 gene product is the mammalian signal transduction factor IkB of the subclass. See dependent PCT application, "NIM1 gene transfer methods for disease resistance in plants", which is incorporated herein by reference.

The NIM1 gene sequence (SEQ ID NO: 1) was used in BLAST studies and maps were identified by the homology of the highly conserved domain and the ankyrin domains in the NIM1 gene sequence, which are found in many proteins such as spectrins, ankyrins, NF-κΒ and IkB. [Michaely and Bennett: Trends Cell. Biol. 2: 127-129 (1992)]. Paired visual observations were made between the NIM1 protein (SEQ ID NO: 2) and 70 known ankyrin-containing proteins and found surprising (striking) identities among members of the ΙκΒα class of transcriptional regulators (Baeuerle and Baltimore 1996; Baldwin 1996). As shown in Figure 1, the NIM1 protein (SEQ ID NO: 2) showed significant homology with the murine, rat and porcine ΙκΒα proteins (SEQ ID NO: 3, 4, and 5). NIM1 contains some essential structural domains of ΙκΒα along the entire length of the protein, including the ankyrin domains (indicated by dotted lines in Figure 1), 2 amino terminals (NIM1 55 and 59), two adjacent lysines (NIM1 99). and 100 amino acids) and an acidic C-terminal. NIM1 and ΙκΒα are identical in 30% of the residues and conservative substitution in 50% of the residues. Therefore, there is an overall 80% homology between ΙκΒα and NIM1 through the two proteins.

One way in which the ΙκΒα protein exerts its effect on signal transduction is by binding to the NF-κΒ cytosolic transcription factor and preventing its entry into the nucleus and altering the transcription of target genes (Baeuerle and Baltimore, 1996). NF-κΒ target genes regulate (activate or inhibit) some cellular processes,

HU 224,480 Β1 including antiviral, antimicrobial, and cell death responses (Baeuerle and Baltimore, 1996). When the signal transduction pathway is activated, ΙκΒα is phosphorylated at the two serine residues (mouse ΙκΒα 32 and

36. amino acid residues). It programs ubiquitination at double lysine (residues 21 and 22 of mouse ΙκΒα). Following attachment, the NF-κΒ complex passes through the proteasome, where ΙκΒα is degraded, and NF-κΒ is transported to the nucleus.

Phosphorylated serine residues important for the function of Ικtakα are conserved in NIM1 in the large adjacent block of the conserved sequence 35 to 84 amino acids (Figure 1). In contrast to ΙκΒα, where double lysine is located at about 15 amino acids toward the N-terminus of the protein, in NIM1, double lysine is located at about 40 amino acids at the C-terminus. Furthermore, a high degree of homology exists between NIM1 and ΙκΒα in the serine / threonine-rich carboxy-terminal region, which appears to be very important in terms of basal turnover [Sun et al., Mol. Cell. Biol. 16: 1058-1065 (1996). According to the present invention, it is expected that NIM1 functions similarly to ΙκΒα and has analogous effects in the regulation of the plant gene, based on the presence of elements known to be important for structural homology and ΙκΙα function.

When plants containing the wild-type NIM1 gene were treated with chemical inducers, it was expected that more NIM1 gene products (IkB homologue) would be produced or that the NIM1 gene product (IkB homologue) would be less phosphorylated. In agreement with this model, we obtained the result that the plant NF-κΒ homologue remains outside the nucleus and leaves SAR gene expression and resistance responses on display. In Nim1 mutant plants, a non-functional NIM1 gene product is present. Therefore, in agreement with this model, the NF-κΒ homologue freely enters the nucleus and represses resistance and SAR gene expression.

Consistent with this concept, salicylic acid-treated animal cells show increased / high levels of IkB and a decrease in active NF-κΒ in the nucleus (Kopp and Ghosh, 1994). Mutations in IkB are known to act as super-repressors or dominant-negative (Britta-Mareen Traenckner et al., 1995, EMBO 14, 2876-2883; Brown et al. (1996) Science 267: 1485-1488; Brockman et al. (1995) Molecular and Cellular Biology 15: 2809-2818; Wang et al., Science 274: 784-787 (1996)]. These mutant forms of ΙκΒ bind to NF-κB but do not phosphorylate or associate and are therefore not degraded. NF-kB remains bound to ΙκΒ and cannot enter the nucleus.

In view of the above, modified forms of NIM1, which act as dominant-negative regulators of the SAR signal transduction pathway, can be prepared. Plants transformed with these dominant-negative forms of NIM1 have the opposite phenotype as the nim1 mutant plants, plants transformed with altered forms of NIM1 have continuous SAR gene expression and therefore a CIM phenotype; i.e., the transgenic plants are immunomodulated. Because of the role of the NIM1 gene in the SAR signal transduction pathway, it is expected that a large number of modifications to the gene, other than those specifically described herein, will result in constitutive expression of SAR genes and therefore a CIM phenotype. The following section, entitled "Recombinant DNA Technology," describes protocols that can be used to recombinantly express altered forms of the NIM1 gene in transgenic plants at "higher than wild type" levels. The following describes some modified forms of the NIM1 gene which act as dominant-negative regulators in the SAR signal transduction pathway.

(a) Replacement of serine residues 55 and 59 with alanine residues

In order to phosphorylate serine residues in human ΒκΒα, stimulation of activated degradation of ΙκΒα and thus activation of NF-κΒ is required. Mutagenesis of serine residues (S32 and S36) in human ΙκΒα inhibits this stimulatory-induced phosphorylation to alanine residues, thereby blocking the proteosome-regulated degradation of ΙκΒα (Traenckner et al., 1995; Brown et al., 1996; Brockman et al., 1996). Wang et al., 1996). This modified form of ΙκΒα is able to function as a dominant-negative form, retaining NF-κΒ-ί in the cytoplasm, thereby blocking downstream signaling events. The amino acid sequences of NIM1 and IkB (see Figs

1), serine 55 (S55) and 59 (S59) in NIM1 are homologous to S32 and S36 to human ΙκΙα. To construct dominant-negative forms of NIM1, the serines at positions 55 and 59 were mutagenized to alanine residues. Thus, in a preferred embodiment, the NIM1 gene is modified to encode a product which contains alanine at amino acid positions 55 and 59 of the Arabidopsis NIM1 amino acid sequence (SEQ ID NO: 2).

b) N-terminal deletion

Human ΙκΒα 1-36. deletion of the amino acid residue (Brockman et al., 1995; Sun et al., 1996) or the amino acids 1-72. deletion of amino acids (Sun et al., 1996), which includes lysine residues at K21 and K22 sites, as well as S32 and S36 phosphorylation sites, results in a dominant-negative ΙκΒα phenotype in human transfected cell cultures. The N-terminal deletion of the first 125 amino acids of the NIM1 gene product will remove eight lysine residues that could serve as ubiquitous sites and putative phosphorylation sites at S55 and S59 mentioned above. Therefore, in a preferred embodiment, the NIM1 gene is modified to encode a product lacking approximately the first 125 amino acids as compared to the native Arabidopsis NIM1 amino acid sequence (SEQ ID NO: 2).

HU 224 480 Β1

c) C-temporal deletion

Human ΙκΒα 261-317. deletion of its amino acids may result in increased internal stability by inhibiting the constitutive phosphorylation of serine and threonine residues at the C-terminus. This modified form of ΙκΒα is expected to function as a dominant-negative form. The serine and threonine rich region at the C terminus of NIM1 is shown in pages 522-593. is present. Thus, in a preferred embodiment, the NIM1 gene is modified to encode a product lacking approximately the C-terminal portion, including the amino acids 522-593. amino acids compared to the native Arabidopsis NIM1 amino acid sequence (SEQ ID NO: 2).

d) N-terminal / C-terminal deletion chimeras and ankyrin domains

Altered forms of the NIM1 gene product may also be produced as a result of C-terminal and N-terminal segment deletions or chimeras. In another embodiment, the invention provides constructs comprising ankyrin domains from the NIM1 gene.

3. Recombinant expression of other SAR genes

Immunomodulated plants for use in the method of the invention may also be produced by recombinant expression of various SAR genes, such as described by Ward et al. (1991). See, for example, U.S. Patent No. 5,614,395, which describes disease-resistant plants obtained by the intensive expression (overexpression) of one or more PR protein genes. Although this relates to the recombinant expression of forms of the NIM1 gene, protocols that are also useful in the recombinant expression of other SAR genes, such as PR protein genes, in transgenic plants at "higher than wild type" levels are particularly described below.

Recombinant DNA technology

The wild-type or modified form of the NIM1 gene, which confers disease resistance to plants through enhanced SAR gene expression, can be incorporated into plant cells using conventional recombinant DNA technology. Typically, this involves inserting a DNA molecule encoding the selected form of NIM1 described above into an expression system that is heterologous to the DNA molecule (i.e., is not normally present) by standard cloning techniques known in the art. The vector contains the elements required for transcription and translation of the coding sequence of the inserted protein. Many vector systems are known which may be used, such as plasmids, bacteriophage viruses, and other modified viruses. Suitable vectors include, but are not limited to, viral vectors such as the XgtH, Xgt10 and Charon 4 lambda vector systems; plasmid vectors such as pB1121, pBR322, pACYCI77, pACYCI84, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101, pCDNA11 and the like. Components of the expression system may also be modified to increase expression. For example, truncated sequences, nucleotide substitutions, or other modifications may be used. The expression systems described herein can be used to transform cells of virtually any crop plant under appropriate conditions. Transformed cells can be regenerated into whole plants by activating SAR in selected plants of the NIM1 gene in transgenic plants.

A) Construction of plant expression cassettes

The gene sequences that you want to express in transgenic plants are first aligned in sequence behind a suitable plant-based promoter. The expression cassettes may also contain additional sequences which are necessary or direct for the expression of the transgenes. Such sequences include, but are not limited to, transcription terminators, foreign expression enhancers, such as introns, vital sequences, and sequences that direct the gene product to specific organs and cellular regions. These expression cassettes can then be readily transferred to the plant transformation vectors as described above. The various components of a typical expression cassette are described below.

1. Promoters

The selection of the promoter used in the expression cassette will be determined by the spatial and temporal expression patterns of the transgenes in the transgenic plant. The selected promoter will express the transgenes in specific cell types (such as leaf epidermal cells, mesophilic cells, root cortex cells) or in specific tissues or organs (such as roots, leaves or flowers) and direct selection to the desired site of gene product accumulation. Alternatively, the selected promoter may direct gene expression under various inducing conditions. Promoters differ in length, i.e. in their ability to stimulate transcription. Depending on the host cell system, any of a number of suitable promoters may be used. The following non-limiting examples of promoters can be used in expression cassettes.

a) Constitutive expression, CaMV 35S promoter

The construction of plasmid pCGN1761 is described in EP 0 392 225, which is incorporated herein by reference. PCGN1761 contains the "double" CaMV 35S promoter and the tml transcriptional terminator with a single Eco RI site between the promoter and the terminator, and a pUC-like framework. A derivative of pCGN1761 was constructed which contains a modified Pollin containing NotI and XhoI sites in addition to the EcoRl site. This derivative was pCGN1761ENX

HU 224 480 Β1 marked. PCGN1761ENX can be used to clone cDNA or gene sequences within the polylinker (including microbial ORF sequences) for expression in transgenic plants under the control of the 35S promoter. The internal 35S promoter gene sequence fm / terminator cassette of such constructs can be cleaved with HindIII, SphI, SalI and XbaI at the 5 'site relative to the promoter, and with XbaI, BamHI and SgI at the 3' site. sites relative to the terminator to transfer to the transformation vectors described below. Further, the double 35S promoter fragment can be removed by 5 'excision with HindIII, SphI, SalI, XbaI or PstI and deletion of the 3' by any of the polylinker restriction sites (EcoRI, NotI or XhoI) to another promoter. If desired, modifications may be made around the cloning sites by introducing sequences that enhance translation. This is especially useful when an intensive expression is needed. For example, pCGN1761ENX may be modified to optimize the translation start point as described in U.S. Patent No. 5,639,949.

Example 37.

b) Expression under the control of a chemically or pathogenically controllable promoter In pCGN1761ENX, the double 35S promoter can be replaced by another promoter selected to produce sufficiently high expression levels. For example, one of the chemically controllable promoters (see US 5,614,395) can replace the double 35S promoter. Preferably, the selected promoter is cleaved from its source by restriction enzymes, but alternatively it can be amplified by PCR using primers with appropriate terminal restriction sites. If PCR amplification is to be performed, the promoter should be re-sequenced to check for amplification errors after cloning of the amplified promoter in the target vector. The chemically or pathogenically controllable tobacco PR-1a promoter is excised from plasmid pCIB1004 (construction see Example 23 of EP 0 332 104) and transferred to plasmid pCGN1761ENX (Uknes et al., 1992). PCIB1004 is cleaved with Ncoll and the 3'-appendix of the resulting linearized fragment is blunt-ended with T4 DNA polymerase. The fragment was then cleaved with HindIII and the resulting PR-1a promoter-containing fragment was gel purified and cloned into plasmid pCGN1761ENX, from which the double 35S promoter was removed. This was done by XhoI digestion and blunt-ended with T4 polymerase, followed by digestion with HindIII and isolation of the larger vector terminator-containing fragment into which the pCIB1004 promoter fragment was cloned. This yields the pCGN1761ENX derivative, which contains the PR-1a promoter, fm / terminator and a single Eco RI and Not I site-bound polylinker. The selected coding sequence can be inserted into this vector and the fusion products (i.e. promoter gene terminator) can then be introduced into any of the selected transformation vectors, including those described below. Various chemical regulators may be used to induce expression of the selected coding sequence in plants transformed according to the invention, such as the benzothiadiazole, isonicotinic acid and salicylic acid derivatives disclosed in U.S. Patent Nos. 5,523,311 and 5,614,395.

c) Constitutive expression, actin promoter

Some actin isoforms are known to be expressed in most cell types and consequently actin promoter is a good choice as a constitutive promoter. Preferably, the promoter from the rice Actl gene has been cloned and characterized [McElroy et al., Plant Cell. 2: 163-171 (1990)]. The 1.3 kb fragment of the promoter was found to contain all the regulatory elements required for expression of protoplasts in rice. Furthermore, several expression vectors based on the Actl promoter have been constructed specifically for use in monocotyledons [McElroy et al., Mol. Gene. Genet. 231: 150-160 (1991)]. These included the Acf / intron, the Adhl 5 'flanking sequence and the Adhl intron 1 (from the maize alcohol dehydrogenase gene) and the sequence from the CaMV 35S promoter. Vectors containing the 35S and AcW intron or Act15 5 'flanking sequence and the Acf / intron fusion show the highest expression. Sequence optimization around the start ATG codon (the GUS reporter gene) also enhances expression. McElroy et al., [Mol. Gene. Genet. 231: 150-160 (1991)] promoter expression cassettes can be easily modified for gene expression and preferably adapted for use in monocotyledonous plants. For example, promoter-containing fragments may be removed from McElroy constructs and used to replace the 35S promoter in pCGN1761ENX, which can then be inserted into specific gene sequences. The fusion genes thus constructed can then be transferred to appropriate transformation vectors. According to a special report, the rice Actl promoter coexisting with its first intron has been found to direct high expression in cultured barley cells [Chibbar et al., Plant Cell. Rep. 12: 506-509 (1993).

d) Constitutive expression, ubiquitin promoter

Ubiquitin is another known gene product that accumulates in many cell types and its promoter has been cloned from some species for use in transgenic plants (e.g. sunflower - Binet et al., Plant Science 79, 87-94 (1991) and maize - Christensen et al., Plant Molec. . Biol. 12: 619-632 (1989)]. The maize ubiquitin promoter has been developed in transgenic monocotyledonous systems and its sequence and constructed vectors for the transformation of monocotyledons have been described in EP 0 342 926 (Lubrizol), which is incorporated herein by reference. Taylor et al., Plant Cell. Rep. 12,

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491-495 (1993) describe a vector (pAHC25) containing the maize ubiquitin promoter and its first intron and having high activity in a suspension of several monocot cells when introduced into the cell by microinjection. 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 herein, modified by the introduction of the appropriate ubiquitin promoter and / or intron sequences.

e) Root-specific expression

Another form of gene expression is root expression. A suitable root promoter is that described by de Framond (1991, FEBS 290, 103-106), which is also disclosed in EP 0 452 269, which is incorporated herein by reference. This promoter is transferred to a suitable vector, such as pCGN1761ENX, for insertion of the selected gene and subsequent transfer of the internal promoter gene terminator cassette into the desired transformation vector.

f) Sebinducible promoters

Wound-inducible promoters are also suitable for gene expression. Many such promoters have been described [e.g., Xu et al., Plant Molec. Biol. 22, 573-588 (1993), Logemann et al., Plant Cell. 1, 151-158 (1989), Rohrmeier and Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al., Plant Molec. Biol. 22, 129-142 (1992), Wagner et al. (1993) Plant J. 3: 191-201], all of which are suitable for use in the present invention. Longemann et al. Described 5 'upstream sequences of the dicotyledonous potato wunl gene. Xu et al. Have shown that a wound-inducible promoter (pin2) of dicot potato is active in monocot rice. Further, Rohrmeier and Lehle have described the cDNA cloning of maize Wipl induced by a wound that can be used to isolate a cognate promoter by standard techniques. Similarly, Firek et al. And Warner et al. Reported a wound-induced gene from the monocotyledon Asparagus officinalis, which was expressed at the wound site and at the pathogen attack sites. Using well known cloning techniques, these promoters can be introduced into suitable vectors, fused to genes permitted by the present invention, and expressed with these genes at sites of plant damage.

g) Gut specific expression

PCT Publication No. WO 93/07278, incorporated herein by reference, describes the isolation of the maize trpA gene, which is preferably expressed in plant gut cells. From the start of transcription to bp 1726, the gene sequence and promoter were provided. By standard molecular biological techniques, this promoter or part thereof can be introduced into a vector, such as pCGN1761, in which the 35S promoter can be replaced and used to direct expression of a foreign gene in a gut-specific manner. Indeed, fragments containing all or part of the gut-specific promoter can be transferred to any vector and modified for use in transgenic plants.

h) Letter-specific expression

The gene encoding maize phosphoenol carboxylase (PEPC) has been described by Hudspeth and Grula [Plant Molec. Biol. 12: 579-589 (1989). By using standard molecular biological techniques, the promoter of this gene can be used to direct any gene expression in leaf-specific transgenic plants.

2. Transcription terminators

A large number of transcription terminators can be used in expression cassettes. They are responsible for transcription termination after transgenes and correct polyadenylation. Suitable transcriptional terminators are those known to be functional in plants, such as the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used for both monocotyledons and dicotyledons.

3. Sequences for enhancing or regulating expression

Within the transcriptional unit, several sequences have been found to enhance gene expression, and these sequences can be used in conjunction with the genes of the invention to increase expression in transgenic plants.

Various intron sequences have also been shown to enhance expression, particularly in monocotyledons. For example, introns of the maize Adhl gene significantly increase expression of the wild-type gene under the cognate promoter when introduced into maize cells. Intron 1 has been shown to be particularly effective and enhances expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al., Genes Develop. 1, 1183-1200 (1987)]. In the same experimental system, the intron from the maize bronze gene is similarly effective in enhancing expression. Intron sequences are routinely incorporated into plant transformation vectors, typically within the untranslated leader sequence.

Numerous viral, untranslated leader sequences are known to enhance expression, and are particularly effective in dicotyledonous cells. In particular, leader sequences derived from tobacco mosaic virus (TMV, " W sequence "), corn staining virus (MCMV), and alfalfa mosaic virus (AMV) have been shown to be highly effective in enhancing expression [e.g., Gallie et al. Nucl. Acids Slit. 15: 8693-8711 (1987); Skuzeski et al .: Plant Molec. Biol. 15: 65-79 (1990).

4. Direction of the gene product inside the cell

Various mechanisms are known that direct gene products in plants and the sequences that regulate the functioning of these mechanisms11

EN 224,480 Β1 to some extent. For example, the targeting of gene products to the chloroplast is controlled by a signal sequence located at the amino terminus of various proteins, which is cleaved during chloroplast import to yield the mature protein [e.g., Comai et al., J. Biol. Chem. 263: 154-105109 (1988)]. These signal sequences may be fused to heterologous gene products to render the import of heterologous products into the chloroplast efficient (van den Broeck et al., Natur. 313, 358-363 (1985)). DNA encoding suitable signal sequences can be isolated from the 5 'end of the cDNAs encoding the RUB1SCO protein, the CAB protein, the EPSP synthase enzyme, the GS2 protein and many other proteins known to be in the chloroplast. See, for example, "Expression With ChloroplastTargeting" in Example 37 of US 5,639,949.

Other gene products are localized in other cellular organs, such as the mitochondria and the peroxisome [e.g., Unger et al., Plant Molec. Biol. 13: 411-418 (1989)]. The cDNAs encoding these products can also be manipulated to be effective in targeting heterologous gene products to these organs. Such sequences include, for example, nucleotide encoding ATPases for the mitochondria and isoforms of specific aspartate amino transferase. The control of cellular protein bodies is described by Rogers et al., Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)].

In addition, sequences have been characterized that direct the gene products to other cellular components. Amino-terminal sequences are responsible for entry into the ER, apoplast, and extracellular secretion from aleurone cells (Koehler and Ho, Plant Cell. 2: 769-783 (1990)]. Furthermore, the amino-terminal sequences coupled to the carboxy-terminal sequences are responsible for the delivery of the gene products into the vacuole [Shinshi et al., Plant Molec. Biol. 14: 357-368 (1990)].

Fusion of the appropriate control sequences with the transgenic sequences of interest described above will allow the transgenic product to be targeted to any cell organ or cell constituent. For example, the chloroplast signal sequence from the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused to the transgenic amino terminal ATG to enter the chloroplast. The selected signal sequence must contain a known cleavage site, and the constructed fusion must take into account any amino acid after the cleavage site that is required for cleavage. In some cases, this desire may be accomplished by adding a small number of amino acids between the transgene ATG and the cleavage site, or alternatively by replacing some amino acids within the transgene sequence. Chloroplast incorporation fusions can be tested for the efficiency of chloroplast uptake by translating in vitro transcribed constructs following in vitro chloroplast uptake using the techniques described by Bartlett et al., Edelmann et al.

(eds.) Methods in Chloroplast Molecular Biology, Elsevier, 1081-1091. She. (1982) and Wasmann et al., Mol. Gene. Genet. 205: 446-453 (1986)]. These construction techniques are well known to those skilled in the art and can be applied to both mitochondria and peroxisomes.

The above-described mechanisms of cellular targeting can be used not only when linked to cognate promoters, but also to heterologous promoters to achieve targeting to a specific cell under transcriptional control of a promoter whose expression pattern differs from that of the promoter control signal comes from.

B) Construction of plant transformation vectors

Numerous transformation vectors suitable for plant transformation are known to those skilled in the art of plant transformation, and the genes of the invention can be linked to any of these vectors. In addition to the preferred transformation technique, the choice of vector will be determined by the plant species to be transformed. Different antibiotic and herbicide selection markers may be preferred for certain target species. Selection markers are routinely used in transformation including the nptll gene which confer kanamycin and similar antibiotic resistance (Messing and Vierra, Gene 19: 259-268 (1982); Bevan et al., Naturre 304, 184-187 (1983)], the bar gene conferring resistance to phosphinothricin herbicide [White et al., Nucl. Acids Slit. 18, 1062 (1990); Spencer et al .: Theor. Appl. Gene. 79, 625-631 (1990)], the hph gene conferring resistance to the antibiotic hygromycin [Blochinger and Digallengann, Mol. Cell. Biol. 4, 2929-2931], and the dhfrgene conferring methotrexate resistance (Bourouis et al., 1983, EMBO J. 2 (7), 1099-1104), and the EPSPS gene conferring resistance to glyphosate (US-PS 4,940,935 and 5,188,642).

1. Vectors suitable for Agrobacterium transformation

Many vectors are suitable for transformation when using Agrobacterium tumefaciens. They typically carry at least one T-DNA flanking sequence (borderline), and include pBIN19 and pXYZ. The construction of two typical vectors for Agrobacterium transformation is described below.

(a) pCIB200 and pCIB2001

The binary vectors pCIB200 and pCIB2001 are used to construct the recombinant vectors in Agrobacteriumma and proceed as follows. PTJS75 [Schmidhauserés Helsinki: J. Bacteriol. 164, 446-455 (1985)] by enzymatic digestion of Narl to generate pTJS75skan, which allows excision of the tetracycline resistance gene, followed by an Accl frag12.

HU 224,480 Β1 months is inserted from pUC4K, which carries an NPTII (Messing and Vierra, Gene 19: 259-268 (1982); Bevan et al., Natura 304: 184-187 (1983); McBridge et al. (1990) Molecular Biology Plant 14: 266-276. Xhol linkers are ligated into the EcoRV fragment of PCIB7, which contains 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 Xho I digested fragment is cloned into Sa I digested pTJS75skan, thereby generating pCIB200 (see also Example 19 of EP-A-0 332 104). PCIB200 contains the following unique polylinker restriction sites: EcoRI, SstI, KpnI, BglII, XbaI and SalI. PCIB2001 is a derivative of pCIB200, which is generated by inserting additional restriction sites into the polylinker. Unique restriction sites on the polylinker of pCIB2001 include EcoRI, SstI, KpnI, BglII, XbaI, SalI, Myl, Bell, AvrII, ApaI, HpaI and StuI. In addition to these unique restriction sites, pCIB2001 contains plant and bacterial kanamycin selection, left and right T-DNA borders for Agro-bacterium-regulated transformation, trfA function from RK2 for mobilization between E. coli and other hosts, and OriT from RK2. and OriV functions. The pCIB2001 polylinker is suitable for cloning plant expression cassettes containing their own regulatory signals.

b) Selection derivatives of pCIBIO and hygromycin

The binary vector pCIBIO contains a gene encoding kanamycin resistance for selection in plants and right and left border sequences of T-DNA, and contains sequences from the wild-type plasmid pRK252 that allow it to replicate in both E. coli and Agrobacterium. The construction of this plasmid has been described by Rothstein et al., Gene 53: 153-161 (1987). Various derivatives of pCIBIO have also been constructed by Gritz et al., Incorporating the hygromycin B phosphotransferase gene (Gene 25: 179-188 (1983)). These derivatives allow selection of transgenic plant cells based only on hygromycin (pCIB743) or hygromycin and kanamycin (pCIB715, pCIB717) resistance.

2. Vectors suitable for transformation of non-Agrobacterium

Transformation without Agrobacterium tumefaciens bypasses the requirement for T-DNA sequences in the selected transformation vector, and consequently, vectors lacking these sequences can be used in conjunction with the vectors carrying the T-DNA sequences described above. Transformation techniques that do not apply to Agrobacterium-containing transformation include particle bombardment, protoplast fusion (e.g., PEG and electroporation), and microinjection. The choice of vector will largely depend on the preferred selection of the species to be transformed. The construction of two typical non-Agrobacterium transformation vectors is described below.

a) pCIB3064

PCIB3064 is a vector derived from pUC that can be used for direct gene transfer techniques in combination with selection by the herbicidal basta (or phosphinothricin). Plasmid pCIB246 contains the CaMV 35S promoter operably fused to the E. coli GUS gene and to the CaMV 35S transcription terminator, which is described in WO 93/07278. In this vector, the 35S promoter carries two ATG sequences at the 5 'start site. These sites are mutated by standard PCR techniques to remove ATGs and create Sspl and Pvull restriction sites. The new restriction sites are 96 and 37 bp away from the sole site and 101 and 42 bp away from the current site. The resulting derivative of pCIB246 is designated pCIB3025. The GUS gene is then excised by SalI and SacI digestion and the resulting ends blunt-ended and re-ligated to give plasmid pCIB3060. Plasmid pJIT82 was obtained from the John Innes Center (Norwich) and the 400 bp Smal fragment containing the bar gene was excised from Streptomyces viridochromogenes and inserted into pCIB060 (Thompson et al., 1987, EMBO J. 6, 2519-2523). ] In place of Hpal. Thus, pCIB3064 is constructed, which, under the control of the CaMv 35S promoter, contains the bar gene and a terminator for herbicidal selection, an ampicillin resistance gene (for selection in E. coli) and unique SphI, PstI, HindIII and BamHI sites. This vector is suitable for the cloning of plant expression cassettes containing their own regulatory signals.

b) pSOG19 and pSOG35

PSOG35 is a transformation vector which utilizes the dihydrofolate reductase (DFR) gene of E. coli as a selection marker to confer resistance to methotrexate. PCR techniques were used to amplify the 35S promoter (-800 bp), the Adhl gene-derived intron 6 (-550 bp) and the 18 bp fragment of the untranslated leader sequence from pSOG10. A 250 bp fragment was also amplified by PCR encoding the dihydrofolate reductase gene of E. coli type II and these two PCR fragments were fused to a Sacl-Pstl fragment from pB1221 (Clontech), which is the backbone of the pUC19 vector. carries the nopaline synthase terminator. Coupling of these fragments results in pSOG19, which contains the 35S promoter in fusion with intron 6, the GUS leader, the DHFR gene, and the nopaline synthase terminator. Replacement of the GUS leader sequence in pSOG 19 with the maize color-spotted virus (MCMV) leader sequence provides the pSOG35 vector. PSOG19 and pSOG35 carry the ampicillin resistance pUC gene and HindIII, SphI, PstI and EcoRI sites suitable for the cloning of foreign substances.

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C) Transformation

Once the coding sequence of interest is cloned into an expression system, it is transformed into a plant cell. Transformation and plant regeneration techniques are well known to those skilled in the art. For example, Ti plasmid vectors are used for the transfer of foreign DNA as well as for direct DNA uptake by liposomes, electroporation, microinjection and microprojection. In addition, bacteria from the genus Agrobacterium can also be used to transform plant cells. The following are important techniques for transforming both dicotyledons and monocotyledons.

1. Transformation of dicotyledons

Transformation techniques for dicotyledons are well known and include Agrobacterium-based and non-Agrobacterium-based techniques. Non-Agrobacterium techniques involve uptake of exogenous genetic material directly by protoplasts and cells. This can be accomplished by PEG or electroporation controlled uptake, particle bombardment controlled delivery, or microinjection. Examples of these techniques can be found in Paszkowski et al., EMBO J. 3, 2717-2722 (1984); Potrykus et al., Mol. Gene. Genet. 199: 169-177 (1985); Reich et al., Biotechnology 4: 1001-1004 (1986) and Klein et al., Natur 327, 7073 (1987). In all cases, transformed cells are regenerated to whole plants using standard techniques known in the art.

Agrobacterium regulated. Transformation is a preferred technique for transforming dicotyledons because of the high efficiency of the transformation and its wide application in many different species. Agrobacterium transformation typically involves the transfer of a binary vector of foreign important DNA (e.g., pCIB200 or pCIB2001) to a suitable Agrobacterium strain dependent on the complement of vir genes, which is carried by the host Agrobacterium strain on a cresistant Ti plasmid or chromosomal 42. for pCIB200 and pCIB2001 - see Uknes et al .: Plant Cell. 5: 159-169 (1993). The transfer of the recombinant binary vector to Agrobacterium is accompanied by a three-parent mating process using E. coli carrying the binary vector, a helper E. coli strain carrying a plasmid such as pRK2013 and capable of mobilizing the recombinant binary vector. Agrobacterium strain. Alternatively, the recombinant binary vector may also be introduced into Agrobacterium by DNA transformation [Hofgen and Willmitzer, Nucl. Acids. Gap. 16, 9877 (1988)].

Transformation of target plant species with recombinant Agrobacterium usually involves the cultivation of Agrobacterium in plant extracts and subsequent protocols which are well known. Transformed tissues are regenerated on selectable media containing antibiotic or herbicide resistance markers between the binary plasmid T-DNA border sequences.

Another way of transforming plant cells with a gene is to propel inhaled or biologically active particles in plant tissues and cells. This technique is described in U.S. Patent Nos. 4,945,050, 5,036,006 and 5,100,792 (Stanford et al.). In general, this method involves folding inert or active particles within the cells under conditions effective to pass through and integrate into the outer surface of the cell. When using inert particles, the vector may be introduced into the cell by coating the particles with the vector carrying the desired gene. Alternatively, the target cell may be surrounded by the vector so that the vector enters the cell by breaking the particle. Biologically active particles (e.g., dried yeast cells, dried bacteria, or a bacteriophage, each containing DNA for introduction) can also be introduced into plant cell tissue.

2. Transformation of monocots

Transformation of most monocot species also occurs by routine procedures. Preferred techniques include direct gene transfer to protoplasts by PEG or electroporation techniques and particle bombardment into callus tissue. Transformations can be accomplished with a single DNA or multiple DNAs (so-called cotransformations), and all of these techniques are suitable for use in the present invention. Co-transformation can be advantageous to remove the complete vector construct and generate transgenic plants with loci unrelated to the gene of interest and selectable marker, and allow removal of the selectable marker in the next generation, which may be desirable. However, cotransformation has the disadvantage that less than 100% of the frequency with which the separated DNA species are integrated into the genome (Schocher et al., Biotechnology 4: 1093-1096 (1986)) is determined.

EP 0 292 435, EP 0 392 225 and WO 93/10278 describe techniques for the preparation of calli and protoplasts from an inbred maize line, transformation of protoplasts with PEG or electroporation and regeneration of maize plants from the transformed protoplast. Gordon-Kamun et al., Plant Cell. 2, 603-618 (1990) and Fromm et al. (Biotechnology 8, 833-839 (1990)) describe techniques for transforming A188-derived maize line by particle bombardment. Further, WO 93/07278 and Ali et al., Biotechnology 11, 194-200 (1993), describe techniques for transforming elite inbred maize lines by particle bombardment. This technique uses immature corn embryos 1.5-2.5 mm long, which were removed from the corn 14-15 days after pollination, and used a PDS-1000He Biolistics device for bombardment.

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Rice transformation has also been accomplished by direct gene transfer techniques using protoplasts or particle bombardment. Protoplast-regulated transformation has been described for Japonica types and / nd / 'ca types [Zhang et al., Plant Cell. Rep. 7: 379-384 (1988); Shimamoto et al., 1989, Nature 338: 274-277; Datta et al. (1990) Biotechnology 8: 736-740]. Both types can also be routinely transformed by particle bombardment (Christou et al., 1991, Biotechnology 9: 957-962).

EP 0 332 581 discloses techniques for the preparation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Further, wheat transformation has been described by Vasil et al. (Biotechnology 10, Q67-674 (1992)), using particle bombardment on long-term regenerated callus cells of type C and using particle bombardment of immature embryos and callus from immature embryos [Biotechnology 11, 1553-1558 (1993) and Weeks et al., Plant Physiol. 102: 1077-1084 (1993). However, a preferred technique for transforming wheat involves transforming wheat by particle bombardment of immature embryos and involves either a high sucrose or a high maltose step prior to gene transfer. Prior to bombardment, embryos (0.75-1 mm in length) were placed on MS medium containing 3% sucrose (Murashiga and Skoog, Physiologia Plantarum 15, 473-497 (1962)) containing 3 mg / l 2,4-D to induce somatic embryos that allow development in the dark. On the day selected for bombardment, the embryos were removed from the induction medium and placed on the osmotic induction medium to which sucrose and maltose were added at the desired concentration, typically 15%. We allow the plasmolysis of the embryos for 2-3 hours before bombarding them. Usually 20 embryos are used per target plate, but this number is not critical. A plasmid carrying a suitable gene (such as pCIB3064 or pSOG35) was cleaved onto gold particles at micrometer thickness using standard procedures. Each plate of embryos was bombarded with DuPont Biolistics® Helium at an explosive pressure of about 1000 psi using a standard 80 mesh screener. After the bombardment, the embryos are returned to the dark and covered for approx. 24 hours (still on the osmotic). After 24 hours, the embryos are removed from the osmotic and returned to the induction medium where they remain for approximately one month before regeneration. Approximately one month later, embryo plants are transferred to the regeneration medium (MS + 1 mg / L NAA, 5 mg / L GA) with the developing embryogenic callus, which further contains a suitable selection agent (10 mg / L basta for pCIB3064 and 2 mg / L GA). methotrexate for pSOG35). Approximately one month later, the developed shoots are transferred to larger sterile containers than "GA7s" containing half-strength MS, 2% sucrose and the same concentration of selection agent.

Transformation of monocots using Agrobacterium has recently been described. See WO

94/00977 and U.S. Patent No. 5,591,616, which are incorporated herein by reference.

Breeding

Immunized plants obtained by transformation with a SAR gene, such as a NIM1 gene, can belong to any of a wide variety of plant species, including monocotyledons and dicotyledons; however, the immuno-modified plants used in the process of the invention are preferably selected from the group of agriculturally significant crop plants listed above. Expression of the selected form of the NIM1 gene, in combination with other traits that are essential for production and quality, can be incorporated into plant cell lines by breeding. Breeding procedures and techniques are well known to those skilled in the art. See, for example, Welsh J. R., Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, NY (1981); Crop Breeding, Wood D. R. (eds.) American Society of Agronomy Madison, Winconsin (1983); Mayo O .: Theory of Plant Breeding, 2nd Edition, Clarendon Press, Oxford (1987); Singh, D.P., 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 introduced into the transgenic seeds and plants described above by genetic engineering can be maintained and propagated in the offspring by sexual reproduction or vegetative growth. Generally, for the said maintenance and propagation, known agricultural methods are used, which have been developed for specific purposes, such as tillage, sowing or harvesting. Special techniques such as hydroponic crop production or greenhouse technology may also be employed. Because the growing plant is susceptible to attack and exposed to damage from insects or infections and competition from weeds, we have performed operations to control weeds, plant diseases, insects and nematodes and other harmful conditions to increase yield. These include mechanical operations such as plowing the soil or removing weeds or infected plants, and the use of agrochemicals such as herbicides, fungicides, gametocides, nematicides, growth regulators, attractants and insecticides.

Advantageous genetic properties of the transgenic plants and seeds of the present invention may also be utilized in plant breeding for the purpose of developing plants with improved properties such as tolerance to pesticides, herbicides or stress, increased nutritional value, increased yield, or improved structure, resulting in less losses. container or crusher. The different breeding steps are well defined

It can be characterized by human intervention, such as selecting the lines to be crossed, directing the pollination of the parental lines, or selecting appropriate progeny plants. Different breeding methods are used depending on the desired properties. Such techniques are well known to those skilled in the art and include hybridization, inbreeding, backcrossing, multi-line cultivation, various combinations, interspecific hybridization, aneuploid techniques, and the like. Hybridization techniques include sterilizing plants to obtain male or female sterile plants by mechanical, chemical, or biochemical means. Cross-pollination of a male-sterile plant with pollen from a different line ensures that the male-sterile, but female plant will uniformly carry the characteristics of both parent lines. Thus, the transgenic seeds and plants of the present invention can be used to breed improved plant lines, which, for example, increase the efficiency of conventional methods such as herbicide or pesticide treatments or allow healing by said methods due to modified genetic traits. Alternatively, new crop plants with increased stress-tolerance may be obtained, which, thanks to optimized genetic "equipment", will produce a higher quality harvest than a crop that is unable to tolerate similar adverse developmental conditions.

In seed production, the quality and uniformity of the germination of the seeds are important product characteristics, while the germination quality and identity of the seeds harvested and sold by the farmer is not important. Because of the problem of keeping the crop free from other crops and weeds, to control seed-borne diseases, and to produce seeds with good germination capacity, fairly extensive and well-defined seed production methods have been developed by seed growers who harvest pure seeds, know how to store and sell. Thus, it is common practice for farmers to purchase certified seed meeting specific quality standards instead of using seed harvested from their own crop. Propagating material, such as seeds, is usually provided with a protective coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, anti-molluscs, or mixtures thereof. Conventional protective coatings include compounds such as captan, carboxin, thiram (TMTD®), metalaxyl (Apron®) and pirimiphos-methyl (Actellic®). If desired, these compounds may be formulated with additional carriers, surfactants, or absorption-promoting adjuvants commonly used in the art to protect against damage caused by bacterial, fungal, or animal pests. Protective coatings may be prepared by impregnating the propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods may be employed, such as treatment of buds or fruits.

Accordingly, the present invention provides novel agricultural methods, such as those described above, characterized in that the transgenic plants, transgenic plant materials or transgenic seeds of the invention are used.

The cores may be disposed in a sachet, container or container made of a suitable packaging material, and the sachet or container may be sealed to retain the cores. A sachet, container or container may be constructed for short or long term storage of the seeds or both. For example, the wrapping material may be paper, such as thick wrapping paper, rigid or flexible plastic or other polymeric material, glass or metal. If desired, the sachet, container or container may comprise multilayer packaging material of the same or different types. In one preferred embodiment, the bag, container, or container is made of a material that excludes or limits water and moisture contacting the core. An example of this is when the pouch, container or container is sealed, for example by soldering it to prevent water or moisture. In another preferred embodiment, water absorbent materials are placed between or adjacent to the layers of the packaging material. In yet another preferred embodiment, the pouch, container, or container, or packaging material thereof, is treated to reduce, suppress, or prevent disease, contamination, or other deleterious effects during storage or transport of the seeds. Such treatment is, for example, sterilization, which may be accomplished by chemical means or by irradiation. A conventional pouch of the invention, together with a suitable carrier and labeled instructions for use, contains the seeds of a transgenic plant carrying a form of the NIM1 gene or NIM1 protein expressed at a higher level in said transformed plant than in wild-type plants, conferring broad-spectrum disease resistance. plants.

Application of a microbicide to immunomodulated plants

As described above, the plant protection method of the invention comprises two steps: first, activating the SAR pathway to create an immuno-modified plant, and second, using a microbicide on the resulting immunomodulated plant to synergistically increase disease resistance.

A. Commonly used microbicides

In the process of the invention, any commercially available or conventional microbicide can be applied to immunomodulated plants obtained by one of the three routes described above. Examples of suitable microbicides include, but are not limited to, the following fungicides: 4- [3- (4-chlorophenyl) -3- (3,4-dimethoxyphe)

HU 224,480 (1'-nyl) -acryloyl] -morpholine ("dimethomorph"), [reference: C. Tomiin (ed.): The Pesticide Manual, 10th edition, Famhan, UK, 1994, 351-352]; 5-methyl-1,2,4-triazolo [3,4-b] [1,3] benzothiazole ("tricyclazole"), [reference: C. Tomiin (ed.): The Pesticide Manual, 10th edition , Famhan, UK, 1994, 1017-1018. old].; 3-Allyloxy-1,2-benzothiazole-1,1-dioxide ("probonazole"), [reference: C. Tomiin (ed.), The Pesticide Manual, 10th edition, Famhan, UK, 1994, 831- 832nd She.]; α- [2- (4-Chloro-phenyl) -ethyl] -α- (1,1-dimethylethyl) -1H-1,2,4-triazole-1-ethanol ("tebuconazole"), EP-A-40,345); 1 - [[3- (2-chlorophenyl) -2- (4-fluorophenyl) oxiran-2-yl] methyl] -1H-1,2,4-triazole ("epoxyconazole"), (ref. EP-A 196 038); α- (4-chlorophenyl) -α- (1-cyclopropylethyl) -1H-1,2,4-triazole-1-ethanol ("cyproconazole"), (US 4,664,696); 5- (4-Chloro-benzyl) -2,2-dimethyl-1- (1H-1,2,4-triazol-1-ylmethyl) -cyclopentanol ("metconazole"), (reference EP-A 267 778) ; 2- (2,4-Dichlorophenyl) -3- (1H-1,2,4-triazol-1-yl) propyl-1,1,2,2-tetrafluoroethyl ether ("tetraconazole"), EP-A 234242); methyl (E) -2- {2- [6- (2-cyanophenoxy) pyrimidin-4-yloxy] phenyl} -3-methoxyacrylate ("ICI A 5504", "azoxystrobin") , (ref. EP-A 382 375); methyl (E) -2-methoxyimino-2- [α- (o-tolyloxy) -o-tolyl] acetate ("BAS 490 F", cresoxime methyl "), (reference EP-A 400 417); 2- (2-phenoxyphenyl) - (E) -2-methoximino-N-methylacetamide, (EP-A-398,692); [2- (2,5-Dimethyl-phenoxymethyl) -phenyl] - (E) -2-methoxy-imino-N-methyl-acetamide (Reference EP-A-398,692); (1R, 3S / 1S, 3R) -2,2-Dichloro-N - [(R) -1 (4-chlorophenyl) ethyl] ethyl-3-methylcyclopropanecarboxamide ("KTU 3616") , (ref. EP-A 341 475); ethylene bis (dithiocarbamate) polymer manganese zinc complex ("mancozeb"), (US 2,974,156); 1- (2- (2,4-Dichlorophenyl) -4-propyl-1,3-dioxolan-2-ylmethyl) -1H-1,2,4-triazole ("propiconazole"), (Reference GB 1522657); 1- {2- [2-Chloro-4- (4-chloro-phenoxy) -phenyl] -4-methyl-1,3-dioxolan-2-ylmethyl} -1H-1,2,4-triazole ("Difenoconazole"), (Reference GB 209860); 1- [2- (2,4-Dichlorophenyl) pentyl] -1H-1,2,4-triazole ("penconazole"), (Reference: GB 1589852; cis-4- [3- (4-tert-butylphenyl) -2-methylpropyl] -2,6-dimethylmorpholine ("fenpropimorph"), (Reference DE 2752135); [3- (4-tert-Butyl-phenyl) -2-methyl-propyl] -piperidine ("fenpropidin"), (Reference DE 2752135); 4-Cyclopropyl-6-methyl-N-phenyl-2-pyrimidine; amine ("cyprodinyl"), (reference EP-A 310550); (RS) -N- (2,6-dimethylphenyl) -N- (methoxyacetyl) -alanine methyl ester ("metalaxyl"), ( (GB 1500581); (R) -N- (2,6-Dimethylphenyl) -N- (methoxyacetyl) -alanine methyl ester ("R-metalaxyl") (Reference GB 1500581); , 2,5,6-tetrahydro-4H-pyrrolo [3,2,1-ij] quinolin-4-one ("pyroquilon"), (Reference GB 1394373); idrogen phosphonate ("fosetyl") (reference: C. Tomiin (ed.): The Pesticide Manual, 10th edition, Famhan, UK, 1994, 530-532); and copper hydroxide (reference: C. Tomiin (ed.), The Pesticide Manual, 10th edition, Famhan, UK, 1994, 229-230).

Preferably, the selected microbicide is applied in the form of a composition to the immunomodulated plant together with other carriers, surfactants or other absorption-promoting adjuvants commonly used in formulation technology. Suitable carriers and adjuvants may be solid or liquid, and may be formulated in a conventional formulation, such as natural or regenerated minerals, solvents, dispersants, wetting agents, thickeners, diluents, binders, and fertilizers.

In one preferred application, the microbicidal composition is applied to parts of the plant which are above the soil, especially to the leaves (foliar application). The frequency and extent of treatment will depend on the biological and climatic conditions of the pathogen. However, the microbicide may also penetrate the plant through the roots in soil or water (systemic action) if the locus of the plant has been impregnated with a liquid formulation (e.g. in rice cultures) or if the microbicide has been applied to the soil in solid form, e.g. ). For the treatment of the seeds, the microbicide may also be applied to the seeds (coating) either by impregnating the tubers or cereal grains with a liquid formulation of the microbicide or by coating with an already combined wet and dry formulation. However, in special cases, other methods may be applied to the plants, such as treatments targeting buds or bunches.

The microbicide may be used in unmodified form or preferably in admixture with adjuvants commonly used in formulation technology, such as emulsifiable concentrates, spreadable pastes, directly sprayed or diluted solutions, dilute emulsions, wettable powders, soluble powders, powdered materials, granules or encapsulation, for example, into polymeric materials. The type of formulation will also determine the mode of administration, such as spraying, atomization (fogging), dusting, scattering, coating or pouring, in accordance with the objects to be treated and the prevailing conditions. Preferably, the microbicide is normally applied in an amount of 50 g to 2 kg of active ingredient / ha, more preferably 100 g to 1000 g of active ingredient / ha, and most preferably 150 g to 700 g of active ingredient / ha. For seed treatment, the amount employed is between 0.5 g and 1000 g, preferably 5 g to 100 g of active ingredient per 100 kg of seed.

The formulation is carried out in a known manner, for example by homogeneous mixing and / or grinding of the microbicide with the excipient, such as a solvent, a solid carrier, where appropriate, a surfactant.

Suitable solvents include: aromatic hydrocarbons, preferably C 8 -C 12 fractions such as 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 esters and their esters such as ethanol, ethylene glycol, ethylene glycol monomethyl or monoethyl ether, ketones such as cyclohexanone, highly polar solvents such as N-methyl-2-pyrrolidone, dimethyl sulfoxide or dimethylformamide, and vegetable

Oils or epoxidized vegetable oils, such as epoxidized coconut oil or soybean oil; or water.

Naturally occurring mineral fillers such as calcite, talc, kaolin, montmorillonite or attapulgite may be used as solid carriers, such as powders and dispersible powders. Highly dispersed silica or highly dispersed absorbent polymers may also be added to improve physical properties. Suitable granulated adsorptive carriers are of the porous type, such as pumice stone, broken brick, sepiolite or bentonite, and suitable non-sorbent carriers such as calcite or sand. In addition, a large number of pre-granulated materials of inorganic or organic nature, such as dolomite or powdered plant residues, may be used.

Depending on the nature of the microbicide, suitable surfactants are nonionic, cationic and / or anionic surfactants with good emulsifying, dispersing and wetting properties. The term "surfactant" also includes mixtures of these agents.

Particularly preferred adjuvants for use are natural or synthetic phospholipids of the cephalin and lecithin series, such as phospholipidyl ethanolamine, phosphatidylserine, phosphatidylglycerol and lysolecithin.

The agrochemical compositions are usually 0.1-99%, preferably 0.1-95%, of the microbicidal active ingredient, 99.9-1%, preferably 99.9-5% of solid or liquid adjuvant and 0-25%, preferably 0.1-95%. They contain 25% surfactant.

The formulations are preferably formulated as concentrates and the end user will normally use the diluted forms.

B) Plant activating microbicides

Alternatively, when applied to an immuno-modified plant obtained by the second or third route (selective breeding or genetic engineering) described above, the microbicide may alternatively be a chemical inducer of SAR (a plant-activating microbicide), such as a benzothiadiazole compound, an isonicotinic acid compound or a salicylic acid compound disclosed in U.S. Patent Nos. 5,523,311 and 5,614,395. Therefore, two methods of immune modification are used simultaneously. Application of the plant-activating microbicide to a plant immunized with selective breeding or genetically engineered results in "extra-immunomodulation" and synergistically increased disease resistance.

As described below, transgenic immunomodulated plants that strongly express the NIM1 gene respond much more rapidly and at much lower doses of BTH than PR-1 gene expression and P. parasitia resistance, and as wild type plants. See Example 35 and Northern blot analysis in Figure 3. The synergistically enhanced disease resistance in NIM1 overexpressing plants can already be achieved with 10 μΜ BTH, which is normally completely inappropriate to induce any effect. Normal phytotoxic or otherwise undesirable concentrations of SAR-inducing chemicals can be eliminated by this synergism. A further advantage of this application is that the duration of SAR activation can be altered when the SAR inducing chemical is applied to an already immunomodulated plant, such as overexpressing NIM1. Further, the economic benefit can be realized by the reduced amount of SAR-inducing chemicals needed to provide plant protection at a given level.

C) Commonly used microbicides together with plant activating microbicides

To provide even greater disease resistance, a conventional microbicide and a plant-activating microbicide can be used in combination with immunomodulated plants obtained either by selective breeding or by genetic engineering. This results in a very high level of synergistic disease resistance when compared to the immune resistance alone or the immune modification only and one microbicide or the disease resistance results obtained when the two microbicides (conventional and plant activator) are used simultaneously. See Table 35 in Example 19.

Evaluation of disease resistance

Evaluation of disease resistance is carried out according to methods known in the art (see Uknes et al., 1993, Molecular Plant Microbe Interactions 6: 680-685; Gorlach et al .: Plant Cell. 8: 629-643 (1996); Alexander et al., Proc. Natl. Acad. Sci. USA 90: 7327-7331 (1993). As an example, some typical disease resistance testing methods are described below.

A) Phytophyhora parasitica (black dehydration) resistance test

Phytophyhora parasiticava, a black dehydrating organism, was tested for resistance on six-week-old plants as described in Alexander et al., Proc. Natl. Acad. Sci. USA 90: 7327-7331 (1993). Plants were watered, allowed to absorb water well, and inoculated with 10 ml of spore suspension (300 spores / ml). The infected plants were kept in a greenhouse at 23-25 ° C at daytime and 20-22 ° C at night. The fading index was used in the test as follows: 0 = no symptoms; 1 = no symptoms; 1 = some signs of withering with reduced swelling; 2 = symptoms of light swelling but no rot or swelling; 3 = clear wilting symptoms with swelling but no rot rot; 4 = severe decay with visible stem rot and some damage to the root system; 5 = like 4, but the plants are almost dead or dead and the root system has been severely reduced. In all studies, plants randomly marked were scored.

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B) Pseudomonas syringae resistance test

A Pseudomonas syringae pv. tabaci strain 155 was injected into the two lower leaves of some 6-7 weeks old plants at a concentration of 10 ⁶ or 3 x 106 ⁶ ml. Six individual plants were evaluated at each time point. Plants infected with Pseudomonas tabaci were evaluated at 5 points depending on the severity of the disease, 5 = 100% dead tissue, 0 = no symptoms. T-test (LSD) was also performed each day and the groups were plotted on the basis of the mean disease severity. For the same letter, the values obtained on the day of the evaluation did not differ statistically significantly.

C) Cercospora nicotianae resistance test

A suspension of spores of Cercospora nicotianae (ATCC 18 366) (100,000-150,000 spores per ml) was sprayed until the liquid flowed to the surface of the leaves. The plants were kept at 100% humidity for 5 days. The plants were then sprayed with water 5-10 times daily. Six plants were evaluated at each time point. Infection with Cercospora nicotianae was evaluated with the% leaf area showing symptoms of the disease. T-test (LSD) was also performed each day and the groups were plotted on the basis of the mean disease severity. For the same letter, the values obtained on the day of the evaluation did not differ statistically significantly.

D) Peronospora parasitica resistance test

Resistance tests against Peronospora parasitica were carried out on plants as described by Uknes et al. (1993). Plants were inoculated with a compatible isolate of P. parasitica by spraying with a conidia suspension (about 5 x 10 ⁴ spores per ml). The infected plants were incubated in humid conditions at 17 ° C in a growing chamber for 14 hours in day / 10 hours in night cycles. The plants are grown for 3-14 days, preferably 7-12. days after inoculation in the presence of conidiophores. In addition, some plants from each treatment were randomly selected and stained with lactophenol trypan blue [Keogh et al., Trans. Br. Mycol. Soc. 74, 329-333 (1980)] for microscopic examination.

Description of figures

Figure 1: Alignment of NIM1 protein sequence with mouse, rat, and porcine ΙκΒα sequences. The vertical lines (|) above the sequences indicate the amino acid identity between the NIM1 and ΙκΒα sequences (matrix score equals 1.5); a colon (:) above the sequences means that the identity score is> 0.5; a single dot (.) above the sequences indicates that the identity is <0.5 but> 0.0; and <0.0 points means no identity and no signal over sequences (see examples). The position of the ankyrin domains of mammalian ΙκΒα was identified according to Martin et al., Gene 152: 253-255 (1995). In-sequence scoring refers to gaps between NIM1 and ΙκΒα proteins. The five ankyrin repeats in ΙκΒα are indicated by the dashed line below the sequence. Amino acid numbering was used to refer to NIM1 protein, introducing gaps where necessary. Plus signs (+) are placed at each of the 10 amino acids above the sequences.

Figure 2: Comparison of a region of NIM1 protein (numbering corresponding to amino acids given in SEQ ID NO: 2) with rice EST protein products (SEQ IDNO: 17-24).

Figure 3 shows the results of Northern analysis over time of PR-1 gene expression in wild type and in lines overexpressing NIM1 after treatment with water or BTH. RNA was prepared from the treated plants and analyzed as described in the Examples. "Ws" is the wild type Arabidopsis thaliana Ws ecotype. "3A", "5B", "6E" and "7C" are plant cell lines overexpressing each NIM1, prepared as in Example 21. "0 BTH" for aqueous treatment; “10 BTH” 10 μΜ BTH treatment; “100 BTH” means 100 μΜ BTH treatment. Day 0 controls; "1", "3" and "5" are samples taken on days 1, 3 and 5.

Description of 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 ΙκΒα amino acid sequence

1

SEQ ID NO: 4 is the rat ΙκΒα amino acid sequence

1

SEQ ID NO: 5 is the porcine ΙκΒα amino acid sequence

1

SEQ ID NO: 6 is the cDNA sequence of the Arabidopsis thaliana NIM1 gene

SEQ ID NOs: 7 and 8 are the DNA coding sequence and the encoded amino acid sequence, which is the dominant-negative form of the NIM1 protein, which contains alanine instead of serine at amino acid positions 55 and 59

The DNA coding sequence of SEQ ID NOs: 9 and 10, respectively, which is the dominant-negative form of the NIM1 protein carrying the N-terminal deletion

SEQ ID NOs: 11 and 12 DNA coding sequence and coded amino acid sequence which is the dominant-negative form of the C-terminal deletion of NIM1 protein

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SEQ ID NOs: 13 and 14, or the encoded amino acid sequence, which is an altered form carrying the N-terminal and C-terminal deletions of the NIM1 gene

SEQ ID NOs: 15 and 16, and the encoded amino acid sequence encoding the ankyrin domain of the NIM1 gene

SEQ ID NO: 17 is a rice-1 33-155 amino acid sequence a

2

SEQ ID NO: 18 is the rice-1 215-328 amino acid sequence from Figure 2

SEQ ID NO: 19 a 33-155 amino acid sequence of rice-2 a

2

SEQ ID NO: 20 is the amino acid sequence 208-288 of rice-2 from Figure 2

SEQ ID NO: 21 is a rice-3 33-155 amino acid sequence a

2

SEQ ID NO: 22 is the rice-3 amino acid sequence 208-288 of Figure 2

SEQ ID NO: 23 is a 33-155 amino acid sequence of rice-4 a

2

SEQ ID NO: 24 is the amino acid sequence 215-271 of rice-4 from Figure 2

SEQ ID NOs: 25 to 32 are oligonucleotide primers

definitions

The following definitions are provided to provide a better understanding of the invention.

Plant cell: A structural and physiological unit of plants consisting of a protoplast and a cell wall. The term "plant cell" refers to any cell that is part of or derived from a plant. Some examples of cells are: differentiated cells that are part of a living plant; differentiated cell cultures; non-differentiated cells in culture; non-differentiated tissue cells such as callus or tumors; differentiated cells of seeds, embryos, flower hoses and pollens.

Plant Tissue: A group of plant cells organized into a structural and functional unit. Includes any tissue of a plant in cuttings or cultures. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture, and any group of plant cells organized into structural and functional units. The term is used in connection with any specific type of plant tissue, either in its absence or otherwise, or is otherwise intended to exclude any other type of plant tissue.

Proroplast: a plant cell without a cell wall. Offspring: the sexually or asexually descended future generation of plants which includes, but is not limited to, progeny plants.

Transgenic Plant: A plant that contains stably incorporated recombinant DNA in its genome.

Recombinant DNA: Any DNA molecule produced by linking DNA segments from various sources and employing recombinant DNA technology.

Recombinant DNA Technology: A technology that produces recombinant DNA in vitro and delivers recombinant DNA into cells where it can be expressed or amplified. See Concise Dictionary of Biomedicine and Molecular Biology, Ed. Juo, CRC Press, Boca Raton (1996)] such as DNA may be introduced into protoplast (s) or cells (s) in various forms such as (1) naked DNA in circular, linear or superspiral form, (2) in nucleosomes or chromosomes, or DNA in nuclei or portions thereof, (3) DNA that is complexed or linked to other molecules, (4) DNA enclosed in liposomes, spheroplasts, cells or protoplasts, or (5) DNA from non-host organisms (ex. Agrobacterium tumefaciens). These and other different methods of introducing recombinant DNA into cells are well known in the art and can be used to produce the transgenic cells or transgenic plants of the invention.

Recombinant DNA technology also includes the homologous recombination method described by Treco et al. (WO 94/12650 and WO 95/31560), which can be used to increase peroxidase activity in a monocot. Specifically, regulatory regions (ex. Promoters) can be introduced into the plant genome to enhance expression of endogenous peroxidase.

Recombinant DNA technology also includes the insertion of a peroxidase coding sequence lacking the expression signals selected for the monocot and the transgenic monocot plant for increased expression of the peroxidase attributable to the endogenous control sequences in the monocot. This would result in an increase in the number of copies of the peroxidase coding sequence within the plant.

Initial incorporation of recombinant DNA into the genome of the R0 plant has not been defined since it is accomplished by conventional plant breeding methods, but more particularly by the technical methods described above. After the initial insertion, the transgenic progeny can be propagated by essentially conventional breeding methods.

Chimeric gene: A DNA molecule comprising at least two heterologous moieties, e.g., portions of pre-deleted DNA sequences that are not linked in their previous position, preferably by recombinant DNA technology.

Expression cassette: a DNA molecule containing a promoter and a terminator with the coding sequence inserted therein.

Encoding sequence: A DNA molecule that, when transcribed and translated, results in the formation of a polypeptide or protein.

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Gene: A discrete chromosomal region containing a regulatory DNA sequence responsible for controlling expression, such as transcription and translation, and the coding sequence that is transcribed and translated to form a precise polypeptide or protein.

acd: accelerated cell death mutant plant

AFLP: amplified fragment length polymorphism AvrRpt2: Rpt2 avirulent gene isolated from Pseudomonas syringae

BAC: artificial bacterial chromosome

BTH: S-methyl ester of benzo [1,2,3] thiadiazole-7-thiocarboxylic acid

TITLE: phenotype with constitutive immunity (SAR is constitutively activated) title: mutant plant with constitutive immunity cM: centimorgan epri: mutant plant constitutively expressing PR genes

Col-O: Arabidopsis ecotype Columbia

ECs: enzyme combinations

Emwa: Peronospora parasitica isolate compatible with Arabidopsis Ws-0 ecotype

EMS: ethyl methanesulfonate

INA: 2,6-dichloroisonicotinic acid

Ler: Arabidopsis ecotype Landsberg erecta

Isd: lesion mimicking f / esions simulating disease) mutant plant nahG: salicylate hydroxylase from Pseudomonas pufid, which converts salicylic acid to catechol

NahG: Arabidopsis line transformed with nahG gene ndr: non-race-specific disease resistance mutant plant nim: non-/ 'nducible zmmunity' mutant plant

NIM1: the wild-type gene contained in the SAR signal transduction cascade

NIM1: protein encoded by the wild-type NIM1 gene nim1: mutant alleles of NIM1 that transmit disease susceptibility to the plant; also applies to mutant Arabidopsis thaliana plants carrying the Nim1 mutant allele of NIM1

Noco: Peronospora parasitica isolate compatible with Arabidopsis Col-0 ecotype

ORF: open reading frame

PCs: primary combinations

PR: pathogenesis-related

SA: Salicylic acid

SARS: systemic acquired resistance

On SAR: immunomodulated plants in which SAR is activated typically have "higher than wild-type SAR gene expression and have a disease-resistant phenotype

SSLP: Simple sequence length polymorphism UDS: Phenotype susceptible to universal disease Wela: Peronospora parasitica isolate compatible with

Arabidopsis Weiningen ecotype

Ws-O: Issilewski of the Arcibidopsis ecotype

WT: wild type

YAC: artificial yeast chromosome

Examples

The invention is illustrated by the following detailed methods, compositions and examples. However, the examples are provided by way of illustration only and are not intended to limit the scope of the invention in any way.

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by Sambrook et al., Molecular Cloning, published by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and Silhavy TJ, Berman ML and Enquist LW Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and Ausbel FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Association and Whileylnterscience (1987).

I. Synergistic Disease Resistance Effects Resulting from the Coordinated Application of the Chemical Inducer of Systemic Acquired Resistance and a Standard Microbicide to Plants

In this series of examples, SAR was induced in plants using a chemical inducer of SAR, such as benzothiadiazole. In addition, conventional microbicides were used for the plants. The plants were then infected with various pathogens. The combination of the two methods (inducing chemical + microbicide) resulted in greater than additive, i.e. synergistic disease resistance. This was defined as the synergistic factor (SF), i.e. the ratio of the observed (O) effect to the expected (E) effect.

The expected effect (E) for a given combination of agents can be described by the so-called Colby formula and calculated as follows [Colby, S. R., "Calculating synergetic and antagonistic responses of herbicide combination". Weeds, Volume 15, pp. 20-22. page (1967)]:

ppm = active ingredient (ha.) milligrams / liter spray mixture

X =% activity of active ingredient I in ppm of active ingredient

Y =% effect of active ingredient II on q ppm of active ingredient

E = expected effect of l + ll active compound combination at p + q ppm (additive effect)

Colby's formula:

E - X + Y - X x Y

100

Example 1: Action against Erysiphe graminis in barley

Residual protective effect: approx. Barley plants, 8 cm long, were spray-sprayed with an aqueous spray mixture (max. 0.02% active ingredient) and sprayed with fungal conidia 3-4 days later. The infected plants were allowed to stand in a greenhouse at 22 ° C. Fungal infection was usually evaluated on the tenth day after infection.

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Systemic protective effect: approx. Barley plants 8 cm long were watered with an aqueous spray mixture (max. 0.02% active ingredient based on soil volume), taking care not to contact the spray mixture with the above-ground parts of the plants. 3-4 days later, the plants were sprayed with fungal conidia. The infected plants were left in a greenhouse at 22 ° C. The fungal infection was evaluated on the tenth day after infection.

Table 1

Effect against Erysiphe graminis in barley

Component I: Benzothiazide-7-carboxylic acid

Component II: Metconazole

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) 1 0.6 0 2 2 40 3 6 89 4 0.6 10 5 2 40 6 6 51 7 20 65 8 0.6 0.6 1: 1 37 10 3.7 9 0.6 2 1: 3 59 40 1.5 10 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

Table 2

Effect against Erysiphe graminis in barley

Component I: Benzothiazide-7-carboxylic acid

Component II: Tetraconazole

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) 1 2 0.6 2 14 27 3 4 0.6 2 45 63 5 6 0.6 0.6 0.6 2 1: 1 1: 3 70 82 53 68 1.3 1.2 7 2 0.6 3: 1 79 60 1.3

Table 3

Effect against Erysiphe graminis in barley

Component I: Benzoyl, 2.3] S-methyl ester of thiadiazole-7-thiocarboxylic acid

Component II: Metconazole

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. 0 (watched) E (expected) 1 2 0.6 2 0 33

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Table 3 (continued)

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) 3 6 17 4 20 33 5 60 50 6 0.6 6 1:10 33 17 1.9 7 0.6 20 1:30 50 33 1.5 8 0.6 60 1: 100 83 50 1.7

Example 2: Action against Colletotrichum lagenarium

Cucumis sativus L.-en

After a 10-14 day cultivation period, the cucumber seedlings were sprayed with a spray mixture of a wettable powder formulation of the test compound. After 3-4 days, the plants were inoculated with a fungal spore suspension (1.0x10 ⁵ spores / ml) and incubated for 30 hours in high humidity at 23 ° C. Incubation was then continued at 22-23 ° C under normal humidity. The protective effect was evaluated on the basis of the fungal infection 7-10 days after infection.

After a cultivation period of 10-14 days, the cucumber seedlings were treated with a spray mixture applied in the soil and prepared from a wettable powder formulation of the test compound. After 3-4 days, the plants were inoculated with a fungal spore suspension (1.0x10 ⁵ spores / ml) and incubated for 30 hours in high humidity at 23 ° C. Incubation was then continued at 22 ° C under normal humidity. The protective effect was evaluated on the basis of the fungal infection 7-10 days after infection.

Table 4

Activity against Colletotrichum lagenarium on Cucumis sativus L. / leaf

Component I: Benzothiazide-7-carboxylic acid

Component II: Azoxystrobin

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) 1 0.06 0 2 0.2 5 3 2 22 4 0.60 5 5 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 10 2 0.6 3: 1 49 31 1.6 11 2 6 1: 3 44 35 1.3

Table 5

Activity against Colletotrichum lagenarium on Cucumis sativus L. / soil

Component I: Benzothiazide-7-carboxylic acid

Component II: Azoxystrobin

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) 1 2 0,006 0.02 0 40

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Table 5 (continued)

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) 3 0.06 49 4 0.2 91 5 0.2 0 6 0.6 9 7 2 28 8 6 66 9 0,006 0.2 1:30 11 0 * 10 0.6 1: 100 30 9 3.3 11 2 1: 300 83 28 3.0 12 0.02 6 1: 300 97 80 1.2 13 0.06 6 1: 100 100 82 1.2

* SF synergistic factor was not calculated

Table 6

Action against Colletotrichum lagenarium on Cucumis sativus L. / Foil application

Component I: Benzothiazide-7-carboxylic acid

Component II: methylcresoxime

Number of dough Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) 1 0.2 3 2 0.62 51 3 2 0 4 20 41 5 0.2 2 1:10 15 3 5 6 0.2 20 1: 100 61 43 1.4

Table 7

Activity against Colletotrichum lagenarium when applied to Cucumis sativus L. / leaf

Component I: S-methyl ester of benzo [1,2,3] thiadiazole-7-thiocarboxylic acid

Component II: Azoxystrobin

Number of dough Ha mg / l (ppm) I: II % effect SF O / E Comp. I Comp. II. O (watched) E (expected) 1 0.06 16 2 0.2 22 3 2 60 4 2 18 5 6 75 6 0.06 2 1:30 43 31 1.4 7 0.2 2 1:10 57 36 1.6

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Table 8

Activity against Colletotrichum lagenarium when applied to Cucumis sativus L. / soil

Component I: S-methyl ester of benzo [1.2.3] thiadiazole-7-thiocarboxylic acid

Component II: Azoxystrobin

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) 1 0.06 0 2 0.02 6 3 0.06 23 4 0.2 36 5 0.02 1 6 0.06 5 7 0.6 27 8 2 61 9 6 93 10 0,006 0.02 1: 3 26 1 26 11 0,006 0.6 1: 100 44 27 1.6 12 0,006 2 1: 300 84 61 1.4 13 0.02 0.02 1: 1 23 7 3.3 14 0.02 2 1: 100 77 64 1.2 15 0.06 0.02 3: 1 42 24 1.8 16 0.06 2 1:30 92 70 1.3 17 0.2 2 1:10 93 75 1.2

Example 3: Action against Cercospora nicotianae on tobacco plants

Tobacco plants (6 weeks) were sprayed with a solution of the test compound formulation (concentration: max. 0.02% active ingredient). Four days after treatment, the plants were inoculated with Cercospora nicotianae spore suspension (150,000 spores / ml) and maintained at high humidity for 4-5 days, and then incubated for a full day. Symptoms were evaluated on the basis of fungal infected leaf surfaces.

Table 9

Effect against Cercospora nicotianae on tobacco plants

Component I: S-methyl ester of benzo [1.2.3] thiadiazole-7-thiocarboxylic acid

Component II: tebuconazole

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) 1 0.2 0 2 2 17 3 6 55 4 20 78 5 2 0 6 6 0 7 0.2 2 1:10 87 0 * 8 0.2 6 1:30 97 0 THE

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Table 9 (continued)

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) 9 2 2 1: 1 87 17 5.1 10 2 6 1: 3 94 17 5.5 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

Table 10

Effect against Cercospora nicotianae on tobacco plants

Component I: S-methyl ester of benzo [1.2.3] thiadiazole-7-thiocarboxylic acid

Component II: cyproconazole

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) 1 0.2 0 2 2 17 3 6 55 4 20 78 5 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 10 2 6 1: 3 94 17 5.5 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 78 1.3

Table 11

Effect against Cercospora nicotianae on tobacco plants

Component I: Benzothiazide-7-carboxylic acid

Component II: fenpropimorph

Test number Ha. Kg per ha I: II % effect SF O / E Comp. I Comp. II. O (watched) E (expected) 0 - - 0 (control) 1 0.2 0 2 0.6 3 3 2 69 4 6 79 5 2 13 6 6 23 7 10 42

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Table 11 (continued)

Test number Ha. Kg per ha I: II % effect SF O / N Comp. I Comp. II. 0 (watched) E (expected) 8 0.2 2 1:10 52 13 4 9 0.2 6 1:30 61 23 2.7 10 0.6 2 1: 3 71 16 4.4 11 6 6 1: 1 100 83 1.2

Table 12

Effect against Cercospora nicotianae on tobacco plants

Component I: Benzothiazide-7-carboxylic acid

Component II: Difenoconazole

Test number Ha. Kg per ha I: II % effect SFO / E Comp. I Comp. II. 0 (watched) E (expected) 0 - - 0 (control) 1 2 69 2 6 79 3 20 100 4 2 3 5 6 23 6 32 7 2 0.6 3: 1 90 70 1.3 8 6 0.6 10:10 100 80 1.3

Example 4: Action against Pyricularia oryzae on rice plants

Rice plants, about 2 weeks old, were placed in a spray-filled container (up to 0.006% active ingredient) together with the soil around their roots. 96 hours 40 later, the rice plants were inoculated with a fungal conidia suspension. The fungal parasite infection was evaluated after the infected plants were exposed to 95-100% relative humidity for 5 days and ca. Incubate at 24 ° C.

Table 13

Effect against Pyricularia oryzae on rice plants

Component I: S-methyl ester of benzo [1.2.3] thiadiazole-7-thiocarboxylic acid

Component II: KTU 3616

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. 0 (watched) E (expected) 1 6 15 2 0.02 0 3 0.06 28 4 0.2 47 5 0.6 79 6 2 83 7 6 91

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Table 13 (continued)

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. 0 (watched) E (expected) 8 6 0.02 300: 1 42 15 2.8 9 6 0.06 100: 1 76 39 1.9 10 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 12 m ² parcel, the rice plants were sprayed with a spray formulation prepared from the wettable powder of the active ingredient. The infection happened naturally. For evaluation, the leaf area infected with the fungus was measured 44 days after treatment. The following results were obtained.

Table 14

Activity against Pyricularia oryzae in rice plants outdoors

Component I: S-methyl ester of benzo [1,2,3] thiadiazole-7-thiocarboxylic acid

Component II: Pyroquilon

Test number Ha. Kg per ha I: II % effect SFO / E 1 comp. Comp. II. O (watched) E (expected) 1 2 0.25 0.5 0 (control) 22 50 3 4 0.75 1.5 46 82 5 6 0.25 0.5 0.75 0.75 1: 3 1: 1.5 80 85 58 73 1.4 1.2

About 2 weeks later, the rice plants, together with the soil around their roots, were placed in a spray-filled container. The fungal infection was evaluated 36 days later. Infection of untreated plants corresponded to a 0% effect.

Table 15

Effect against Pyricularia oryzae on rice plants

Component I: S-methyl ester of benzo [1,2,3] thiadiazole-7-thiocarboxylic acid

Component II: Tricyclazole

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) 1 2 3 4 0.5 0.25 0.1 0.05 65 39 18 5

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Table 15 (continued)

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. 0 (watched) E (expected) 5 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 10 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

Example 5: Colletotrichum sp. (Anthracnose) and at intervals of about 7 days, per hectare

Cercospora sp. (Leaf Spot) with 500-700 liters of spray formulation. Three days on

Effects on the crop: the area is approx. After spraying the plants were first (test site mestersége100 m ² plots: Cikampek, 25 sen infected with the fungus.

Java, Indonesia) The chili plants were sprayed on Monday. spreadsheet

Effect against Colletotrichum: evaluation of the infestation of the chilli fruit after the fifth spray with estimation of infection

Component I: S-methyl ester of benzo [1.2.3] thiadiazole-7-thiocarboxylic acid

Component II: mancozeb

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) 1 5 55 2 100 12 3 5 100 1:20 77 59 1.3

Table 17

Effect against Cercospora: Evaluation of leaves by estimating infection after sixth spraying

Component I: S-methyl ester of benzo [1.2.3] thiadiazole-7-thiocarboxylic acid

Component II: mancozeb

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) 1 5 76 2 100 8 3 5 100 1:20 87 78 1.1

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Table 18

Effect on crop: the dill was harvested after the sixth spray

Component I: S-methyl ester of benzo [1.2.3] thiadiazole-7-thiocarboxylic acid

Component II: mancozeb

Test number Ha mg / l (ppm) I: II %-ancestor effect SFO / E Comp. I Comp. II. O (watched) E (expected) 1 2 5 100 459 8 3 5 100 1:20 1400 approx. 460 approx. 3

Example 6: Action against Puccinia recondita in wheat Seven-day-old wheat plants were spray-sprayed with a spray formulation of the active ingredient or combination of active ingredients. After 4 days, the treated plants were inoculated with a suspension of fungal conidia and then the treated plants were incubated for 2 days at 90-100% relative atmospheric humidity at 20 ° C. Ten days after infection, the fungal infection was evaluated.

Table 19

Effect against Puccinia recondita in wheat

Component I: S-methyl ester of benzo [1.2.3] thiadiazole-7-thiocarboxylic acid

Component II: Propiconazole

Test number Ha mg / l (ppm) I: II %-ancestor effect SFO / E Comp. I Comp. II. O (watched) E (expected) 1 100 - 0 (control) 51 2 5 10 3 100 5 20: 1 79 56 1.4

Table 20

Effect against Puccinia recondita in wheat

Component I: S-methyl ester of benzo [1.2.3] thiadiazole-7-thiocarboxylic acid

Component II: fenpropidine

Test number Ha. Kg per ha I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) 1 2 6 20 0 (control) 20 40 3 4 20 60 40 60 5 6 6 6 20 20 1: 3 1:10 73 75 52 68 1.4 1.1

Example 7: Effect against Erysiphe graminis on wheat In a growing area of 10 m ^2, winter wheat was sprayed with a spray formulation prepared from a wettable powder of the active ingredient. The infection occurred naturally. Ten days after infection, the fungal infection was evaluated. The following results were obtained.

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Table 21

Effect against Erysiphe graminis in wheat

Component I: S-methyl ester of benzo [1.2.3] thiadiazole-7-thiocarboxylic acid

Component II: Propiconazole

Test number Ha g per ha I: II % effect SF O / E Comp. I Comp. II. 0 (watched) E (expected) 1 5 - 0 (control) 29 2 3 50 100 2 31 4 5 5 5 50 100 1:10 1:20 49 59 32 51 1.5 1.2

Table 22

Effect against Erysiphe graminis in wheat

Component I: S-methyl ester of benzo [1.2.3] thiadiazole-7-thiocarboxylic acid

Component II: cyprodinil

Test number Ha .g per ha I: II % effect SF O / E Comp. I Comp. II. O (watched) E (expected) 1 5 - 0 (control) 29 2 3 50 100 2 31 4 5 5 5 50 100 1:10 1:20 49 59 32 51 1.5 1.2

Example 8: Mycosphaerella fijensis banana 300 m ² plots were sprayed with 40 banana plants in a 17 to 19-day intervals with a spray mixture prepared with a wettable powder of the active ingredient, a total of six times. The infection happened naturally. Fungal infected leaves were measured for evaluation. The following results were obtained.

Table 23

Effect against Mycosphaerella fijensis in bananas

Component I: S-methyl ester of benzo [1.2.3] thiadiazole-7-thiocarboxylic acid

Component II: Propiconazole

Test number Ha .g per ha I: II %-ancestor effect SF O / E Comp. I Comp. II. 0 (watched) E (expected) 1 5 - 0 (control) 19 2 50 26 3 5 50 1: 1 46 40 1.15 |

Example 9: Action against Alternaria solani in tomato

In a 7 m ² plot, tomato plants were sprayed at 7 day intervals with a spray formulation of the active ingredient wettable powder for a total of nine times. The infection happened naturally. Fungal infected leaves were measured for evaluation. The following results were obtained.

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Table 24

Action against Alternaria solani on tomato plants outdoors

Component I: S-methyl ester of benzo [1,2,3] thiadiazole-7-thiocarboxylic acid

Component II: cyprodinil

Test number Ha .g per ha I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) 1 2.5 - 0 (control) 32 2 3 12.5 25 30 51 4 5 2.5 2.5 12.5 25 1: 5 1:10 79 80 53 67 1.5 1.2

Example 10: Action against Phytophthora infestans in tomato 20

Tomato plants called "Roter

Gnom 'was spray-dripped with a spray formulation made from the active ingredient or combination of active ingredients. After four days, the treated plants were inoculated with a suspension of the fungal spore and then incubated for 2 days at 18-20 ° C and 90-100% relative atmospheric humidity. On day 5 post-infection, the fungal infection was evaluated. The following results were obtained.

Table 25

Effect against Phytophthora infestans in tomato

Component I: S-methyl ester of benzo [1,2,3] thiadiazole-7-thiocarboxylic acid

Component II: Metalaxyl

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) - - - 0 (control) 1 5 14 2 25 36 3 100 61 4 500 72 5 0.1 13 6 1 23 7 10 35 8 50 68 9 5 0.1 50: 1 50 25 2.0 10 5 1 5: 1 62 34 1.8 11 5 10 1: 2 87 44 2.0 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 15 100 50 2: 1 95 88 1.1 16 500 10 50: 1 97 82 1.2

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Table 26

Effect against Phytophthora infestans in tomato

Component I: Benzothiazide-7-carboxylic acid

Component II: Metalaxyl

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) - - - 0 (control) 1 0.1 0 2 0.5 9 3 1 22 4 5 45 5 1 13 6 10 33 7 50 63 8 100 83 9 0.1 1 1:10 36 13 2.8 10 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 on cucumber

16-19 day-old cucumber plants ("Winconsin") were spray-sprayed with a spray formulation prepared from the active ingredient or combination of active ingredients. After four days, the treated plants were inoculated with a suspension of Pseudoperonospora cubenswas spore (strain 365, Ciba; max. 5000 / ml) and then incubated for 1-2 days at 18-20 ° C and 70-90% relative atmospheric humidity. . On the 10th day after infection, fungal infection was estimated and compared with that of untreated plants. The following results were obtained.

Table 27

Effect against Pseudoperonospora cubensis on cucumber

Component I: Benzothiazide-7-carboxylic acid

Component II: Metalaxyl

Test number Ha mg / l (ppm) I: II % effect SF O / E Comp. I Comp. II. O (watched) E (expected) - - - 0 (control) 1 0.05 0 2 0.5 6 3 5 66 4 0.5 31 5 5 66 6 50 91

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Table 27 (continued)

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) 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 10 0.5 5 1:10 83 68 1.2

Example 12: Peronospora tabacina 15 on tobacco plants

Six-week tobacco plants were sprayed with a solution of the test compound formulation. Four days after treatment, the plants were inoculated with a fungal spore suspension, kept at high humidity for 4-5 days, and then incubated for a full day. In the tests, the symptoms were evaluated on the basis of the fungal infected leaf surface. The infection rate of untreated plants corresponds to a 0% effect.

Table 28

Effect against Phytophthora infestans in tomato

Component I: S-methyl ester of benzo [1.2.3] thiadiazole-7-thiocarboxylic acid

Component II: Metalaxyl

Test number Ha mg / l (ppm) I: II % effect SFO / E Comp. I Comp. II. O (watched) E (expected) 1 0.03 14 2 0.1 34 3 0.3 88 4 0.3 52 5 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

Example 13: Activity against Peronospora parasitica

Arabidopsis in thaliana

Metalaxyl, fosetyl and copper hydroxide fungicides and the SAR activator benzo [1.2.3] thiadiazole-7-carbothioic acid S-50 methyl ester (BTH) are 25%, 80%, 70% and 25%, respectively. (ha.) formulation with a wettable powder carrier and applied as a fine mist to the leaves of 3-week-old plants.

The wettable powder itself served as a control. Three days later, the plants were inoculated with Peronospora parasitica conidia suspension as described by Delaney (1995). Ws plants were inoculated with compatible P. parasitica Emwa isolate (1-2x10 ⁵ spores / ml); Col plants are compatible with P. parasitica 60

Inoculated with NoCo2 isolate (0.5-1 x 10 10 spores / ml). Following infection, the plants were covered to maintain high humidity and placed in a Percival growth chamber at 17 ° C for a 14 hour daily / 10 hour night cycle (Uknes et al., 1993). The tissue was harvested 8 days after infection.

The progress of the fungal infection was monitored for 12 days under a preparative microscope, observing the development of conidiophore dots (Delaney et al., 1994; Dietrich et al., 1994). Each leaf was stained with lactophenol trypan blue to observe fungal growth within the leaf tissue. Fungal growth was quantified using an rRNA fungal probe obtained by PCR on White and

(1990; PCR Protocol: A Guide to Methods and Application, 315-322) using NS1 and NS2 primers and P. parasitica EmWa DNA as templates. RNA was purified from frozen tissue obtained by phenol / chloroform extraction and subsequent lithium chloride precipitation (Lagrimini et al., PNAS 84: 7542-7546 (1987)). Samples (7.5 µg) were separated by electrophoresis on formaldehyde agarose gel and transferred to nylon membranes (Hybond-N +, Amersham), as described by Ausbel et al. (1987).

Hybridization and washes were performed according to Church and Gilbert (PNAS 81, 1991-1995, 1984). Relative amounts of transcripts were determined using a Phosphor Imager (Molecular Dynamics, Sunnyvale, CA) following the manufacturer's instructions. The sample was normalized by probing the striped filter blots with continuously expressed b-tubulin Arabidopsis cDNA. Fungal infection of untreated plants corresponded to a 0% inhibition of fungal growth. The following results were obtained.

Table 29

Activity against Peronospora parasitica NoCo2 in Arabidopsis thaliana (Col-O)

Component I: S-methyl ester of benzo [1,2,3] thiadiazole-7-thiocarboxylic acid

Component II: Metalaxyl

Test number components Inhibition of fungal growth% SFO / E BTH metalaxyl O (watched) E (expected) Ref. - - 0 1 0.01 mM - 0 2 - 0.1 mg / l 0 3 0.01 mM 0.1 mg / l 40.7 0 oo |

Table 30

Effect of Peronospora parasitica EmWa on Arabidopsis thaliana (Ws)

Component I: S-methyl ester of benzo [1,2,3] thiadiazole-7-thiocarboxylic acid

Component II: Metalaxyl

Test number components Inhibition of fungal growth% SFO / E BTH metalaxyl O (watched) E (expected) Ref. - - 0 1 0.01 mM 20 2 0.003 mM 0 3 2.5 mg / L 75 4 0.5 mg / L 50 5 0.1 mg / l 50 6 0.01 mM 2.5 mg / L 100 90 1.1 7 0.01 mM 0.5 mg / L 95 70 1.4 8 0.01 mM 0.1 mg / l 88 70 1.3 9 0.003 mM 2.5 mg / L 100 75 1.3

Table 31

Action against Peronospora parasitica Emwa in Arabidopsis thaliana (Ws)

Component I: S-methyl ester of benzo [1,2,3] thiadiazole-7-thiocarboxylic acid

Component II: fosetyl

Test number components Inhibition of fungal growth% SFO / E BTH fosetyl O (watched) E (expected) Ref. - - 0 1 0.01 mM 30

HU 224 480 Β1

Table 31 (continued)

Test number components Inhibition of fungal growth% SFO / E BTH fosetyl O (watched) E (expected) 2 1.0 g / l 40 3 0.2 g / l 10 4 0.04 g / l 0 5 0.01 mM 1.0 g / l 100 70 1.4 6 0.01 mM 0.2 g / l 100 40 2.5 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: S-methyl ester of benzo [1,2,3] thiadiazole-7-thiocarboxylic acid

Component II: Copper (II) hydroxide

Test number components Inhibition of fungal growth% SFO / E BTH Cu (OH) ₂ O (watched) E (expected) Ref. - - 0 1 0.01 mM - 30 2 - 0.01 g / l 0 3 0.01 mM 0.01 g / l 85 30 2.8

As shown in Table 29, synergistic disease resistance effects were demonstrated in wild-type Arabidopsis Col-O plants. No inhibition of fungal growth was observed when either 0.01 mM BTH or 0.0001 g / L metalaxyl alone was applied to the plants since these 40 concentrations were normally insufficient for efficacy. However, when both compounds were applied together to plants at concentrations that were normally inadequate, a 40.7% inhibition of fungal growth was observed, clearly showing 45 synergistic effects. 30-32. Tables 1 to 4 show synergistic disease resistance effects in wild-type Arabidopsis Ws plants. Only 20-30% inhibition of fungal growth was observed when 0.01 mM BTH was applied to Ws plants. However, synergistic disease resistance was observed when BTH and one of metalaxyl, fosetyl or copper hydroxide were used concomitantly. These combined antifungal effects, which result in a reduction in the effective concentration of the fungicide and BTH required to control the pathogen, allow the chemical dose to stop fungal growth to be reduced and therefore reduce the frequency of leaf damage attributable to chemical tolerance. 60

II. Synergistic Disease Resistance Effects Obtained by Application of Standard Microbicides and / or Chemical Inducers of Systemic Acquired Resistance to Constitutive Immunity (CIM) Mutant Plants

A Northern blot screening method was developed to identify mutant plants with high levels of PR-1 mRNA in normal growth, with the idea that these mutants also have acquired systemic resistance. A large number of mutants were isolated by this screening procedure and showed that not only PR-1 but also PR-2 and PR-5 accumulate mRNA [Lawton et al. (1993); Dietrich et al. (1994); and Weymann et al. (1995)]. These mutants also have elevated levels of SA and are resistant to pathogen infections, confirming that this approach can be used to isolate SAR signal transduction mutants.

Two classes of SAR signal transduction mutants were isolated by this screening. One class is designated as Isd mutants (lsd = / esion simulating disease), hereinafter referred to as "class I mutants" (see WO 94/16077, which is incorporated herein by reference in its entirety). This is the Isd class (aka

EN 224,480 cím1 Title I) spontaneously lesions on leaves, accumulates SA at elevated concentrations, has high levels of PR-1, PR-2, and PR-5 mRNA and is resistant to fungal and bacterial pathogens (Dietrich et al. ., 1994; Weymann et al., 1995).

The other class is designated as title mutants (cim = constitutive / mmunity) and, as described below, has all the features of Isd mutants except spontaneous lesions. This second class (title) corresponds to the "class II class" mutants described in WO 94/16077. The cim3 mutant plant line described below belongs to this class (cim class II) and is the dominant mutant of the wild type, which expresses SA, SAR gene mRNA at a stable, elevated level and has broad spectrum disease resistance.

Example 14: Isolation and characterization of cim mutants constitutively expressing the SAR gene

1100 unique M2 mutagenized (EMS) Arabidopsis plants were grown on Aracon plates (Lehie Seeds, Round Rock, TX) in about 100 series. Plants were grown as described by Uknes et al. (1993, supra), with particular attention to avoid over-irrigation and pathogen infection. More specifically, Metro Mix 360 was saturated with water and autoclaved three times for 70 minutes in 10 liter portions. The disinfected mix was mixed between each autoclave. The seed surface was sterilized in 20% Clorox for 5 minutes and washed seven times with sterile water before sowing. The seeded seeds were germinated for 3-4 days, then grown in chambers at 22 ° C for 9 hours during the day and 15 hours during the night. When the plants were 3-4 weeks old with 1-2 leaves weighing 50-100 mg, they were harvested and total RNA was isolated by rapid mini-RNA preparation [Verwoerd et al., Nuc. Acid. Gap. 17, 2361 (1989)]. Expression of the PR-1 gene by Northern blot analysis (Lagrimini et al., Proc. Natl. Acad. Sci. USA 84: 7542-7546 (1987); Ward et al. (1991)]. Each plant series also contained an untreated A. thaliana Col-O and a 2-day INA-treated (0.25 mg / ml) control. All plants were maintained as described by Weymann et al. (1995).

a putative mutant accumulating PR-1 mRNA was identified. The progeny were then tested and five were selected for further characterization. The putative cim mutants did not show increased SAR gene expression in the absence or pathogenicity of the pathogen. Testing the progeny of putative cim mutants is consistent with heritable constitutive PR-1 expression. Two of the cim mutants, cim2 and cim3, which showed the highest and most stable PR-1 expression, were further characterized.

Crossbreeds to Columbia have used the recessive hairline trait as a marker to identify F1 offspring. Col-gl1 flower buds were anaesthetized prior to pollen release and pollen obtained from the mutants was used immediately and the following day. F1 plants were grown in soil and crossed plants were identified by the presence of hairs.

After crossing cim2 and cim3 to the Col-0 or La-er ecotype, a high percentage of F1 plants were identified by high SAR gene expression, assuming that these traits were dominant. For cim2, some, but not all, F1 plants continuously expressed the SAR gene. Such an outcome would be expected if the cim2 mutant was dominant and carried as a parent heterozygote. Further genetic testing of cim2 showed a continuously changing segregation in the F2 generation, consistent with incomplete entry.

Cim3 showed a 1: 1 segregation in the F1 generation, which resulted in two self-pollinating F1 plants expressing high levels of PR-1 mRNA to generate a F2 population. F2 segregation obtained by PR-1 mRNA accumulation showed 93 F2 plants with high PR-1 mRNA accumulation and 25 F2 plants without significant PR-1 mRNA accumulation, yielding a 3.7: 1 ratio (c ² = 1.77). ; 0.5>P> 0.1), which is consistent with the hypothesis that cim3 is a dominant single gene mutation. Subsequent crosses confirmed that cim3 is inherited as a dominant mutation.

For cim3, the original M2 plant was identified in the screening procedure and the M3 population appeared normally. However, since cim3 plants are self-pollinating, some of the best expressing lines are less fertile. After cross-linking with Col-gl1, plants with normal appearance and fertility and high expression of PR-1 were obtained.

When initially identified, cim3 also appeared, only dwarf, with thin, twisted leaves. However, F2 plants resulting from crossing with the Col-gl 1 ecotype retained high SAR gene expression and could not be distinguished from the wild type. This suggests that the dwarf twisted leaf phenotype is caused by an independent mutant that is unrelated to constant SAR gene expression. The cim3 mutant phenotype was also observed when the plants were grown under sterile conditions, in agreement with the fact that PR-1 mRNA accumulation is not caused by a pathogen.

Example 15: SAR gene expression

In addition to PR-1, two other SAR genes, PR-2 and PR-5, were also highly expressed in cim3 mutants. SAR gene expression levels differed in offspring, but were always more than ten times higher than untreated controls, and were comparable to levels obtained after treatment of wild type with resistance-inducing INA (0.25 mg / ml).

Example 16: Salicylic acid analysis

Endogenous SA concentrations showed an increase in Arabidopsis following pathogen-induced necrosis

HU 224,480 Β1 (Uknes et al., 1993, supra). Salicylic acid and its glucose conjugate were analyzed as described by Uknes et al. (1993). Leaf tissues were collected from 10 cim3 and 10 control 4-week-old plants. The leaves from each plant were collected and the expression of the PR-1 gene was analyzed. SA levels were measured in plants expressing PR-1. Free SA concentration was 3-4 times higher than in uninfected wild-type Arabidopsis (233 ± 35 and 69 ± 8 ng / g fresh weight, respectively). SA-glucose conjugate (SAG) is 13.1-fold higher in cim3 than in uninfected Arabidopsis (4519 ± 473 and 344 ± 58 ng / g fresh weight, respectively). These elevated levels of SA and SAG are comparable to levels reported for pathogen-infected or cpr mutants.

Example 17: Disease resistance

Cim3 was evaluated for resistance to Peronospora parasitica (NoCo2), a causative agent of flour mildew in Arabidopsis. Thirty cim3 (identified by Pr-1 RNA expression) and thirty control plants (Columbia ecotype), each 4 weeks old, were inoculated with P. parasitica as described by Uknes et al. (1992, supra). Seven days later, the plants were analyzed for sporulation and stained with trypan blue to visualize fungal structures as described by Keogh et al., Trans. Br. Mycol. Soc. 74, 329-333 (1980)] and Koch and Slusarenko (Plant Cell. 2: 437-445 (1990). Wild-type (Col-O) plants supported growth of hyphae, conidia and oospores, whereas INA-treated (0.25 mg / ml) wild-type plants and cim3 plants showed no fungal growth. Cim3-regulated 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 is induced (Uknes et al. , 1992, supra). Occasionally, moderate resistance phenotypes, including creeping necrosis, have been observed to stimulate the hyphal end of cim3 plants. This creeping 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 in the cim3 plants, whereas all control plants showed sporulation. No spontaneous lesions were observed on non-inoculated cim3 leaves when stained with trypan blue.

In addition to resistance to the fungal pathogen P. parasitica, cim3 is also resistant to infection with the bacterial pathogen Pseudomonas syringae DC3000. Six-week-old wild-type (+ INA treatment) and cim3 plants were infected with a suspension of P. syringae DC3000, and the development of the disease was monitored by monitoring the growth of bacteria extracted from infected leaves over time. Differences in bacterial titers between Col-O, Col-O + INA and cim3 on day zero or day two are not statistically significant. However, by day 4, a 31-fold decrease in bacterial growth was observed between wild-type and cim3 plants (P <0.003; Sokai and Rohlf, 1981). Plants were also visually examined for these symptoms. Leaves from wild-type plants were severely chlorotic (yellowing) with symptoms of disease extending well beyond the initial zone of infection. In contrast, plants pretreated with INA or cim3 were almost free of disease symptoms.

For example, Pseudomonas syringae pv. Tomato DC3000 strains were grown in King's B medium (agar plates or liquid) at 28 ° C containing 50 pg / ml rifampicin [Walen et al., Plant Cell. 3: 49-59 (1991)]. We diluted with the overnight culture and resuspended in 10 mM MgCl ₂ in 2 to 5 x 10 ⁵ cells / ml density and is injected into Arabidopsis leaves. Injection was done by creating a small cavity in the middle of the leaf with a 28-gauge needle and then injecting about 250 μΙ diluted bacterial solution with a 1 cm syringe. At different times, 10 samples were randomly taken, consisting of 3 randomly punched leaf samples taken with a 1 cork drill, from 10 plants for each treatment. The 3 punctured leaf samples were placed in an eppendorf tube in 300 μΙ MgCl ₂ and crushed with a pestle. The resulting bacterial suspension was diluted appropriately and added to King's B medium plus rifampicin (50 pg / ml) and cultured for 4 days at 28 ° C. Bacterial colonies were counted and the data analyzed by Student's statistical analysis (Sokai and Rohlf, Biometry, 2nd edition, New York: WH Freeman and Company, 1981).

For this example, 2,6-dichloroisonicotinic acid was also suspended in sterile distilled water and formulated as a 25% active ingredient in a wettable powder [0.25 mg / ml, 325 μΜ; Kessmann et al .: Annu. Port. Phytopathol. 32: 439-59 (1994). All plants were sprayed with water or INA until confluent.

Example 18: Role of SA in SAR Gene Expression and Disease Resistance To study the relationship between SA, SAR gene expression and resistance, Arabidopsis plants expressing the salicylate hydroxylase (nahG) gene were cross-labeled in cim3 (Delaney et al., 1994). ). These "NahG plants" were produced by transforming the 35S-led nahG gene into Arabidopsis by Agrobacterium-regulated transformation. See Huang H. and Ma H., Plant Mole. Biol. Rep. 10: 372-383 (1992); Gaffney et al., 1993, Science 261: 754-756; and Delaney et al., Science 266: 1247-1250 (1994), which are incorporated herein by reference. Col-nahG in Arabidopsis carries a dominant kanamycin resistance gene in addition to the dominant nahG gene, so Col-nahG was used as a pollendor. The F1 core was hydrated in water for 30 minutes to give 38

After 222,480 Β1, the surface was disinfected with 10% Clorox and 0.5% Tween 20 for 5 minutes and rinsed thoroughly in sterile water. The seeds were then placed on germination medium (GM, Murashige and Skoog medium containing 10 g / l sucrose buffered with 0.5 g / l of 2- (N-morpholino) ethanesulfonic acid, pH 5.7 KOH) at 25 mg. / ml kanamycin for selection of F1 plants [see Valvekens et al., Proc. Natl. Acad. Sci. USA 85: 5536-5540 (1988)]. The presence of the nahG gene and PR-1 expression were confirmed by Northern blot in each experiment.

Since both the cim3 mutant and nahG phenotypes are dominant, epistasis between the two genes could be analyzed in F1 plants. Seventy F1 plants obtained from cim3xnahG crossing were analyzed for PR-1 and nahG gene expression. In the Northern blot analysis of mRNA expression, the presence of the nahG gene was correlated with the suppressed SAR gene expression. The presence of cim3 in each F1 was confirmed by measurement of PR-1 mRNA in the resulting F2 segregants.

To determine whether the cim3 mutation is epistatic to nahG in terms of disease resistance, 5 F1 plants obtained from cim3xnahG cross-linking with the presence of nahG and PR-1 mRNA were self-pollinated and 20-30 F2 seeds were planted. The mRNA expression of nahG and PR-1 was analyzed in individuals from this F2 population, which were then infected with P. parasitica (NoCo2) to assess their susceptibility to the disease. Disease resistance mediated by cim3 was eliminated in the presence of the nahG gene, demonstrating that nahG is epistatic to cim3 in terms of SAR gene expression and disease resistance phenotypes.

Example 19: Synergistic Disease Resistance Using Microbicides and / or BTH for Cim Mutants

Three days before the pathogen challenge, a 25% active ingredient (ha.) Formulation of the chemical inducer of systemic acquired resistance, benzo [1.2.3] thiadiazole-7-thiocarboxylic acid (BTH), was formulated in a wettable powder form. vehicle (Metraux et al., 1991) and / or 25% ha-containing metalaxyl microbicide (CGA 48 988) and these or the wettable powder alone were applied to the leaves of the 4-week-old plants in the form of a fine spray. Plants were inoculated with compatible Peronospora parasitica NoCo2 spore suspension (1.8x10 ⁵ spores / ml). Following infection, the plants were covered to maintain high humidity and placed in a Percival growth chamber at 17 ° C for a 14 hour day / 10 hour night cycle (Uknes et al., 1993). Tissue was harvested 8 days after infection.

Fungal growth was determined using a rRNA fungal assay obtained by PCR according to White et al. (1990; PCR Protocol: A Guide to Methods and Application, 315-322) using NS1 and NS2 primers and P. parasitica EmWa DNA. as templates. RNA was purified from frozen tissue obtained by phenol / chloroform extraction and subsequent lithium chloride precipitation (Lagrimini et al., PNAS 84: 7542-7546 (1987)). Samples (7.5 µg) were separated by electrophoresis on formaldehyde agarose gel and transferred to nylon membranes (Hybond-N +, Amersham), as described by Ausbel et al. (1987). Hybridization and washes were performed according to Church and Gilbert (PNAS 81, 1991-1995, 1984). Relative amounts of transcripts were determined using a Phosphor Imager (Molecular Dynamics, Sunnyvale, CA) following the manufacturer's instructions. The sample was normalized by testing the striped filter biotypes with continuously expressed b-tubulin Arabidopsis cDNA. Fungal infection of untreated plants corresponded to a 0% inhibition of fungal growth.

Treatment with metalaxyl alone, the plant activator BTH alone, or the combination of metalaxyl and BTH with the cim3 mutants resulted in a greater than additive, i.e. synergistic, disease resistance. This effect was defined as the synergistic factor (SF), which is the quotient of the observed (O) and the expected effect (E). The following results were obtained.

Table 33

Activity against Peronospora parasitica in Arabidopsis

Component I: cim3 mutant

Component II: Metalaxyl

Test number components Inhibition of fungal growth% SFO / E Cim3 metalaxyl O (watched) E (expected) Ref. Wt - 0 1 Cim3 - 12.5 2 Wt 12.5 mg / L 52.7 3 Wt 2.5 mg / L 0 4 Wt 0.1 mg / l 0 5 Wt 0.02 mg / l ND

HU 224 480 Β1

Table 33 (continued)

Test number components Inhibition of fungal growth% SFO / E Cim3 metalaxyl O (watched) E (expected) 6 Cim3 12.5 mg / L ND ND ND 7 Cim3 0.5 mg / L 82.2 12.5 6.6 8 Cim3 0.1 mg / l 57.8 12.5 4.6 _ Cim3 0.02 mg / l 55.6 ND ND

Wt = wild-type Col-0 ND = not determined

Table 34

Activity against Peronospora parasitica in Arabidopsis

Component I: cim3 mutant

Component II: BTH

Test number components Inhibition of fungal growth% SFO / E Cim3 BTH O (watched) E (expected) Ref. 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 5 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 35

Activity against Peronospora parasitica in Arabidopsis

Component I: cim3 mutant

Component II: BTH and metalaxyl (M)

Test number components Inhibition of fungal growth% SFO / E Cim3 BTH + M O (watched) E (expected) Ref. Wt - 0 1 Cim3 - 12.5 2 Wt BTH 0.01 mM 100 + M 0.5 mg / L 3 Wt BTH 0.01 mM 40.7 + M 0.1 mg / L 4 Wt BTH 0.01 mM ND

HU 224 480 Β1

Table 35 (continued)

Test number components Inhibition of fungal growth% SFO / E Cim3 BTH + M O (watched) E (expected) 5 Cim3 + M 0.02 mg / l BTH 0.01 mM ND 100 ND 6 Cim3 + M 0.5 mg / L BTH 0.01 mM 100 53.2 1.9 7 Cim3 + M 0.1 mg / L BTH 0.01 mM 77.7 ND ND + M 0.02 mg / l

Wt = wild-type Col-O; ND = not determined

As shown in the tables above, synergistic disease resistance effects were observed in the cim3 plants, either metalaxyl or BTH alone or in combination with metalaxyl and BTH. For example, 12.5% inhibition of fungal growth was seen in the untreated cim3 plant relative to the untreated wild-type plant; this confirms that constitutive SAR gene expression in the cim3 mutant plant is associated with patient resistance. However, as shown in Table 30, when metalaxyl was applied at a concentration of 0.0001 g / L (this concentration is normally ineffective) to the immunomodulated (SAR-on) cim3 plant, the observed level of inhibition of fungal growth was increased to 57.8%. From these data, a synergistic factor of 4.6 was calculated, which clearly demonstrates the synergistic effect achieved by the use of a microbicide in an immunomodulated plant.

The data in Table 31 show that synergism can also be achieved with a chemical inducer of systemic acquired resistance, such as BTH, in an immunomodulated (SAR-on) cim3 mutant. For example, in wild-type plants, BTH at a concentration of 0.03 mM is normally inadequate to render disease resistance effective by providing only 20.8% inhibition of fungal growth. However, in cim3 mutant plants, this normally deficient concentration of BTH inhibits 73.1% of fungal growth, which is almost as high as the 0.1 mM BTH recommended for efficacy. The synergistic factor of 2.2 calculated from the data in Table 31 clearly confirms the synergistic effect achieved with BTH on a plant that has already been immuno-modified by other means.

The effects on disease resistance were much more dramatic when BTH and metalaxyl were co-administered to cim3 plants. As shown in Example 13 (Table 29), fungal inhibition was not achieved in wild-type plants when administered alone with 0.01 mM BTH or 0.0001 g / L metalaxyl because these concentrations are normally ineffective. However, when these compounds were applied to plants at these normally ineffective concentrations, a 40.7% inhibition of fungal growth was observed, a synergistic effect compared to wild-type plants. In cim3 plants, 100% inhibition was observed when 0.01 mM BTH and 0.0001 g / L metalaxyl were co-administered, which clearly demonstrates further synergistic activity.

Thus, the combined use of immunomodulated label plants and normally ineffective low-concentration chemicals provides disease resistance, the benefits of which will be apparent to those skilled in the art. Concentrations of chemicals that are normally toxic or otherwise undesirable can be eliminated by the synergy shown here. In addition, economic benefits can be realized as a result of the reduced amount of chemicals needed to provide a certain level of plant protection.

III. Synergistic disease resistance effects achieved by the use of conventional microbicides and / or chemical inducers of systemic acquired resistance to transgenic plants containing NIM1 gene forms

A key component of the NIM1 gene is the systemic acquired resistance (SAR) pathway in plants (Ryals et al., 1996). The NIM1 gene is involved in the activation of SAR by chemical and biological inducers and is linked to such inducers necessary for SAR and SAR gene expression. The position of the NIM1 gene has already been determined by molecular biological analysis of the genome of mutant plants known to be mutant nim1

They carry a gene which renders host plants extremely sensitive to a wide range of pathogens and renders them unable to respond to pathogens and chemical inducers of SAR. The Arabidopsis NIM1 gene has been mapped and sequenced (SEQ ID NO: 1). The wild-type NIM1 gene product (SEQ ID NO: 2) is present in both the SAR and the gene-to-gene disease-resistance signal transduction cascade in Arabidopsis (Ryals et al., 1997). Recombinant intensive expression of the wild-type form of NIM1 leads to immuno-modified plants with a constitutive immunity (CIM) phenotype and therefore transfers disease resistance to transgenic plants. Increased levels of active NIM1 protein result in the same disease resistance activity as induction with chemicals such as BTH, INA and SA (see dependent U.S. Patent Application Serial No. 08 / 880,179).

Furthermore, the NIM1 gene product has been shown to be a structural homologue of the IkB mammalian signal transduction factor of the subclass (Ryals et al., 1997). Mutations in IkB have been reported to act as super-repressors or dominant-negatives of the NF-κΒ / ΙκΒ regulatory system. Thus, certain modified forms of NIM1 act as dominant-negative regulators of the SAR signal transduction pathway. These modified forms of NIM1 create opposing phenotypes in plants transformed with this nim1 mutant; that is, immunomodulatory plants that have constitutive SAR gene expression transformed with modified forms of NIM1 and have CIM phenotypes. See dependent PCT application "Methods of using the NIM1 gene to confer disease resistance in plants", which is incorporated herein by reference.

Example 20: Plant transformation of wild type

Cosmid clones containing the NIM1 gene

Cosmid D7 (deposited at ATCC 1996).

on September 25, ATCC Accession No. 97736) is derived from a clone that spans the NIM1 region and therefore includes the wild-type NIM1 gene (SEQ ID NO:

1). Cosmid E1 is also derived from a clone spanning the NIM1 region and therefore contains the wild-type NIM1 gene (SEQ ID NO: 1). The cosmids D7 and E1 were introduced into Agrobacterium tumefaciens AGL-1 by conjugate transfer in a three-parent pair using the HB101 helper strain (pRK2013) as described in U.S. Patent Application No. 08 / 880,179. These cosmids were then used to transform a kanamycin-sensitive nim1 mutant Arabidopsis line by vacuum infiltration [Mindrinos et al., Cell.

78: 1089-1099 (1994). The seed from the infiltrated plants was harvested and allowed to germinate on GM agar plates containing 50 mg / ml kanamycin as the selection agent. The seedlings that survived the selection were transferred to the soil about two weeks after plating.

Plants transferred to soil were grown in a phytotron for approx. for one week after the transfer. 300 mM INA was used in the form of a fine mist to completely cover the plants with the aid of a chromister. After two days, the leaves were harvested for RNA extraction and PR-1 expression analysis. The plants were then sprayed with Peronospora parasitica (EmWa isolate) and grown under high humidity in a growing chamber at 19 ° C daytime and 17 ° C night time in 8-hour day / 16-hour night cycles. 8-10 days after the fungal infection, the plants were evaluated and the fungal growth was scored with a positive or negative sign. Ws and nim1 plants were treated in the same manner to serve as controls in each experiment.

Total RNA from the harvested tissue was extracted with a LiCl / phenol extraction buffer [Verwoerd et al., Nuc. Acid Slit. 2362 (1989)]. RNA samples were run on formaldehyde agarose gel and blotted onto GeneScreen Plus (DuPont) membranes. The biota were hybridized with ^{20 * * * * * * * * * * * 32} P-labeled PR-1 cDNA probes. The resulting biotypes were plated to determine which transformants are capable of inducing PR-1 expression after INA treatment.

To see if the D7 and E1 transformants express NIM1 intensively due to the insertion site position, the primary transformants carrying the cosmids D7 and E1 were self-pollinated and the T2 core was harvested. Seeds from the 95 D7 line and an E1 line were planted in soil and grown as described above. When at least four true leaves were obtained on T2 plants, one leaf was harvested separately from each plant. RNA from this tissue was extracted and analyzed for PR-1 and NIM1 expression. Plants were then inoculated with P. parasitica (EmWa) and examined for fungal growth 10 days after infection. A large number of transformants showed sub-normal fungal growth, and four of them, namely lines D7-2, D7-74, D7-89 and E1-1, did not show any visible fungal growth. Plants showed higher than normal expression of NIM1 and PR-1 and marked fungal resistance, indicating that disease expression is a consequence of intensive expression of NIM1.

Example 21: Intense expression of NIM1 under its native promoter

Plants expressing the NIM1 gene constitutively were derived from the transformation of wild-type plants with the BamHI-H / ndIIII NIM1 genomic fragment (bases of SEQ ID NO: 1: 1249-5655), which contains the 1.4 kb promoter sequence. This fragment was cloned into pSGCG01 and Agrobacterium strain GV3101 [pMP90, Koncz and Shell, Mol. Gene. Genet. 204: 383-396 (1986)]. Ws plants were infiltrated as described above. The resulting seeds were harvested and placed on GM agar plates containing 50 pg / ml kanamycin. Surviving plant blues were transferred to soil as described above

HU 224,480 Β1 was tested for resistance to Peronospora parasitica Emwa isolate. Selected plants were self-pollinated and two consecutive generations were selected to produce homozygous lines. Seeds from some of these lines were planted in soil and 15-18 plants per line were grown for 3 weeks and tested for Emwa resistance without prior treatment with any chemical inducer. Approximately 24, 48 hours and 5 days after fungal treatment, tissue from each line was harvested, pooled, and frozen. Plants were left in the growth chamber for 10 days after inoculation and resistance to Emwa was then scored.

RNA was prepared from all collected samples and analyzed as described above (Delaney et al., 1995). The blot was hybridized to the Arabidopsis PR-1 gene probe (Uknes et al., 1992). Of the 13 transgenic lines tested, five showed early induction of PR-1 gene expression. In these lines, PR-1 mRNA was evident 24 or 48 hours after fungal infection. There was no visible increase in fungus at these five lines. The leaves were stained with lactophenol blue (Dietrich et al., 1994) to determine the absence of fungal hyphae in the leaves. PR-1 gene expression was not induced in the other 8 lines even after 48 hours, and these plants were not resistant to Emwa.

A subset of resistant lines was also tested for increased resistance to the bacterial pathogen Pseudomonas syringae DC3000 to determine the resistance spectrum as described by Uknes et al. (1993). These experiments were performed essentially as described by Lawton et al. (1996). Bacterial growth was lower in lines showing constitutive resistance to Emwa. This indicates that plants strongly expressing the NIM1 gene under the control of their native promoter have constitutive immunity to pathogens.

To evaluate further features of the CIM phenotype in these lines, uninfected plants were examined for free and glucose-conjugated salicylic acid, and the leaves were stained with lactophenol blue for the presence of microscopic lesions. Resistant plants were sexually crossbred with SAR mutants, such as NahG and ndr1, to determine the episthetic relationship of the resistant phenotype to other mutants and whether these dominant-negative mutants of NIM1 might influence the salicylic acid-dependent feedback mechanism.

Example 22: Intense expression of NIM1 35S

The full-length NIM1 cDNA (SEQ ID NO: 6) is pCGN1761 ENX [Comai et al .: Plant Mole. Biol. 15: 373-381 (1990)]. The resulting plasmid yielded an Xba I fragment containing the activated CaMV 35S promoter, the NIM1 cDNA in the orientation appropriate for transcription, and a tml 3 'terminator. This fragment was cloned into the binary vector pCIB200 and transformed into GV3101. Ws plants were infiltrated as described above. The resulting seeds were harvested and placed on GM agar plates containing 50 pg / ml kanamycin. Surviving plant blues were transferred to soil and tested as described above. Selected plants were self-pollinated and two consecutive generations were selected to produce homozygous lines. Nine of 58 lines were tested for resistance when treated with Emwa without prior chemical treatment. Thus, intensive expression of NIM1 cDNA also results in disease resistance in plants.

Example 23: NIM1 is a homologue of ΙκΒα

The sequence sequences of the NIM1 and IkB protein gene products were prepared and it was determined that the NIM1 gene product is a homologue of ΙκΒα (Fig. 1). Sequence homology research was performed by BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)]. Multiple sequence sequences were constructed using the Clustal V program (Higgins et al. (1989) CABIOS 5: 151-153) as part of the Lasergene Biocomputing Software package available from DNASTAR (Madison, WI). The sequences were as follows: NIM1 (SEQ ID NO: 2), mouse Ικ SEα (SEQ ID NO: 3, GenBank accession No. 1022734), rat ΙκΒα [SEQ ID NO: 4, GenBank accession No. 57674 and X63594; Tewari et al .: Nucleic Acids Slit. 20, 607 (1992)] and porcine ΙκΒα [SEQ ID NO. 5, GenBank Accession No. Z21968; de Martin et al. (1993) EMBO J. 12, 2773-2779; GenBank Accession No. 517193, de Martin et al., 1995, Gene 152: 253-255. The parameters used in the Clustal analysis were 10 error points and 10 error points. Evolutionary divergence distances were calculated using the PAM250 mass table [Dayhoff et al., A Model of Evolutionary Change in Proteins. Matrices for Detecting Distant Relationships ”. Atlas Protein Sequence and Structure, Vol. 5, Suppl. 3, Dayhoff Μ. O. (published by National Biomedical Research Foundation, Washington, D.C.), pp. 345-358. (1978); Gribskov and Burgess: Nucleic Acids Slit. 14: 6745-6763 (1986)].

Homology research has shown that NIM1 is similar to ankyrin domains of certain proteins, including ankyrin, NF-κB and ΙκΒ. The most comprehensive is homology to ΙκΒ and related molecules (Fig. 1). NIM1 contains two positions at positions 55 and 59; serine at position 59 is located in the D / ExxxxS environment and at the N-terminal, in agreement with its role in phosphorylation-dependent, ubiquitin-regulated, inducible degradation. All ΙκΒα possess these N-terminal serines, which are required for the inactivation of IkB and subsequent release of NF-κΒ. The ankyrin domains of NIM1 are shown in pages 263-290. 323-371. amino acids. Ankyrin domains are believed to play a role in protein-to-protein interaction43

They have a ubiquitous property toward the IkB and NF-κΒ molecules. The C-terminals of ΙκΒs may be different. NIM1 is also somewhat homologous to the QL-rich region (amino acids 491-499), which is located at the C-terminus of some IkBs.

Example 24: Generation of modified forms of NIM1 in which serines 55 and 59 were replaced with alanine

In human ΙκΒα, phosphorylation of serine residues is required for stimulatory activation of IkB, resulting in the activation of NF-κΒ. Mutagenesis of serine residues (S32-S36) in human ΙκΒα to alanine residues inhibits stimulus-induced phosphorylation, thereby blocking the proteosome-regulated degradation of ΙκΒα [Britta-Mareen Traenckner E. et al., EMBO J. 14, 2967-2883 (1995); Brown et al. (1996) Science 267: 1485-1488; Brockman et al. (1995) Molecular and Cellular Biology 15: 2809-2818; Wang et al., Science 274: 784-787 (1996)].

This altered form of ΙκΒα functions as a dominant-negative form, retaining NF-κB in the cytoplasm, thereby blocking downstream signaling. Based on sequence comparison between NIM1 and IkB, serine 55 (S55) and serine 59 (S59) are homologous to human ΙκΒα serine 32 (S32) and 36 (S36). To create dominant-negative forms of NIM1, serines 55 and 59 are mutagenized to alanine residues. This can be accomplished by any method known to those skilled in the art, such as the QuikChange Site Directed Mutagenesis Kit (No. 200518, Strategene).

Using a full-length NIM1 cDNA (SEQ ID NO: 6) containing the 42 bp untranslated UTR sequence and the 187 bp 3 'UTR sequence, the mutagenized construct can be constructed using the following primers (SEQ ID NO: 6, Positions 192-226): 5'-CAA CAG CTT CGA AGC CGT CTT TGA CGC GCC GGA TG-3 '(SEQ ID NO: 25) and 5'-CAT CCG GCG CGT CAA AGA CGG CTT CGA AGC TGT TG- 3 '(SEQ ID NO: 26), where the underlined bases indicate the mutation. The strategy was to clone the NIM1 cDNA into vector pSE936 [Elledge et al., Proc. Natl. Sci. USA 88: 1731-1735 (1991)] and primers containing altered bases were heat-treated. DNA polymerase (Pfu) was extended to the primers by non-strand replacement to give "nicked" circular filaments. The DNA was subjected to Dpn1 restriction endonuclease digestion, which cleaves only at the methylated sites (non-mutagenized template DNA). The remaining circular dsDNA was transformed into E. coli strain XL1-Blue. From the colonies thus obtained, the plasmids were extracted and sequenced for the presence of the mutated bases and for the absence of any other mutation.

The mutagenized NIM1 cDNA was digested with EcoRI restriction endonuclease and cloned into pCGN1761 under transcriptional control of the cauliflower mosaic virus dual 35S promoter. The transformation cassette containing the 35S promoter, NIM1 cDNA and tml terminator was recovered from PCGN1761 by partial restriction digestion with XbaI and ligated into the Xba I site of the dephosphorylated pCIB200. SEQ ID NOs: 7 and 8 show the DNA coding sequence and the coded amino acid sequence of the modified form of the NIM1 gene.

Example 25: Construction of N-terminal Deletion Modified NIM1 Forms of human ΙκΒα as shown in Figures 1-36. amino acid deletion (Brockman et al., Hedet et al.); deletion of amino acids (Sun et al.), which include the amino acid residues K21, K22, S32 and S36, results in a dominant-negative ΙκΒα phenotype in transferred human cell cultures. The N-terminal deletion of approximately 125 first amino acids of the product encoded by the NIM1 cDNA removes 8 lysine residues that serve as potential ubiquitination sites and also removes putative phosphorylation sites (S55 and S59) (see Example 2). This modified gene construct can be generated by any known method. For example, using the method of Ho et al., Gene 77, 51-59 (1989), it is possible to form a NIM1 in which approx. DNA encoding the first 125 amino acids was deleted. The following primers resulted in a 1612 bp PCR product (SEQ ID NO: 6: 418-2011): 5'-gg aa tca-ATG GAT TCG GTT GTG ACT GTT TTG-3 '(SEQ ID NO: 27) '-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 the EcoRI linker sequence is written in lowercase. The following reaction mixture was used to amplify the fragments: 0.1-100 ng DNA template, 10 mM Tris pH 8.3 / 50 mM KCl / 2 mM MgCl ₂ / 0.001% gelatin / 0.25 mM each dNTP / 0.2 mM each primer and 1 unit rTth DNA polymerase in a final volume of 50 ml and a Perkin Elmer Cetus 9600 PCR apparatus. The PCR parameters were as follows: 94 ° C for 3 min: 35 * (94 ° C for 30 s: 52 ° C for 1 min: 72 ° C for 2 min): 72 ° C for 10 min. The PCR product was directly cloned into vector pCR2.1 (Invitrogen). The PCR insert was released in the PCR vector by EcoRI restriction endonuclease digestion and ligated to the EcoRI site of the dephosphorylated pCGN1761 under transcriptional control of the double 35S promoter. The resulting construct was sequenced to confirm the presence of the synthetic ATG start codon and to make sure no other mutations occurred during the PCR. The transformation cassette containing the 35S promoter, the modified NIM1 cDNA and the tml terminator was released by partial restriction digestion with XbaI and ligated into the Xba I site of pCIB200. SEQ ID NOs: 9 and 10 show the DNA coding sequence and the encoded amino acid sequence of a modified form of the NIM1 gene carrying the N-terminal deletion.

HU 224 480 Β1

Example 26: Creating modified forms of NIM1 containing a C-terminal deletion

It is believed that human ΙκΒα 261-317. deletion of the amino acids at position C-2 results in enhanced internal stability by inhibiting the constitutive phosphorylation of the serine and threonine residues at the C-terminus. NIM1 at the C-terminus is shown in pages 522-593. amino acid residues present a serine and threonine rich region. The region encoding the C-terminus of the NIM1 gene can be modified as described in pages 522-593. by deleting nuleotides encoding amino acids. Using the method of Ho et al. (1989), the C-terminal coding region and the 3 'UTR region of NIM1 CDNS (SEQ ID NO: 6: 1606-2011) were deleted by PCR, creating a 1623 bp fragment with the following primers: cggaattcGATCTCTTTAATTTGTGAATTT C-3 '(SEC ID NO: 29) and 5'-qqaattcTCAACAGTT CATAATCTGGTCG-3' (SEQ ID NO: 30), in which the synthetic stop codon is underlined (TGA on the complementary strand) and the EcoRI linker. The components of the PCR reaction were as described above and the cyclization parameters were as follows: 94 ° C for 3 min: 35 (94 ° C for 30 s: 52 ° C for 30 s: 72 ° C for 2 min): 72 ° C for 10 min. The PCR product was directly cloned into vector pCR2.1 (Invitrogen). The PCR insert was released in the PCR vector by EcoRI restriction endonuclease digestion and ligated into the EcoRI site of the dephosphorylated pCGN1761 containing the double 35S promoter. The resulting construct was sequenced to confirm the presence of the synthetic open frame stop codon and to make sure no other mutations occurred during the PCR. The transformation cassette containing the 35S promoter, the modified NIM1 cDNA and the tml terminator was released by partial restriction digestion with XbaI and ligated into the Xba I site of pCIB200. SEQ ID NOs: 11 and 12 show the DNA coding sequence and the encoded amino acid sequence of a modified form of the NIM1 gene carrying the C-terminal deletion.

Example 27: Creating Modified Chimeric Forms of NIM1 Containing N-terminal / C-Term Deletion

The N-terminal and C-terminal deletions of NIM1 were prepared using the unique Kpn1 restriction site (SEQ ID NO: 6) at position 819. The N-terminal deletion form (Example 25) was cleaved with Eco RI / Kpn I restriction endonucleases and the 415 bp fragment corresponding to the modified N-terminal was recovered by gel electrophoresis. Similarly, the C-terminal deletion form (Example 26) was cleaved with EcoRI / KpnI restriction endonucleases and the 790 bp fragment corresponding to the modified C-terminal was recovered by gel electrophoresis. The fragments were ligated at 15 ° C, digested with Eco RI to eliminate Eco RI, and cloned into the Eco RI site of dephosphorylated pCGN1761. The N / C-terminal deletion form of NIM1 is under transcriptional control of the double 35S promoter. Similarly, the chimeric form of NIM1 containing the putative phosphorylation sites of S55 / S59 (Example 24) was fused to the C-terminal deletion form (Example 26). This structure was prepared as described above. The resulting constructs were sequenced to confirm the presence of start and stop codons and that no further mutations occurred during cloning. Transformation cassettes containing the 35S promoter, NIM1 chimera and tml terminator were released by partial restriction digestion with XbaI and ligated into the Xba I site of pCIB200. SEQ ID NO; 13 and NO: 14 show the DNA coding sequence and the encoded amino acid sequence of a modified form of the NIM1 gene carrying both N-terminal and C-terminal deletions.

Example 28: Creation of modified forms of NIM1 containing ankyrin domains

NIM1 exhibits homology to ankyrin motifs of about 103-362. amino acids. Using the method of Ho et al. (1989), the DNA sequence encoding putative ankyrin domains (SEQ ID NO: 1: 3093-3951) was obtained from the NIM1 cDNA (SEQ ID NO: 6: 349-1128) by PCR. (conditions: 94 ° C for 3 min: 35 ° (94 ° C for 30 s: 72 ° C for 2 min): 72 ° C for 10 min) using the following primers: 5'-qqaattcaATGGCTCAACAACACCGCCGC-3 '(SEQ ID NO: 31) and 5'-qqaattcTCAACCTTCCAAAGTTGCTTCTGATG-3 '(SEQ ID NO: 32). The resulting product was digested with EcoRI restriction endonuclease and then ligated into the EcoRI site of dephosphorylated pCGN1761 under transcriptional control of the double 35S promoter. The resulting construct was sequenced to confirm the presence of the synthetic ATG start codon and the open frame TGA stop codon and to make sure no other mutations occurred during the PCR. The transformation cassette containing the 35S promoter, the ankyrin domains, and the tml terminator was released by partial restriction digestion with XbaI and ligated into the Xba I site of pCIB200. SEQ ID NOs: 15 and NO: 16 show the DNA sequence encoding the ankyrin domain of the NIM1 gene and the encoded amino acid sequence.

Example 29: Construction of chimeric genes

A 4407 bp HindIII / BamHI fragment (bases of SEQ ID NO: 1: 1249-5655) and / or a 5655 bp EcoRV / BamHI fragment (SEQ ID NO: 1: 1) were used to enhance the appropriate static and temporal expression of modified NIM1 forms. Bases -5655), which contained the NIM1 promoter and gene, were used as described in paragraphs 24-28. to create the modified NIM1 forms described in the examples. Although the design steps may differ, the concepts are similar to those described above. Strong overexpression of modified forms can be potentially deadly. Therefore, NIM1 gene 24-28. The modified forms described in Examples 1 to 4 were placed under the control of promoters other than the endogenous NIM1 promoter, including but not limited to the nos promoter or a small subunit of the Rubisco promoter. Ha45

Similarly, modified NIM1 forms may also be expressed under the control of the pathogen-sensitive PR-1 promoter (U.S. Patent No. 5,614,395). Such expression allows for strong expression of modified NIM1 forms only under pathogen attack or other SAR activating conditions. Further, disease resistance may develop in transformants expressing modified NIM1 forms under the control of the PR-1 promoter when treated with concentrations of SAR activating compounds (such as BTH or INA) that normally do not activate SAR, thereby activating the feedback loop [ Weymann et al .: Plant Cell. 7, 2013-2022 (1995)].

Example 30: Transformation of modified forms of NIM1 into Arabidopsis thaliana

24-29. The constructs constructed in Example 1A were introduced into Agrobacterium tumefaciens strain GV3101 by electroporation. These constructs were then used to transform the Arabidopsis Col-O and Ws-O ecotypes by vacuum infiltration [Mindrinos et al., Cell. 78, 1089-1099 (1994)] or standard root transformation. Seeds from these plants were harvested and allowed to germinate on agar plates containing kanamycin (or another suitable antibiotic) as the selection agent. Selection of surviving seedlings was transferred to soil and tested for the CIM (constitutive immunity) phenotype. Observed phenotypic differences were evaluated in comparison to wild-type plants.

Example 31: Evaluation of the CIM phenotype in plants transformed with the wild-type NIM1 gene or a modified form of the NIM1 gene A leaf was isolated from each of the first transformants, RNA isolated [Verwoerd et al., Nuc. Acid Slit. 2362 (1989)] and tested by RNA blot for constitutive PR-1 expression (Uknes et al., 1992). Individual transformants were tested for enhanced disease resistance response as a sign of constitutive SAR expression analysis (Uknes et al., 1992). Two compatible P. parasitica isolates were prepared at 5-10 x 10 ⁴ spores / ml conidial suspension (i.e., these fungal strains cause disease on wild-type Ws-0 and Col-0 plants) and the transformants were sprayed with a suitable isolate depending on the transformant ecotype. The infected plants were incubated at high humidity for 7 days. The degree of disease in the plants at day 7 was determined by single leaf RNA blot analysis with a fungal infection assay.

Transformants that were CIM phenotypes were fitted to the T1 generation and homozygous plants were identified. The transformants were subjected to the disease resistance test series described below. The fungal infection was repeated with Noco and Emwa and the leaves were stained with lactophenol blue to detect the presence of fungal hyphae (Dietrich et al., 1994). The transformants were infected with a bacterial pathogen, Pseudomonas syringae

DC3000 to evaluate the resistance spectrum as described by Uknes et al. (1993). In uninfected plants, both free and glucose bound SA were determined and the leaves were stained with lactophenol blue for microscopic lesions. Resistant plants were sexually crossbred with SAR mutants, such as NahG (US 5,614,395) and ndrf, to examine the epistatic relationship of the resistant phenotype with other mutants and to determine how these dominant-negative mutants affect the SA-dependent feedback loop. .

Example 32: Isolation of NIM1 homologs

NIM1 homologs can be obtained by hybridizing under moderate stringency to at least 10 consecutive nucleotides of the entire NIM1 gene from Arabidopsis, or preferably an oligonucleotide probe from the Arabidopsis NIM1 gene. Factors that influence the stability of hybrids determine the stringency of hybridization. One such factor is the melting temperature T _m which is easily calculated according to the specified DNA PROBES In formula (George H. Keller and Mark M. Manak, Macmillan Publishers Ltd, 1993, Section one: Molecular Hybridization Technology; page 8 ff). The preferred hybridization temperature is about 10 minutes. C compared to 25 ° in the range below the calculated melting temperature _Tm, preferably 12-15 ° C below the T _m below, and by 5 to 10 ° C below the T _m for oligonucleotides.

Using NIM1 cDNA (SEQ ID NO: 6) as a probe, Arabidopsis NIM1 homologs can be identified by screening genomic or cDNA libraries from different crop plants; these are listed but are not limited to Example 33. DNA libraries [or plaques or colonies; see, for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989), including hybridization by standard techniques and PCR amplification with oligonucleotides (see, e.g., Innis et al., PCR Protocols, Guide to Methods and Applications, Academic Press (1990)]. Homologues are identified by introducing it into an expression vector by genetic engineering and transforming it into the crop plants listed above. Transformants are assayed for increased disease resistance using the plant-relevant pathogens to be tested.

NIM1 homologs were detected in the genome of cucumber, tomato, tobacco, maize, wheat and barley by DNA blot analysis. Genomic DNA was isolated from cucumber, tomato, tobacco, corn, wheat, and barley by digestion with BamHI, HindIII, Xbal or SalI restriction enzymes, electrophoretically separated on 0.8% agarose gels, and transferred to a nylon membrane. Subsequently, the DNA was fixed by UV crosslinking, the membrane was hybridized under less stringent conditions46

HU 224,480 [1 [(1% BSA; 520 mM NaPO ₄ , pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride) at 55 ° C for 18-24 hours] ³² P-radiolabeled Arabidopsis thaliana NIM1 cDNA. Following hybridization, the biota were washed under mild conditions [6 * SSC for 15 min (3X), 3 * SSC for 15 min (X1) at 55 ° C; 1 * SSC 0.15 M NaCl, 15 mM Na citrate (pH 7.0)] and exposed to X-ray film to visualize bands corresponding to NIM1.

In addition, expressed sequence fags (ESTs) similar to the NIM1 gene can be used to isolate homologs. For example, some rice expressed sequence sequences (ESTs) have been identified based on similarity to the NIM1 gene. Multiple sequence sequences were constructed using Clustal V (Higgins, Desmond G. and Sharp PM (1989), Fast and Sensitive Parallel Sequences on a Microcomputer, CABIOS 5, 151-153), which is part of the DNA * (1228 South Park Street, Madison Wisconsin). , 53715) to the Lasergene Biocomputing Software package (Macintosh, 1994). Certain regions of the NIM1 protein are homologous to the amino acid sequence of 4 different rice cDNA protein products. Homologies were identified by NIM1 sequences in a GenBank BLAST study. Comparison of homologous regions in NIM1 and rice cDNA products is shown in Figure 2 (see also SEQ ID NO: 2 and SEQ ID NO: 17-24). NIM1 protein fragments are 36-48% identical to the amino acid sequence of the 4 rice products. These rice ESTs can be advantageously used to isolate NIM1 homologs from other monocotyledons.

Homologs can also be obtained by PCR. In this method, comparisons are made between known homologs (e.g., rice and Arabidopsis). Substantially similar or identical amino acid and DNA regions are then used as PCR primers. The amino acid residue regions M and W are most closely followed by the amino acid residue regions F, Y, C, H, Q, K and E, since these amino acids are encoded by a limited number of codons. One such suitable region was identified using primers having a diversity of substitution at the third codon position. In the third position, this diversity of substitution may be forced, depending on the target species. Thus, for example, since maize is rich in GC, primers have been designated to utilize a G or C in the third position, if possible. The PCR reaction was performed from cDNA or genomic DNA under various standard conditions. Once a band has appeared, it has been cloned and / or sequenced to determine whether it is a NIM1 homologue.

Example 33: Expression of a form of NIM1 in crop plants

In Col-O or Ws-O, constructs conferring the TITLE phenotype were transformed into crop plants for evaluation. Alternatively, the modified native NIM1 genes isolated from the plants obtained in the previous example were returned to the respective plants. Although the NIM1 gene can be introduced into any broad class of plant cells, the cells of the following plants may be advantageously used, such as rice, wheat, barley, rye, oats, potatoes, carrots, sweet potatoes, sugar beets, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip (parsnips), radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, spoon, pumpkin, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, autumn , nectarines, apricots, strawberries, grapes, raspberries, blackberries, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugar cane. Transformants were tested for increased disease resistance. In a preferred embodiment of the invention, the expression of the NIM1 gene is at a level that is at least twice that of the native NIM1 gene in wild-type plants, and is preferably ten times higher than that of the wild-type expression.

Example 34: Synergistic Disease Resistance Obtained by Application of a Standard Microbicide to Transgenic Plants Highly Expressing NIM1

The plant lines (6E and 7C) used in this example are derived from the transformation of wild-type Arabidopsis thaliana plants (Ws ecotype) with the BamHI-HindIII NIM1 genomic fragment (bases of SEQ ID NO: 1: 1249-5655) as in Example 21. We described. Metalaxyl, fosetyl, and copper hydroxide fungicides were formulated with 25%, 80%, and 70% active ingredient (ha.) In a wettable powder carrier and applied as a fine spray to leaves of 3-week-old transgenic Ws plants, which continuously expressed NIM1 gene. The wettable powder was used alone as a control. Three days later, the plants suspension Emwa (1-2 x 10 ⁵ spores / ml) was inoculated with Peronospora parasitica isolate spores as described by Delaney et al (1995). Following inoculation, the plants were covered to maintain high humidity and placed in a Percival growing chamber at 17 ° C for a 14 hour day / 10 hour night cycle (Uknes et al., 1993). Tissue was harvested 8 days after inoculation.

The progression of fungal growth was monitored for 12 days under a preparative microscope to score for the development of conidiophores [Delaney et al. (1994); Dietrich et al. (1994)]. Staining each leaf with lactophenol-trypan blue, we observed fungal growth within the leaf tissue. Fungal growth was quantified using an rRNA fungal assay obtained by PCR according to White et al. (1990; PCR Protocols: A Guide to Methods and Application, 315-322), NS1 and NS2 primers, and P. parasitica EmWa DNA. applied as templates. RNA was purified from frozen tissue obtained by phenol / chloroform extraction and subsequent lithium chloride precipitation (Lagrimini et al., PNAS 84: 7542-7546 (1987)). Samples (7.5 pg) were separated by electrophoresis on formaldehyde agarose gel and transferred to nylon47

HU 224,480 Β1 membranes (Hybond-N +, Amersham), as described by Ausbel et al. (1987). Hybridization and washes were performed according to Church and Gilbert (PNAS 81: 1991-1995, 1984). Relative amounts of transcripts were determined using a Phosphor Imager (Molecular Dynamics, Sunnyvale, CA) following the manufacturer's instructions. The sample was normalized by testing the striped filter biotypes with continuously expressed b-tubulin Arabidopsis cDNA. Fungal infection of untreated plants corresponded to a 0% inhibition of fungal growth.

The use of metalaxyl, fosetyl, or copper hydroxide on plant lines expressing NIM1 strongly resulted in greater than additive, i.e. synergistic, disease resistance. This effect was characterized by the synergistic factor (SF), which is the quotient of the observed effect (0) and the expected effect (E). The following results were obtained.

Table 36

Activity against Peronospora parasitica in Arabidopsis

Component I: NIM1 Strong Expression (Line 6E)

Component II: Metalaxyl

Test number components Inhibition of fungal growth% SFO / E NIM1 metalaxyl O (watched) E (expected) Ref. Wt - 0 1 NIM1 - 10 2 Wt 0.0125 g / l 59 3 Wt 0.0012 g / l 27 4 NIM1 0.0125 g / l 76 69 1.1 5 NIM1 0.0012 g / l 56 37 1.5

Wt = wild type Ws

Table 37

Activity against Peronospora parasitica in Arabidopsis

Component I: NIM1 Strong Expression (Line 6E)

Component II: fosetyl

Test number components Inhibition of fungal growth% SFO / E NIM1 fosetyl O (watched) E (expected) Ref. Wt - 0 1 NIM1 - 10 2 Wt 5.0 g / l 7 3 Wt 0.5 g / l 1 4 Wt 0.05 g / l 0 5 NIM1 5.0 g / l 93 17 5.5 6 NIM1 0.5 g / l 83 12 6.9 7 NIM1 0.05 g / l 42 10 4.2

Wt = wild type Ws

Table 38

Activity against Peronospora parasitica in Arabidopsis

Component I: NIM1 High Expression (Line 7C)

Component II: fosetyl

Test number components Inhibition of fungal growth% SFO / E NIM1 fosetyl O (watched) E (expected) Ref. Wt - 0 1 NIM1 - 14

HU 224 480 Β1

Table 38 (continued)

Test number components Inhibition of fungal growth% SFO / E NIM1 fosetyl O (watched) E (expected) 2 Wt 5.0 g / l 7 3 Wt 0.5 g / l 2 4 NIM1 5.0 g / l 80 21 3.8 5 NIM1 0.5 g / l 56 16 3.5

Wt = wild type Ws

Table 39

Activity against Peronospora parasitica in Arabidopsis

Component I: NIM1 Strong Expression (Line 6E)

Component II: Copper hydroxide

Test number components Inhibition of fungal growth% SFO / E NIM1 Cu (OH) ₂ O (watched) E (expected) Ref. Wt - 0 1 NIM1 - 10 2 Wt 2.0 g / l 0 3 Wt 0.2 g / l 0 4 Wt 0.02 g / l 0 5 NIM1 2.0 g / l 66 10 6.6 6 NIM1 0.2 g / l 14 10 1.4 7 NIM1 0.02 g / l 20 10 2.0

Wt = wild type Ws

Table 40

Activity against Peronospora parasitica in Arabidopsis

Component I: NIM1 High Expression (Line 7C)

Component II: Copper hydroxide

Test number components Inhibition of fungal growth% SFO / E NIM1 Cu (OH) ₂ O (watched) E (expected) Ref. Wt - 0 1 NIM1 - 14 2 Wt 2.0 g / l 0 3 Wt 0.2 g / l 0 4 Wt 0.02 g / l 0 5 NIM1 2.0 g / l 77 14 5.5 6 NIM1 0.2 g / l 51 14 3.6 7 NIM1 0.02 g / l 55 14 3.9

Wt = wild type Ws

As shown in the tables above, synergistic disease resistance was demonstrated in plants expressing NIM1 intensively with metalaxyl, fosetyl and copper hydroxide. For example, untreated NIM1 (line 6E) showed 10% fungal growth inhibition compared to untreated wild type; this demonstrates that constitutive SAR gene expression in this NIM1 overexpressor is associated with disease resistance. However, as shown in Table 37, fosetyl is 5.0 g / l

When applied at a concentration of 480 Β1 (normally inactive) to a plant overexpressing immunomodulated (SAR-οπ) NIM1, the observed level of inhibition of fungal growth was increased to 93%. From these data, a synergistic factor of 5.5 was calculated, which clearly confirms the synergistic effect achieved by the use of a microbicide on an immunomodulated plant (SAR). In another example, the untreated NIM1 plant (line 7C) showed a 14% inhibition of fungal growth compared to the untreated wild type, demonstrating that constitutive SAR gene expression in this NIM1 overexpressor was associated with disease resistance.

However, as shown in Table 40, when applied at a concentration of 2.0 g / l copper hydroxide (normally inactive) to a plant overexpressing immunomodulated (SAR-οπ) NIM1, the observed level of inhibition of fungal growth was increased to 77%. . From these data, a synergistic factor of 5.5 was calculated, which clearly confirms the synergistic effect achieved by the use of a microbicide on an immunomodulated plant (SAR).

Thus, the combination of immunomodulated plants overexpressing NIM1 with low levels of normally ineffective microbicides provides disease resistance, the benefits of which will be apparent to those skilled in the art. High concentrations of microbicides that are normally toxic or otherwise undesirable can be eliminated by the advantage of synergism presented herein. In addition, economic benefits can be realized from the reduced amounts of microbicides required to achieve a given level of plant protection.

Example 35 Synergistic Disease Resistance Obtained to Transgenic Plants Highly Expressing NIM1 Using 40 Chemical Inducers of SAR The transgenic plants containing the NIM1 genomic DNA fragment under its own promoter (Example 21) were also tested for different concentrations of BTH. responses to wild-type Ws line 45. Seeds were sown from each line and allowed to grow as described above. After approximately 3 weeks, leaf samples were collected from each line (0 day controls) and the remaining plants were treated with water, 10 μΜ BTH or 100 μΜ BTH. Further samples were collected at 1, 3, and 5 na10 post-treatment. On the day after collection, 3 samples, one subunit of the plants for each line, were removed and treated with Peronospora parasitica Emwa isolate as described above. RNA was harvested from the harvested tissue and subjected to Northern analysis using the Arabidopsis PR-1 gene probe. Plants were scored for fungal resistance 8 days after infection.

The results of the Northern analysis for the Ws and the four NIM1 overexpressing lines (3A, 5B, 6E and 7C) are shown in Figure 3. PR-1 gene expression in the wild-type Ws line is barely detectable after low-level 10 μΜ BTH treatment (normally 100-300 μΜ BTH concentration is required for effect). Plants resulting from this treatment are still susceptible to P. parasitica (Emwa) fungal infection. However, all lines expressing strongly NIM1 gave a much stronger response to PR-1 gene expression after low-level BTH treatment. Furthermore, all lines expressing NIM1 intensively, treated with 10 μΜ BTH, showed complete or almost complete resistance to P. parasitica. The leaves were stained with lactophenol blue to identify the presence of fungal hyphae (Dietrich et al., 1994), which is consistent with the lack of fungal growth in NIM1 overexpressing lines. PR-1 gene expression in leaf tissue after 100 μΜ BTH treatment is also much stronger and faster in NIM1 overexpressing lines compared to wild type. Therefore, immunomodulated plants are able to respond much faster to much lower doses of BTH, as shown by PR-1 gene expression and P. parasitica resistance than wild-type. These data demonstrate that synergistic disease resistance has been achieved by applying a chemical inducer of systemic acquired resistance (SAR), such as BTH, to an immuno-modified plant (SAR), such as a plant overexpressing NIM1.

Thus, the combination of immunomodulated plants overexpressing NIM1 with low levels of normally ineffective SAR inducers, such as BTH, provides disease resistance, the benefits of which will be apparent to those skilled in the art. Concentrations of chemicals that are normally toxic or otherwise undesirable for SAR can be eliminated by the benefit of synergism presented herein. Further, economic benefits can be realized from the reduced amounts of SAR inducers required to achieve a given level of plant protection.

SEQUENCE LIST (1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Novartis AG (B) STREET: Schwarzwaldallee 215 (C) CITY: Basel (E) COUNTRY: Switzerland (F) POSTAL CODE (ZIP): 4002

HU 224,480 Β1 (G) TELEPHONE: +41 61 69 11 11 (H) TELEFAX: +41 61 696 79 76 (I) TELEX: 962 991 (v) COMPUTER READABLE FORM:

(A) MEDIA ΤΥΡΕ: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Release # 1.0, Version # 1.30 (ii) TITLE OF INVENTION: METHOD FORUM PROTECTING PLANTS (iii) NUMBER OF SEQUENCES: 32 (2) INFORMATION FORUM SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5655 base pairs (Β) ΤΥΡΕ: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE ΤΥΡΕ: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:

(A) NAME / KEY: exon (B) LOCATION: 2787 ... 3347 (D) OTHER INFORMATION: / product = "1 st exon of NIM1" (ix) FEATURE:

(A) NAME / KEY: exon (B) LOCATION: 3427 ... 4162 (D) OTHER INFORMATION: / product = 2nd exon of NIM1 (ix) FEATURE:

(A) NAME / KEY: exon (B) LOCATION; 4271 ... 4474 (D) OTHER INFORMATION: / product = 3rd Exon of NIM1 (ix) FEATURE:

(A) NAME / KEY: exon (B) LOCATION: 4586 ... 4866 (D) OTHER INFORMATION: / product = "4th exon of NIM1" (ix) FEATURE:

(A) NAME / KEY: CDS (B) LOCATION: Good (2787 ... 3347, 3427 ... 4162, 4271 ... 4474. 4586 ... 4866) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :

TGTGATGCAA GTCATGGGAT ATTGCTTTGT GTTAAGTATA CAAAACCATC ACGTGGATAC 60

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

TTCTTCCATA CTCCGAACTC TGATGTTTTC TCAGGATTAG TCAGATACGA AGGGAAGCTA 480

GGTGCTATTC GTCAGTGGAC AAACAAAGAT CAAGAAGATG TTCACGAGTT ATGGGTTTTA 540

AAGAGCAGTT TTGAAAAGTC GTGGGTTAAA GTGAAAGATA TTAAAAGCAT TGGAGTAGAT 600

TTGATTACGT GGACTCCAAG CAACGACGTT GTATTGTTTC GTAGTAGTGA TCGTGGTTGC 660

CTCTACAACA TAAACGCAGA GAAGTTGAAT TTAGTTTATG CAAAAAAAGA GGGATCTGAT 720

TGTTCTTTCG TTTGTTTTCC GTTTTGTTCT GATTACGAGA GGGTTGATCT GAACGGAAGA 780

AGCAACGGGC CGACACTTTA AAAAAAAAAT AAAAAAAATG GGCCGACAAA TGCAAACGTA 840

GTTGACAAGG ATCTCAAGTC TCAAGTCTCA ATTGGCTCGC TCATTGTGGG GCATAAATAT 900

ATCTAGTGAT GTTTAATTGT TTTTTATAAG GTAAAAAGGA ATATTGAATT TTGTTTCTTA 960

GGTTTATGTA ATAATACCAA ACATTGTTTT ATGAATATTT AATCTGATTT TTTGGCTAGT 1020

TATTTTATTA TATCAAGGGT TCCTGTTTAT AGTTGAAAAC AGTTACTGTA TAGAAAATAG 1080

TGTCCCAATT TTCTCTCTTA AATAATATAT TAGTCATTAAT AAGATATTTT AATATATTAG 1140

ATATACATAA TATCTAAAGC AACACATATT TAGACACAAC ACGTAATATC TTACTATTGT 1200

TTACATATAT TTATAGCTTA CCAATATAAC CCGTATCTAT GTTTTATAAG CTTTTATACA 1260

HU 224 480 Β1

ATATATGTAC GGTATGCTGT CCACGTATAT ATATTCTCCA AAAAAAACGC ATGGTACACA 1320 AlAATTTATTA AATATTTGGC AATTGGGTGT TTATCTAAAG TTTATCACAA TATTTATCAA 1380 CTATAATAGA TGGTAGAAGA TAAAAAAATT ATATCAGATT GATTCAATTA AATTTTATAA 1440 TATATCATTT TAAAAAATTA ATTAAAAGAA AACTATTTCA TAAAATTGTT CAAAAGATAA 1500 TTAGTAAAAT TAATTAAATA TGTGATGCTA TTGAGTTATA GAGAGTTATT GTAAATTTAC 1560 TTAAAATCAT AGAAATCTTA TCCTAATTTA ACTTATCATT TAAGAAATAC AAAAGTAAAA 1620 AACGCGGAAA GCAATAATTT ATTTACCTTA TTATAACTCC TATATAAAGT ACTCTGTTTA 1680 TTCAACATAA TCTTACGTTG TTGTATTCAT AGGCATCTTT AACCTATCTT TTCATTTTCT 1740 GATCTCGATC GTTTTCGATC CAACAAAATG AGTCTACCGG TGAGGAACCA AGAGGTGATT 1800 ATGCAGATTC CTTCTTCTTC TCAGTTTCCA GCAACATCGA GTCCGGAAAA CACCAATCAA 1860 GTGAAGGATG AGCCAAATTT GTTTAGACGT GTTATGAATT TGCTTTTACG TCGTAGTTAT 1920 TGAAAAAGCT GATTTATCGC ATGATTCAGA ACGAGAAGTT GAAGGCAAAT AACTAAAGAA 1980 GTCTTTTATA TGTATACAAT AATTGTTTTT AAATCAAATC CTAATTAAAA AAATATATTC 2040 ATTATGACTT TCATGTTTTT AATGTAATTT ATTCCTATAT CTATAATGAT TTTGTTGTGA 2100 AGAGC GTTTT CATTTGCTAT AGAACAAGGA GAATAGTTCC AGGAAATATT CGACTTGATT 2160 TAATTATAGT GTAAACATGC TGAACACTGA AAATTACTTT TTCAATAAAC GAAAAATATA 2220 ATATACATTA CAAAACTTAT GTGAATAAAG CATGAAACTT AATATACGTT CCCTTTATCA 2280 TTTTACTTCA AAGAAAATAA ACAGAAATGT AACTTTCACA TGTAAATCTA ATTCTTAAAT 2340 TTAAAAAATA ATATTTATAT ATTTATATGA AAATAACGAA CCGGATGAAA AATAAATTTT 2400 ATATATTTAT ATCATCTCCA AATCTAGTTT GGTTCAGGGG CTTACCGAAC CGGATTGAAC 2460 TTCTCATATA CAAAAATTAG CAACACAAAA TGTCTCCGGT ATAAATACTA ACATTTATAA 2520 CCCGAACCGG TTTAGCTTCC TGTTATATCT TTTTAAAAAA GATCTCTGAC AAAGATTCCT 2580 TTCCTGGAAA TTTACCGGTT TTGGTGAAAT GTAAACCGTG GGACGAGGAT GCTTCTTCAT 2640 ATCTCACCAC CACTCTCGTT GACTTGACTT GGCTCTGCTC GTCAATGGTT ATCTTCGATC 2700 TTTAACCAAA TCCAGTTGAT AAGGTCTCTT CGTTGATTAG CAGAGATCTC TTTAATTTGT 2760 GAATTTCAAT TCATCGGAAC CTGTTG ATG GAC ACC ACC ATT GAT GGA TTC GCC in 2813

Met Asp Thr Thr Ile Asp Gly Phe Alá 1 5

DAM asp 10 TCT Ser TAT Tyr GAA Glu ATC Ile AGC Ser 15 AGC Ser ACT Thr AGT Ser TTC Phe GTC With 20 GCT below ACC Thr DAM asp AAC Asn ACC Thr 25 2861 GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA GTA CTC ACC GGA CCT 2909 asp Ser Ser Ile With Tyr Leu below below Glu Gin With Leu Thr Gly Pro 30 35 40 DAM GTA TCT GCT CTG CAA TTG CTC TCC AAC AGC TTC GAA TCC GTC TTT 2957 asp With Ser below Leu Gin Leu Leu Ser Asn Ser Phe Glu Ser With Phe 45 50 55 GAC TCG CCG DAM DAM TTC TAC AGC GAC GCT AAG CTT GTT CTC TCC GAC 3005 asp Ser Pro asp asp Phe Tyr Ser asp below Lys Leu With Leu Ser asp 60 65 70 GGC CGG GAA GTT TCT TTC CAC CGG TGC GTT TTG TCA GCG AGA AGC TCT 3053 Gly Arg Glu With Ser Phe His Arg Cys With Leu Ser below Arg Ser Ser 75 80 85 TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAG GAG AAA GAC TCC AAC 3101 Phe Phe Lys Ser below Leu below below below Lys Lys Glu Lys asp Ser Asn 90 95 100 105 AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG ATT GCC AAG DAM TAC 3149 Asn Thr below below With Lys Leu Glu Leu Lys Glu Ile below Lys asp Tyr 110 115 120 GAA GTC GGT TTC DAM TCG GTT GTG ACT GTT TTG GCT TAT GTT TAC AGC 3197 Glu With Gly Phe asp Ser With With Thr With Leu below Tyr With Tyr Ser 125 130 135

HU 224 480 Β1

AGC Ser AGA Arg GTG With 140 AGA Arg CCG Pro CCG Pro CCT Pro AAA Lys 145 GGA GTT Gly Val TCT Ser GAA Glu TGC Cys 150 GCA below GAC asp GAG Glu AAT TGC TGC CAC GTG GCT TGC CGG CCG GCG GTG DAM TTC ATG TTG GAG Asn Cys Cys His With below Cys Arg Pro below With asp Phe Met Leu Glu 155 160 165 GTT CTC TAT TTG GCT TTC ATC TTC AAG ATC CCT GAA TTA ATT ACT CTC With Leu Tyr Leu below Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu 170 175 180 185 TAT CAG GTAAAACACC ATCTGCATTA AGCTATGGTT ACACATTCAT GAATATGTTC Tyr Gin TTACTTGAGT ACTTGTATTT GTATTTCAG AGG CAC TTA TTG GAC GTT GTA GAC Arg His Leu Leu asp With With asp 190 195 AAA GTT GTT OVER THE GAG GAC ACA TTG GTT OVER THE CTC AAG CTT GCT AAT OVER THE Lys With With Ile Glu asp Thr Leu With Ile Leu Lys Leu below Asn Ile 200 205 210 TGT GGT AAA GCT TGT ATG AAG CTA TTG DAM AGA TGT AAA GAG ATT ATT Cys Gly Lys below Cys Met Lys Leu Leu asp Arg Cys Lys Glu Ile Ile 215 220 225 GTC AAG TCT AAT GTA DAM ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA With Lys Ser Asn With asp Met With Ser Leu Glu Lys Ser Leu Pro Glu 230 235 240 GAG CTT GTT AAA GAG OVER THE ATT DAM AGA CGT AAA GAG CTT GGT TTG GAG Glu Leu With Lys Glu Ile Ile asp Arg Arg Lys Glu Leu Gly Leu Glu 245 250 255 GTA CCT AAA GTA AAG AAA CAT GTC TCG AAT GTA CAT AAG GCA CTT GAC With Pro Lys With Lys Lys His With Ser Asn With His Lys below Leu asp 260 265 270 275 TCG DAM DAM ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG DAM CAC ACC Ser asp asp Ile Glu Leu With Lys Leu Leu Leu Lys Glu asp His Thr 280 285 290 AAT CTA DAM DAM GCG TGT GCT CTT CAT TTC GCT GTT GCA TAT TGC AAT Asn Leu asp asp below Cys below Leu His Phe below With below Tyr Cys Asn 295 300 305 GTG AAG ACC GCA ACA DAM CTT TTA AAA CTT DAM CTT GCC DAM GTC AAC With Lys Thr below Thr asp Leu Leu Lys Leu asp Leu below asp With Asn 310 315 320 CAT AGG AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG His Arg Asn Pro Arg Gly Tyr Thr With Leu His With below below Met Arg 325 330 335 AAG GAG CCA CAA TTG OVER THE CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA Lys Glu Pro Gin Leu Ile Leu Ser Leu Leu Glu Lys Gly below Ser below 340 345 350 355

3245

3293

3341

3397

3450

3498

3546

3594

3642

3690

3738

3786

3834

3882

3930

HU 224 480 Β1

TCA GAA GCA ACT TTG GAA GGT AGA ACC GCA CTC ATG ATC GCA AAA CAA 3978

Ser Glu Alá Thr Leu Glu Gly Arg Thr Alá Leu Met He Alá Lys Gin

360 365 370

GCC ACT ATG GCG GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT 4026

Alá Thr Met Alá Val Glu Cys Asn Asn Ile Pro Glu Gin Cys Lys His

375,380,385

TCT CTC AAA GGC CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA 4074

Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gin Glu Asp Lys

390 395 400

CGA GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TTT GCA GTG GCG GCC 4122

Arg Glu Gin Ile Pro Arg Asp Val Pro Pro Ser Phe Alá Val Alá Alá

405 410 415

GAT GAA TTG AAG ATG ACG CTG CTC GAT CTT GAA AAT AGA G 4162

Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg 420 425 430

GTATCTATCA AGTCTTATTT CTTATATGTT TGAATTAAAT TTATGTCCTC TCTATTAGGA 4222

AACTGAGTGA ACTAATGATA ACTATTCTTT GTGTCGTCCA CTGTTTAG TT GCA CTT 4278

Val Alá Leu 435

GCT CAA CGT CTT TTT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC 4326

Alá Gin Arg Leu Phe Pro Thr Glu Alá Gin Alá Met Glu Ile Alá

440,445,450

GAA ATG AAG GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC 4374

Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu Glu Pro Asp

455 460 465

CGT CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAG ATA GCA CCT 4422

Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys Ile Alá Pro

470,475,480

TTC AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA 4470

Phe Arg Ile Leu Glu Glu His Gin Ser Arg Leu Lys Alá Leu Ser Lys

485,490,495

ACC G GTATGGATTC TCACCCACTT CATCGGACTC CTTATCACAA AAAACAAAAC 4524

Thr

500

TAAATGATCT TTAAACATGG TTTTGTTACT TGCTGTCTGA CCTTGTTTTT TTTATCATCA 4584

G TG GAA CTC GGG AAA CGA TTC TTC CCG CGC TGT TCG GCA GTG CTC 4629

Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Al Val Leu 505 510 515

GAC CAG ATT ATG AAC TGT GAG GAC TTG ACT CAA CTG GCT TGC GGA GAA 4677

Asp Gin Ile Met Asn Cys Glu Asp Leu Thr Gin Leu Alá Cys Gly Glu

520,525,530

GAC GAC ACT GCT GAG AAA CGA CTA CAA AAG AAG CAA AGG TAC ATG GAA 4725

Asp Asp Thr Alá Glu Lys Arg Leu Gin Lys Lys Gin Arg Tyr Met Glu

HU 224 480 Β1

OVER THE Ile CAA GAG ACA Thr CTA Leu AAG Lys AAG Lys GCC below 555 TTT Phe AGT Ser GAG GAC Glu Asp AAT Asn 560 TTG Leu GAA Glu TTA Leu 4773 Gin Glu 550 GGA AAT TCG TCC CTG ACA DAM TCG ACT TCT TCC ACA TCG AAA TCA ACC 4821 Gly Asn Ser Ser Leu Thr asp Ser Thr Ser Ser Thr Ser Lys Ser Thr 565 570 575 GGT GGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT CGT CGG TGA 4866 Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg Arg ★ 580 585 590

GACTCTTGCC TCTTAGTGTA ATTTTTGCTG TACCATATAA TTCTGTTTTC ATGATGACTG 4926

TAACTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT 4986

ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG 5046

ATTTGTAATA TATATTTATG TACATCAACA ATAACCCATG ATGGTGTTAC AGAGTTGCTA 5106

GAATCAAAGT GTGAAATAAT GTCAAATTGT TCATCTGTTG GATATTTTCC ACCAAGAACC 5166

AAAAGAATAT TCAAGTTCCC TGAACTTCTG GCAACATTCA TGTTATATGT ATCTTCCTAA 5226

TTCTTCCTTT AACCTTTTGT AACTCGAATT ACACAGCAAG TTAGTTTCAG GTCTAGAGAT 5286

AAGAGAACAC TGAGTGGGCG TGTAAGGTGC ATTCTCCTAG TCAGCTCCAT TGCATCCAAC 5346

ATTTGTGAAT GACACAAGTT AACAATCCTT TGCACCATTT CTGGGTGCAT ACATGGAAAC 5406

TTCTTCGATT GAAACTTCCC ACATGTGCAG GTGCGTTCGC TGTCACTGAT AGACCAAGAG 5466

ACTGAAAGCT TTCACAAATT GCCCTCAAAT CTTCTGTTTC TATCGTCATG ACTCCATATC 5526

TCCGACCACT GGTCATGAGC CAGAGCCCAC TGATTTTGAG GGAATTGGGC TAACCATTTC 5586

CGAGCTTCTG AGTCCTTCTT TTTGATGTCC TTTATGTAGG AATCAAATTC TTCCTTCTGA 5646

CTTGTGGAT 5655 (2) INFORMATION FORUM SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 594 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Asp Thr Thr Ile Asp Gly Phe Alá Asp Ser Tyr Glu Ile Ser Ser 15 10 15

Thr Ser Phe Val Al Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu 20 25 30

Alá Alá Glu Gin Val Leu Thr Gly Pro Asp Val Ser Alá Leu Gin Leu 35 40 45

Leu Ser Asn Ser Phe Glu Ser Val Phe Asp Ser Pro Asp Asp Phe Tyr 50 55 60

Ser Asp Alá Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His

70 75 80

Arg Cys Val Leu Ser Alá Arg Ser Ser Phe Phe Lys Ser Alá Leu Alá

90 95

Alá Alá Lys Lys Glu Lys Asp Ser Asn Asn Thr Alá Alá Val Lys Leu 100 105 110

Glu Leu Lys Glu Ile Alá Lys Asp Tyr Glu Val Gly Phe Asp Ser Val 115 120 125

Val Thr Val Leu Alá Tyr Val Tyr Tyr Ser Ser Arg Val Arg Pro Pro 130 135 140

HU 224 480 Β1

Lys Gly Val Ser Glu Cys Alá Asp Glu Asn Cys Cys His Val Alá Cys

145 150 155 160

Arg Pro Alá Val Asp Phe Met Leu Glu Val Leu Tyr Leu Alá Phe Ile

165 170 175

Phe Lys Ile Pro Glu Leu He Thr Leu Tyr Gin Arg His Leu Leu Asp 180 185 190

Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu 195 200 205

Alá Asn Ile Cys Gly Lys Alá Cys Met Lys Leu Leu Asp Arg Cys Lys 210 215 220

Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser

225 230 235 240

Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu

245 250 255

Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys 260 265 270

Alá Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu 275 280 285

Asp His Thr Asn Leu Asp Asp Alá Cys Alá Leu His Phe Alá Val Alá 290 295 300

Tyr Cys Asn Val Lys Thr Alá Thr Asp Leu Leu Lys Leu Asp Leu Alá

305 310 315 320

Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Alá

325 330 335

Alá Met Arg Lys Glu Pro Gin Leu Ile Leu Ser Leu Leu Glu Lys Gly 340 345 350

Alá Ser Alá Ser Glu Al Thr Leu Glu Gly Arg Thr Al Al Leu Met Ile 355 360 365

Alá Lys Gin Alá Thr Met Alá Val Glu Cys Asn Asn Ile Pro Glu Gin 370 375 380

Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gin

385,390,395,400

Glu Asp Lys Arg Glu Gin Ile Pro Arg Asp Val Pro Pro Ser Phe Alá

405 410 415

Val Alá Ala Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg 420 425 430

Val Alá Leu Alá Gin Arg Leu Phe Pro Thr Glu Alá Gin Alá Alá Met 435 440 445

Glu Ile Alá Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu 450 455 460

HU 224 480 Β1

Glu 465 Pro Asp Arg Leu Thr Gly Thr 470 Lys Arg Thr 475 Ser Pro Gly With Lys 480 Ile below Pro Phe Arg Ile Leu Glu Glu His Gin Ser Arg Leu Lys below 485 490 495 Leu Ser Lys Thr With Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser 500 505 510 below With Leu asp Gin Ile Met Asn Cys Glu asp Leu Thr Gin Leu below 515 520 525 Cys Gly Glu asp asp Thr below Glu Lys Arg Leu Gin Lys Lys Gin Arg 530 535 540 Tyr Met Glu Ile Gin Glu Thr Leu Lys Lys below Phe Ser Glu asp Asn 545 550 555 560 Leu Glu Leu Gly Asn Ser Ser Leu Thr asp Ser Thr Ser Ser Thr Ser 565 570 575 Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg

580,585,590

Arg * (2) INFORMATION FORUM SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 314 amino acids (Β) ΤΥΡΕ: amino acid (C) STRANDEDNESS: woman relevant (D) TOPOLOGY: female relevant (ii) MOLECULE ΤΥΡΕ: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Met 1 Phe Gin Pro below 5 Gly His Gly Gin Asp Trp 10 below Met Glu Gly 15 Pro Arg Asp Gly Leu 20 Lys Lys Glu Arg Leu Val Asp 25 asp Arg His 30 asp Ser Gly Leu Asp 35 Ser Met Lys asp Glu 40 Glu Tyr Glu Gin Met 45 With Lys Glu Leu Arg Glu 50 Ile Arg Leu Gin 55 Pro Gin Glu Alá Pro 60 Leu below below Glu Pro 65 Trp Lys Gin Gin Leu 70 Thr Glu Asp Gly Asp 75 Ser Phe Leu His Leu 80 below Ile Ile His Glu 85 Glu Lys Pro Leu Thr Met 90 Glu With Ile Gly 95 Gin With Lys Gly asp 100 Leu below Phe Leu Asn Phe Gin 105 Asn Asn Leu 110 Gin Gin Thr Pro Leu 115 His Leu below With Ile 120 Thr Asn Gin Pro Gly 125 Ile below Glu below Leu Leu Lys below Gly Cys asp Pro Glu Leu Arg asp Phe Arg Gly

130 135 140

HU 224 480 Β1

Asn 145 Thr Pro Leu His Leu 150 below Cys Glu Gin Gly 155 Cys Leu below Ser With 160 below With Leu Thr Gin Thr Cys Thr Pro Gin His Leu His Ser With Leu 165 170 175 Gin below Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu below Ser Thr 180 185 190 His Gly Tyr Leu below Ile With Glu His Leu With Thr Leu Gly below asp 195 200 205 With Asn below Gin Glu Pro Cys Asn Gly Arg Thr below Leu His Leu below 210 215 220 With asp Leu Gin Asn Pro asp Leu With Ser Leu Leu Leu Lys Cys Gly 225 230 235 240 below asp With Asn Arg With Thr Tyr Gin Gly Tyr Ser Pro Tyr Gin Leu 245 250 255 Thr Trp Gly Arg Pro Ser Thr Arg Ile Gin Gin Gin Leu Gly Gin Leu 260 265 270 Thr Leu Glu Asn Leu Gin Met Leu Pro Glu Ser Glu asp Glu Glu Ser 275 280 285 Tyr asp Thr Glu Ser Glu Phe Thr Glu asp Glu Leu Pro Tyr asp asp 290 295 300 Cys With Phe Gly Gly Gin Arg Leu Thr Leu

305 310 (2) INFORMATION FORUM SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 314 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: woman relevant (D) TOPOLOGY: female relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Trp below Met Glu Gly 15 Pro Met 1 Phe Gin Pro Down Gly His 5 Gly Gin asp 10 Arg asp Gly Leu 20 Lys Lys Glu Arg Leu 25 With asp asp Arg His 30 asp Ser Gly Leu asp 35 Ser Met Lys Asp Glu 40 asp Tyr Glu Gin Met 45 With Lys Glu Leu Arg 50 Glu Ile Arg Leu Gin 55 Pro Gin Glu below Pro 60 Leu below below Glu Pro 65 Trp Lys Gin Gin Leu Thr 70 Glu asp Gly asp 75 Ser Phe Leu His Leu 80 below Ile Ile His Glu Glu Lys 85 Thr Leu Thr 90 Met Glu With Ile Gly 95 Gin

HU 224 480 Β1

With Lys Gly asp 100 Leu below Phe Leu Asn 105 Phe Gin Asn Asn Leu 110 Gin Gin Thr Pro Leu His Leu below With He Thr Asn Gin Pro Gly He below Glu 115 120 125 below Leu Leu Lys below Gly Cys asp Pro Glu Leu Arg asp Phe Arg Gly 130 135 140 Asn Thr Pro Leu His Leu below Cys Glu Gin Gly Cys Leu below Ser With 145 150 155 160 below With Leu Thr Gin Thr Cys Thr Pro Gin His Leu His Ser With Leu 165 170 175 Gin below Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu below Ser He 180 185 190 His Gly Tyr Leu Gly He With Glu His Leu With Thr Leu Gly below asp 195 200 205 With Asn below Gin Glu Pro Cys Asn Gly Arg Thr below Leu His Leu below 210 215 220 With asp Leu Gin Asn Pro asp Leu With Ser Leu Leu Leu Lys Cys Gly 225 230 235 240 below asp With Asn Arg With Thr Tyr Gin Gly Tyr Ser Pro Tyr Gin Leu 245 250 255 Thr Trp Gly Arg Pro Ser Thr Arg He Gin Gin Gin Leu Gly Gin Leu 260 265 270 Thr Leu Glu Asn Leu Gin Thr Leu Pro Glu Ser Glu asp Glu Glu Ser 275 280 285 Tyr asp Thr Glu Ser Glu Phe Thr Glu asp Glu Leu Pro Tyr asp asp 290 295 300 Cys With Phe Gly Gly Gin Arg Leu Thr Leu 305 310

(2) INFORMATION FORUM SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 314 amino acids (Β) ΤΥΡΕ: amino acid (C) STRANDEDNESS: female relevant (D) TOPOLOGY: female relevant (ii) MOLECULE ΤΥΡΕ: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Met 1 Phe Gin Pro below 5 Glu Pro Gly Gin Glu 10 Trp below Met Glu Gly 15 Pro Arg asp below Leu Lys Lys Glu Arg Leu Leu asp asp Arg His asp Ser 20 25 30 Gly Leu asp Ser Met Lys asp Glu Glu Tyr Glu Gin Met With Lys Glu

40 45

HU 224 480 Β1

Leu Arg 50 Glu Ile Arg Leu Glu 55 Pro Gin Glu Alá Pro 60 Arg Gly Ala Glu Pro Trp Lys Gin Gin Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu 65 70 75 80 Down Ile Ile His Glu Glu Lys Alá Leu Thr Met Glu With Val Arg Gin 85 90 95 Val Lys Gly asp Leu Al Phe Leu Asn Phe Gin Asn Asn Leu Gin Gin 100 105 110 Thr Pro Leu His Leu Ala Val Ile Thr Asn Gin Pro Glu Ile Ala Glu 115 120 125 Down Leu Leu Glu below Gly Cys Asp Pro Glu Leu Arg asp Phe Arg Gly 130 135 140 Asn Thr Pro Leu His Leu Alá Cys Glu Gin Gly Cys Leu Alá Ser With 145 150 155 160 Gly Val Leu Thr Gin Pro Arg Gly Thr Gin His Leu His Ser Ile Leu 165 170 175 Gin Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Alá Ser Ile 180 185 190 His Gly Tyr Leu Gly Ile Val Glu Leu Leu Val Ser Leu Gly Ala asp 195 200 205 Val Asn below Gin Glu Pro Cys Asn Gly Arg Thr below Leu His Leu below 210 215 220 Val Asp Leu Gin Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly 225 230 235 240 Under Asp With Asn Arg Val Thr Tyr Gin Gly Tyr Ser Pro Tyr Gin Leu 245 250 255 Thr Trp Gly Arg Pro Ser Thr Arg Ile Gin Gin Gin Leu Gly Gin Leu 260 265 270 Thr Leu Glu Asn Leu Gin Met Leu Pro Glu Ser Glu asp Glu Glu Ser 275 280 285 Tyr Asp Thr Glu Ser Glu Phe Thr Glu Asp Glu Leu Pro Tyr Asp asp 290 295 300 Cys Val Leu Gly Gly Gin Arg Leu Thr Leu 305 310 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2011 base pairs (B) TYPE: i nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Arabidopsis thaliana

HU 224 480 Β1 (ix) FEATURE:

(x) NAME / KEY: misc_feature (B) LOCATION: 1 ... 2011 (D) OTHER INFORMATION: / note = * NIM1 cDNA sequence * (ix) FEATURE:

(A) NAME / KEY: CDS (B) LOCATION: 43 ... 1824 (D) OTHER INFORMATION: / product = * NIM1 protein * (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC 54

Met Asp Thr Thr

ATT Ile 5 DAM asp GGA Gly TTC Phe GCC below DAM asp 10 TCT Ser TAT Tyr GAA Glu ATC Ile AGC Ser 15 AGC Ser ACT Thr AGT Ser TTC Phe GTC With 20 102 GCT ACC DAM AAC ACC GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA 150 below Thr asp Asn Thr asp Ser Ser Ile With Tyr Leu below below Glu Gin 25 30 35 GTA CTC ACC GGA CCT DAM GTA TCT GCT CTG CAA TTG CTC TCC AAC AGC 198 With Leu Thr Gly Pro asp With Ser below Leu Gin Leu Leu Ser Asn Ser 40 45 50 TTC GAA TCC GTC TTT GAC TCG CCG DAM DAM TTC TAC AGC GAC GCT AAG 246 Phe Glu Ser With Phe asp Ser Pro asp asp Phe Tyr Ser asp below Lys 55 60 65 CTT GTT CTC TCC GAC GGC CGG GAA GTT TCT TTC CAC CGG TGC GTT TTG 294 Leu With Leu Ser asp Gly Arg Glu With Ser Phe His Arg Cys With Leu 70 75 80 TCA GCG AGA AGC TCT TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAG 342 Ser below Arg Ser Ser Phe Phe Lys Ser below Leu below below below Lys Lys 85 90 95 100 GAG AAA GAC TCC AAC AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG 390 Glu Lys asp Ser Asn Asn Thr below below With Lys Leu Glu Leu Lys Glu 105 110 115 ATT GCC AAG DAM TAC GAA GTC GGT TTC DAM TCG GTT GTG ACT GTT TTG 438 Ile below Lys asp Tyr Glu With Gly Phe asp Ser With With Thr With Leu 120 125 130 GCT TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT 486 below Tyr With Tyr Ser Ser Arg With Arg Pro Pro Pro Lys Gly With Ser 135 140 145 GAA TGC GCA GAC GAG AAT TGC TGC CAC GTG GCT TGC CGG CCG GCG GTG 534 Glu Cys below asp Glu Asn Cys Cys His With below Cys Arg Pro below With 150 155 160 DAM TTC ATG TTG GAG GTT CTC TAT TTG GCT TTC ATC TTC AAG ATC CCT 582 asp Phe Met Leu Glu With Leu Tyr Leu below Phe Ile Phe Lys Ile Pro 165 170 175 180 GAA TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA 630 Glu Leu Ile Thr Leu Tyr Gin Arg His Leu Leu asp With With asp Lys 185 190 195

HU 224 480 Β1

GTT With GTT With OVER THE Ile GAG Glu 200 GAC asp ACA Thr TTG Leu GTT With OVER THE Ile 205 CTC Leu AAG Lys CTT Leu GCT below AAT Asn 210 OVER THE Ile TGT Cys 678 GGT AAA GCT TGT ATG AAG CTA TTG DAM AGA TGT AAA GAG ATT ATT GTC 726 Gly Lys below Cys Met Lys Leu Leu asp Arg Cys Lys Glu Ile Ile With 215 220 225 AAG TCT AAT GTA DAM ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA GAG 774 Lys Ser Asn With asp Met With Ser Leu Glu Lys Ser Leu Pro Glu Glu 230 235 240 CTT GTT AAA GAG OVER THE ATT DAM AGA CGT AAA GAG CTT GGT TTG GAG GTA 822 Leu With Lys Glu Ile Ile asp Arg Arg Lys Glu Leu Gly Leu Glu With 245 250 255 260 CCT AAA GTA AAG AAA CAT GTC TCG AAT GTA CAT AAG GCA CTT GAC TCG 870 Pro Lys With Lys Lys His With Ser Asn With His Lys below Leu asp Ser 265 270 275 DAM DAM ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG DAM CAC ACC AAT 918 asp asp Ile Glu Leu With Lys Leu Leu Leu Lys Glu asp His Thr Asn 280 285 290 CTA DAM DAM GCG TGT GCT CTT CAT TTC GCT GTT GCA TAT TGC AAT GTG 966 Leu asp asp below Cys below Leu His Phe below With below Tyr Cys Asn With 295 300 305 AAG ACC GCA ACA DAM CTT TTA AAA CTT DAM CTT GCC DAM GTC AAC CAT 1014 Lys Thr below Thr asp Leu Leu Lys Leu asp Leu below asp With Asn His 310 315 320 AGG AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG AAG 1062 Arg Asn Pro Arg Gly Tyr Thr With Leu His With below below Met Arg Lys 325 330 335 340 GAG CCA CAA TTG OVER THE CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA 1110 Glu Pro Gin Leu Ile Leu Ser Leu Leu Glu Lys Gly below Ser below Ser 345 350 355 GAA GCA ACT TTG GAA GGT AGA ACC GCA CTC ATG ATC GCA AAA CAA GCC 1158 Glu below Thr Leu Glu Gly Arg Thr below Leu Met Ile below Lys Gin below 360 365 370 ACT ATG GCG GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT TCT 1206 Thr Met below With Glu Cys Asn Asn Ile Pro Glu Gin Cys Lys His Ser 375 380 385 CTC AAA GGC CGA CTA TGT GTA GAA OVER THE CTA GAG CAA GAA GAC AAA CGA 1254 Leu Lys Gly Arg Leu Cys With Glu Ile Leu Glu Gin Glu asp Lys Arg 390 395 400 GAA CAA ATT CCT AGA DAM GTT CCT CCC TCT TTT GCA GTG GCG GCC DAM 1302 Glu Gin Ile Pro Arg asp With Pro Pro Ser Phe below With below below asp 405 410 415 420 GAA TTG AAG ATG ACG CTG CTC DAM CTT GAA AAT AGA GTT GCA CTT GCT 1350 Glu Leu Lys Met Thr Leu Leu asp Leu Glu Asn Arg With below Leu below 425 430 435

HU 224 480 Β1

CAA Gin CGT Arg CTT Leu TTT Phe 440 CCA Pro ACG Thr GAA GCA CAA GCT GCA below ATG Met GAG Glu ATC Ile 450 GCC below GAA Glu 1398 Glu Alá Gin 445 below ATG AAG GGA ACA TGT GAG TTC OVER THE GTG ACT AGC CTC GAG CCT GAC CGT 1446 Met Lys Gly Thr Cys Glu Phe Ile With Thr Ser Leu Glu Pro asp Arg 455 460 465 CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAG OVER THE GCA CCT TTC 1494 Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly With Lys Ile below Pro Phe 470 475 480 AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA ACC 1542 Arg Ile Leu Glu Glu His Gin Ser Arg Leu Lys below Leu Ser Lys Thr 485 490 495 500 GTG GAA CTC GGG AAA CGA TTC TTC CCG CGC TGT TCG GCA GTG CTC GAC 1590 With Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser below With Leu asp 505 510 515 CAG ATT ATG AAC TGT GAG GAC TTG ACT CAA CTG GCT TGC GGA GAA GAC 1638 Gin Ile Met Asn Cys Glu asp Leu Thr Gin Leu below Cys Gly Glu asp 520 525 530 GAC ACT GCT GAG AAA CGA CTA CAA AAG AAG CAA AGG TAC ATG GAA OVER THE 1686 asp Thr below Glu Lys Arg Leu Gin Lys Lys Gin Arg Tyr Met Glu Ile 535 540 545 CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GAA TTA GGA 1734 Gin Glu Thr Leu Lys Lys below Phe Ser Glu asp Asn Leu Glu Leu Gly 550 555 560 AAT TTG TCC CTG ACA DAM TCG ACT TCT TCC ACA TCG AAA TCA ACC GGT 1782 Asn Leu Ser Leu Thr asp Ser Thr Ser Ser Thr Ser Lys Ser Thr Gly 565 570 575 580 GGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT CGT CGG TGA 1824 Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg Arg k 585 590

GACTCTTGCC TCTTAGTGTA ATTTTTGCTG TACCATATAA TTCTGTTTTC ATGATGACTG 1884

TAACTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT 1944

ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG 2004

ATTTGTA 2011 (2) INFORMATION FORUM SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2011 base pairs (Β) ΤΥΡΕ: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE ΤΥΡΕ: cDNA (ix) FEATURE:

(A) NAME / KEY: CDS (B) LOCATION: 43 ... 1824 (D) OTHER INFORMATION: / product = "altered form of NIM1" / note = "Serine residues at amino acid positions 55 and 59 in wild-type NIM1 gene product has changed to Alanine residues. "(Ix) FEATURE:

(A) NAME / KEY: misc_feature (B) LOCATION: 205 ... 217

HU 224,480 Β1 (D) OTHER INFORMATION: / note =, nucleotides 205 and 217 changed from T to G compared to wild-type mixture.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC 54 Met. 1 Asp Thr Thr ATT GAT GGA TTC GCC GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC GTC 102 Ile Asp Gly Phe Alá Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val 5 10 15 20 GCT ACC GAT AAC ACC GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA 150 Alá Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu Alá Alla Glu Gin 25 30 35 GTA CTC ACC GGA CCT GAT GTA TCT GCT CTG CAA TTG CTC TCC AAC AGC 198 Val Leu Thr Gly Pro Asp Val Ser Alá Leu Gin Leu Leu Ser Asn Ser 40 45 50 TTC GAA GCC GTC TTT GAC GCG CCG GAT GAT TTC TAC AGC GAC GCT AAG 246 Phe Glu Alá Val Phe Asp Alá Pro Asp Asp Phe Tyr Ser Asp Alá Lys 55 60 65 CTT GTT CTC TCC GAC GGC CGG GAA GTT TCT TTC CAC CGG TGC GTT TTG 294 Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His Arg Cys Val Leu 70 75 80 TCA GCG AGA AGC TCT TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAG 342 Ser Alá Arg Ser Ser Phe Phe Lys Ser Alá Leu Ala Ala Under Lys Lys 85 90 95 100 GAG AAA GAC TCC AAC AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG 390 Glu Lys Asp Ser Asn Asn Thr Alá Alá Val Lys Leu Glu Leu Lys Glu 105 110 115 ATT GCC AAG GAT TAC GAA GTC GGT TTC GAT TCG GTT GTG ACT GTT TTG 438 Ile Alá Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu 120 125 130 GCT TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT 486 Alá Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser 135 140 145 GAA TGC GCA GAC GAG AAT TGC TGC CAC GTG GCT TGC CGG CCG GCG GTG 534 Glu Cys Alá Asp Glu Asn Cys Cys His Val Alá Cys Arg Pro Alá Val 150 155 160 GAT TTC ATG TTG GAG GTT CTC TAT TTG GCT TTC ATC TTC AAG ATC CCT 582 Asp Phe Met Leu Glu Val Leu Tyr Leu Alá Phe Ile Phe Lys Ile Pro 165 170 175 180 GAA TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA 630 Glu Leu Ile Thr Leu Tyr Gin Arg His Leu Leu Asp Val Val Asp Lys 185 190 195 GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT 678 Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Alá Asn Ile Cys 200 205 210

HU 224 480 Β1

GGT Gly AAA Lys GCT below 215 TGT Cys ATG Met AAG Lys CTA Leu TTG Leu 220 DAM asp AGA Arg TGT Cys AAA Lys GAG Glu 225 ATT Ile ATT Ile GTC With 726 AAG TCT AAT GTA DAM ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA GAG 774 Lys Ser Asn With asp Met With Ser Leu Glu Lys Ser Leu Pro Glu Glu 230 235 240 CTT GTT AAA GAG OVER THE ATT DAM AGA CGT AAA GAG CTT GGT TTG GAG GTA 822 Leu With Lys Glu Ile Ile asp Arg Arg Lys Glu Leu Gly Leu Glu With 245 250 255 260 CCT AAA GTA AAG AAA CAT GTC TCG AAT GTA CAT AAG GCA CTT GAC TCG 870 Pro Lys With Lys Lys His With Ser Asn With His Lys below Leu asp Ser 265 270 275 DAM DAM ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG DAM CAC ACC AAT 918 asp asp Ile Glu Leu With Lys Leu Leu Leu Lys Glu asp His Thr Asn 280 285 290 CTA DAM DAM GCG TGT GCT CTT CAT TTC GCT GTT GCA TAT TGC AAT GTG 966 Leu asp asp below Cys below Leu His Phe below With below Tyr Cys Asn With 295 300 305 AAG ACC GCA ACA DAM CTT TTA AAA CTT DAM CTT GCC DAM GTC AAC CAT 1014 Lys Thr below Thr asp Leu Leu Lys Leu asp Leu below asp With Asn His 310 315 320 AGG AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG AAG 1062 Arg Asn Pro Arg Gly Tyr Thr With Leu His With below below Met Arg Lys 325 330 335 340 GAG CCA CAA TTG OVER THE CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA 1110 Glu Pro Gin Leu Ile Leu Ser Leu Leu Glu Lys Gly below Ser below Ser 345 350 355 GAA GCA ACT TTG GAA GGT AGA ACC GCA CTC ATG ATC GCA AAA CAA GCC 1158 Glu below Thr Leu Glu Gly Arg Thr below Leu Met Ile below Lys Gin below 360 365 370 ACT ATG GCG GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT TCT 1206 Thr Met below With Glu Cys Asn Asn Ile Pro Glu Gin Cys Lys His Ser 375 380 385 CTC AAA GGC CGA CTA TGT GTA GAA OVER THE CTA GAG CAA GAA GAC AAA CGA 1254 Leu Lys Gly Arg Leu Cys With Glu Ile Leu Glu Gin Glu asp Lys Arg 390 395 400 GAA CAA ATT CCT AGA DAM GTT CCT CCC TCT TTT GCA GTG GCG GCC DAM 1302 Glu Gin Ile Pro Arg asp With Pro Pro Ser Phe below With below below asp 405 410 415 420 GAA TTG AAG ATG ACG CTG CTC DAM CTT GAA AAT AGA GTT GCA CTT GCT 1350 Glu Leu Lys Met Thr Leu Leu asp Leu Glu Asn Arg With below Leu below 425 430 435 CAA CGT CTT TTT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC GAA 1398 Gin Arg Leu Phe Pro Thr Glu below Gin below below Met Glu Ile below Glu 440 445 450

HU 224 480 Β1

ATG Met AAG Lys GGA Gly 455 ACA Thr TGT Cys GAG Glu TTC Phe ATA GTG ACT Thr AGC Ser CTC Leu GAG Glu 465 CCT Pro GAC asp CGT Arg 1446 Ile 460 With CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAG OVER THE GCA CCT TTC 1494 Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly With Lys Ile below Pro Phe 470 475 480 AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA ACC 1542 Arg Ile Leu Glu Glu His Gin Ser Arg Leu Lys below Leu Ser Lys Thr 485 490 495 500 GTG GAA CTC GGG AAA CGA TTC TTC CCG CGC TGT TCG GCA GTG CTC GAC 1590 With Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser below With Leu asp 505 510 515 CAG ATT ATG AAC TGT GAG GAC TTG ACT CAA CTG GCT TGC GGA GAA GAC 1638 Gin Ile Met Asn Cys Glu asp Leu Thr Gin Leu below Cys Gly Glu asp 520 525 530 GAC ACT GCT GAG AAA CGA CTA CAA AAG AAG CAA AGG TAC ATG GAA OVER THE 1686 asp Thr below Glu Lys Arg Leu Gin Lys Lys Gin Arg Tyr Met Glu Ile 535 540 54 5 CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GAA TTA GGA 1734 Gin Glu Thr Leu Lys Lys below Phe Ser Glu asp Asn Leu Glu Leu Gly 550 555 560 AAT TTG TCC CTG ACA DAM TCG ACT TCT TCC ACA TCG AAA TCA ACC GGT 1782 Asn Leu Ser Leu Thr asp Ser Thr Ser Ser Thr Ser Lys Ser Thr Gly 565 570 575 580 GGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT CGT CGG TGA 1824 Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg Arg * 585 590

GACTCTTGCC TCTTAGTGTA ATTTTTGCTG TACCATATAA TTCTGTTTTC ATGATGACTG 1884 TAACTGTTTA TGTCTATCGT TGTTTCGCTTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 594 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Met 1 asp Thr Thr Ile 5 asp Gly Phe Below Asp 10 Ser Tyr Glu Ile Ser 15 Ser Thr Ser Phe With 20 below Thr asp Asn Thr 25 asp Ser Ser Ile With 30 Tyr Leu below below Glu 35 Gin With Leu Thr Gly 40 Pro asp With Ser below 45 Leu Gin Leu Leu Ser 50 Asn Ser Phe Glu below 55 With Phe asp below Pro 60 asp asp Phe Tyr

HU 224 480 Β1

Ser Asp 65 below Lys Leu Val 70 Leu Ser asp Gly Arg 75 Glu With Ser Phe His 80 Arg Cys With Leu Ser below Arg Ser Ser Phe Phe Lys Ser below Leu below 85 90 95 below below Lys Lys Glu Lys asp Ser Asn Asn Thr below below With Lys Leu 100 105 110 Glu Leu Lys Glu Ile below Lys asp Tyr Glu With Gly Phe asp Ser With 115 120 125 With Thr With Leu below Tyr With Tyr Ser Ser Arg With Arg Pro Pro Pro 130 135 140 Lys Gly With Ser Glu Cys below asp Glu Asn Cys Cys His With below Cys 145 150 155 160 Arg Pro below With asp Phe Met Leu Glu With Leu Tyr Leu below Phe Ile 165 170 175 Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gin Arg His Leu Leu asp 180 185 190 With With asp Lys With With Ile Glu asp Thr Leu With Ile Leu Lys Leu 195 200 205 below Asn Ile Cys Gly Lys below Cys Met Lys Leu Leu asp Arg Cys Lys 210 215 220 Glu Ile Ile With Lys Ser Asn With asp Met With Ser Leu Glu Lys Ser 225 230 235 240 Leu Pro Glu Glu Leu With Lys Glu Ile Ile asp Arg Arg Lys Glu Leu 245 250 255 Gly Leu Glu With Pro Lys With Lys Lys His With Ser Asn With His Lys 260 265 270 below Leu asp Ser asp asp Ile Glu Leu With Lys Leu Leu Leu Lys Glu 275 280 285 asp His Thr Asn Leu asp asp below Cys below Leu His Phe below With below 290 295 300 Tyr Cys Asn With Lys Thr below Thr asp Leu Leu Lys Leu asp Leu below 305 310 315 320 asp With Asn His Arg Asn Pro Arg Gly Tyr Thr With Leu His With below 325 330 335 below Met Arg Lys Glu Pro Gin Leu Ile Leu Ser Leu Leu Glu Lys Gly 340 345 350 below Ser below Ser Glu below Thr Leu Glu Gly Arg Thr below Leu Met Ile 355 360 365 below Lys Gin below Thr Met below With Glu Cys Asn Asn Ile Pro Glu Gin 370 375 380

HU 224 480 Β1

Cys 385 Lys His Ser Leu Lys 390 Gly Arg Leu Cys With 395 Glu Ile Leu Glu Gin 400 Glu asp Lys Arg Glu Gin Ile Pro Arg asp With Pro Pro Ser Phe below 405 410 415 With below below asp Glu Leu Lys Met Thr Leu Leu asp Leu Glu Asn Arg 420 425 430 With below Leu below Gin Arg Leu Phe Pro Thr Glu below Gin below below Met 435 440 445 Glu Ile below Glu Met Lys Gly Thr Cys Glu Phe Ile With Thr Ser Leu 450 455 460 Glu Pro asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly With Lys 465 470 475 480 Ile below Pro Phe Arg Ile Leu Glu Glu His Gin Ser Arg Leu Lys below 485 490 495 Leu Ser Lys Thr With Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser 500 505 510 below With Leu asp Gin Ile Met Asn Cys Glu asp Leu Thr Gin Leu below 515 520 525 Cys Gly Glu asp asp Thr below Glu Lys Arg Leu Gin Lys Lys Gin Arg 530 535 540 Tyr Met Glu Ile Gin Glu Thr Leu Lys Lys below Phe Ser Glu asp Asn 545 550 555 560 Leu Glu Leu Gly Asn Leu Ser Leu Thr asp Ser Thr Ser Ser Thr Ser 565 570 575 Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg 580 585 590

Arg * (2) INFORMATION FORUM SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1597 base pairs (Β) ΤΥΡΕ: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE ΤΥΡΕ: cDNA (ix) FEATURE:

(A) NAME / KEY: CDS (B) LOCATION: 1 ... 1410 (D) OTHER INFORMATION: / product = "Altered form of NIM1" / note = "N-terminal deletion compared to wild-type NIM1 sequence." (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

ATG GAT TCG GTT GTG ACT GTT TTG GCT TAT GTT TAC AGC AGC AGA GTG 48

Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val

10 15

HU 224 480 Β1

AGA Arg CCG Pro CCG Pro CCT Pro 20 AAA Lys GGA Gly GTT With TCT Ser GAA Glu 25 TGC Cys GCA below GAC asp GAG Glu AAT Asn 30 TGC Cys TGC Cys 96 CAC GTG GCT TGC CGG CCG GCG GTG DAM TTC ATG TTG GAG GTT CTC TAT 144 His With below Cys Arg Pro below With asp Phe Met Leu Glu With Leu Tyr 35 40 45 TTG GCT TTC ATC TTC AAG ATC CCT GAA TTA ATT ACT CTC TAT CAG AGG 192 Leu below Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gin Arg 50 55 60 CAC TTA TTG GAC GTT GTA GAC AAA GTT GTT OVER THE GAG GAC ACA TTG GTT 240 His Leu Leu asp With With asp Lys With With Ile Glu asp Thr Leu With 65 70 75 80 OVER THE CTC AAG CTT GCT AAT OVER THE TGT GGT AAA GCT TGT ATG AAG CTA TTG 288 Ile Leu Lys Leu below Asn Ile Cys Gly Lys below Cys Met Lys Leu Leu 85 90 95 DAM AGA TGT AAA GAG ATT ATT GTC AAG TCT AAT GTA DAM ATG GTT AGT 336 asp Arg Cys Lys Glu Ile Ile With Lys Ser Asn With asp Met With Ser 100 105 110 CTT GAA AAG TCA TTG CCG GAA GAG CTT GTT AAA GAG OVER THE ATT DAM AGA 384 Leu Glu Lys Ser Leu Pro Glu Glu Leu With Lys Glu Ile Ile asp Arg 115 120 125 CGT AAA GAG CTT GGT TTG GAG GTA CCT AAA GTA AAG AAA CAT GTC TCG 432 Arg Lys Glu Leu Gly Leu Glu With Pro Lys With Lys Lys His With Ser 130 135 140 AAT GTA CAT AAG GCA CTT GAC TCG DAM DAM ATT GAG TTA GTC AAG TTG 480 Asn With His Lys below Leu asp Ser asp asp Ile Glu Leu With Lys Leu 145 150 155 160 CTT TTG AAA GAG DAM CAC ACC AAT CTA DAM DAM GCG TGT GCT CTT CAT 528 Leu Leu Lys Glu asp His Thr Asn Leu asp asp below Cys below Leu His 165 170 175 TTC GCT GTT GCA TAT TGC AAT GTG AAG ACC GCA ACA DAM CTT TTA AAA 576 Phe below With below Tyr Cys Asn With Lys Thr below Thr asp Leu Leu Lys 180 185 190 CTT DAM CTT GCC DAM GTC AAC CAT AGG AAT CCG AGG GGA TAT ACG GTG 624 Leu asp Leu below asp With Asn His Arg Asn Pro Arg Gly Tyr Thr With 195 200 205 CTT CAT GTT GCT GCG ATG CGG AAG GAG CCA CAA TTG OVER THE CTA TCT CTA 672 Leu His With below below Met Arg Lys Glu Pro Gin Leu Ile Leu Ser Leu 210 215 220 TTG GAA AAA GGT GCA AGT GCA TCA GAA GCA ACT TTG GAA GGT AGA ACC 720 Leu Glu Lys Gly below Ser below Ser Glu below Thr Leu Glu Gly Arg Thr 225 230 235 240 GCA CTC ATG ATC GCA AAA CAA GCC ACT ATG GCG GTT GAA TGT AAT AAT 768 below Leu Met Ile below Lys Gin below Thr Met below With Glu Cys Asn Asn 245 250 255

HU 224 480 Β1

816

ATC CCG GAG CAA TGC AAG CAT TCT CTC AAA GGC CGA CTA TGT GTA GAA Ile Pro Glu Gin 260 Cys Lys His Ser Leu 265 Lys Gly Arg Leu Cys 270 With Glu OVER THE CTA GAG CAA GAA GAC AAA CGA GAA CAA ATT CCT AGA DAM GTT CCT Ile Leu Glu 275 Gin Glu asp Lys Arg 280 Glu Gin Ile Pro Arg 285 asp With Pro CCC TCT TTT GCA GTG GCG GCC DAM GAA TTG AAG ATG ACG CTG CTC DAM Pro Ser 290 Phe below With below below 295 asp Glu Leu Lys Met 300 Thr Leu Leu asp CTT GAA AAT AGA GTT GCA CTT GCT CAA CGT CTT TTT CCA ACG GAA GCA Leu 305 Glu Asn Arg With below 310 Leu below Gin Arg Leu 315 Phe Pro Thr Glu below 320 CAA GCT GCA ATG GAG ATC GCC GAA ATG AAG GGA ACA TGT GAG TTC OVER THE Gin below below Met Glu 325 Ile below Glu Met Lys 330 Gly Thr Cys Glu Phe 335 Ile GTG ACT AGC CTC GAG CCT GAC CGT CTC ACT GGT ACG AAG AGA ACA TCA With Thr Ser Leu 340 Glu Pro asp Arg Leu 345 Thr Gly Thr Lys Arg 350 Thr Ser CCG GGT GTA AAG OVER THE GCA CCT TTC AGA ATC CTA GAA GAG CAT CAA AGT Pro Gly With 355 Lys Ile below Pro Phe 360 Arg Ile Leu Glu Glu 365 His Gin Ser AGA CTA AAA GCG CTT TCT AAA ACC GTG GAA CTC GGG AAA CGA TTC TTC Arg Leu 370 Lys below Leu Ser Lys 375 Thr With Glu Leu Gly 380 Lys Arg Phe Phe CCG CGC TGT TCG GCA GTG CTC GAC CAG ATT ATG AAC TGT GAG GAC TTG Pro 385 Arg Cys Ser below With 390 Leu asp Gin Ile Met 395 Asn Cys Glu asp Leu 400 ACT CAA CTG GCT TGC GGA GAA GAC GAC ACT GCT GAG AAA CGA CTA CAA Thr Gin Leu below Cys 405 Gly Glu asp asp Thr 410 below Glu Lys Arg Leu 415 Gin AAG AAG CAA AGG TAC ATG GAA OVER THE CAA GAG ACA CTA AAG AAG GCC TTT Lys Lys Gin Arg 420 Tyr Met Glu Ile Gin 425 Glu Thr Leu Lys Lys 430 below Phe AGT GAG GAC AAT TTG GAA TTA GGA AAT TTG TCC CTG ACA DAM TCG ACT Ser Glu asp 435 Asn Leu Glu Leu Gly 440 Asn Leu Ser Leu Thr 445 asp Ser Thr TCT TCC ACA TCG AAA TCA ACC GGT GGA AAG AGG TCT AAC CGT AAA CTC Ser Ser 450 Thr Ser Lys Ser Thr 455 Gly Gly Lys Arg Ser 460 Asn Arg Lys Leu

TCT CAT CGT

CGT

CGG

TGA GACTCTTGCC TCTTAGTGTA ATTTTTGCTG

864

912

960

1008

1056

1104

1152

1200

1248

1296

1344

1392

1440

Ser His Arg Arg Arg * 465,470

TACCATATAA TTCTGTTTTC ATGATGACTG TAACTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATAG

1500

1560

1597

HU 224 480 Β1

(2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 470 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Met Asp Ser 1 With With 5 Thr With Leu Alá Tyr Val 10 Tyr Ser Ser Arg 15 Arg Pro Pro Pro Lys Gly With Ser Glu Cys Alá asp Glu Asn Cys 20 25 30 His Val Alá Cys Arg Pro below With Asp Phe Met Leu Glu With Leu 35 40 45 Leu Alá Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gin 50 55 60 His Leu Leu asp With With asp Lys Val Val Ile Glu asp Thr Leu 65 70 75 Ile Leu Lys Leu below Asn Ile Cys Gly Lys Alá Cys Met Lys Leu 85 90 95 Asp Arg Cys Lys Glu Ile Ile With Lys Ser Asn With asp Met With 100 105 110 Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile asp 115 120 125 Arg Lys Glu Leu Gly Leu Glu With Pro Lys Val Lys Lys His With 130 135 140 Asn Val His Lys below Leu asp Ser Asp Asp Ile Glu Leu With Lys 145 150 155 Leu Leu Lys Glu asp His Thr Asn Leu Asp Asp below Cys below Leu 165 170 175 Phe Alá Val below Tyr Cys Asn With Lys Thr Alá Thr asp Leu Leu 180 185 190 Leu Asp Leu below asp With Asn His Arg Asn Pro Arg Gly Tyr Thr 195 200 205 Leu His Val below below Seven Arg Lys Glu Pro Gin Leu Ile Leu Ser 210 215 220 Leu Glu Lys Gly below Ser below Ser Glu Alá Thr Leu Glu Gly Arg 225 230 235 Alá Leu Met Ile below Lys Gin below Thr Met Alá With Glu Cys Asn 245 250 255 Ile Pro Glu Gin Cys Lys His Ser Leu Lys Gly Arg Leu Cys With 260 265 270 Ile Leu Glu Gin Glu asp Lys Arg Glu Gin Ile Pro Arg asp With 275 280 285

With

Cys

Tyr

Arg

With

Leu

Ser

Arg

Ser

Leu

160

His

Lys

With

Leu

Thr

240

Asn

Glu

Pro

HU 224 480 Β1

Pro Ser 290 Phe Alá Val below below 295 asp Glu Leu Lys Met 300 Thr Leu Leu asp Leu Glu Asn Arg With below Leu below Gin Arg Leu Phe Pro Thr Glu below 305 310 315 320 Gin below below Met Glu Ile below Glu MeC Lys Gly Thr Cys Glu Phe Ile 325 330 335 With Thr Ser Leu Glu Pro asp Arg Leu Thr Gly Thr Lys Arg Thr Ser 340 345 350 Pro Gly With Lys Ile below Pro Phe Arg Ile Leu Glu Glu His Gin Ser 355 360 365 Arg Leu Lys below Leu Ser Lys Thr With Glu Leu Gly Lys Arg Phe Phe 370 375 380 Pro Arg Cys Ser below With Leu asp Gin Ile Met Asn Cys Glu asp Leu 385 390 395 400 Thr Gin Leu below Cys Gly Glu asp asp Thr below Glu Lys Arg Leu Gin 405 410 415 Lys Lys Gin Arg Tyr Met Glu Ile Gin Glu Thr Leu Lys Lys below Phe 420 425 430 Ser Glu asp Asn Leu Glu Leu Gly Asn Leu Ser Leu Thr asp Ser Thr 435 440 445 Ser Ser Thr Ser Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu 450 455 460 Ser His Arg Arg Arg ★

465 470 (2) INFORMATION FORUM SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1608 base pairs (Β) ΤΥΡΕ: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE ΤΥΡΕ: cDNA (ix) FEATURE:

(A) NAME / KEY: CDS (B) LOCATION: 43 ... 1608 (D) OTHER INFORMATION: /product=,.Altered form of NIM1 "/ note =" C-terminal deletion compared to wild-type NIM1. " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC 54

Met Asp Thr Thr

ATT Ile 5 DAM asp GGA Gly TTC Phe GCC below DAM asp 10 TCT Ser TAT Tyr GAA Glu ATC Ile AGC Ser 15 AGC Ser ACT Thr AGT Ser TTC Phe GTC With 20 102 GCT ACC DAM AAC ACC GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA 150 below Thr asp Asn Thr 25 asp Ser Ser Ile With 30 Tyr Leu below below Glu 35 Gin

HU 224 480 Β1

GTA With CTC Leu ACC Thr GGA Gly 40 CCT Pro DAM asp GTA With TCT Ser GCT Ala 45 CTG Leu CAA Gin TTG Leu CTC Leu TCC Ser 50 AAC Asn AGC Ser 198 TTC GAA TCC GTC TTT GAC TCG CCG DAM DAM TTC TAC AGC GAC GCT AAG 246 Phe Glu Ser With Phe asp Ser Pro asp asp Phe Tyr Ser asp Ala Lys 55 60 65 CTT GTT CTC TCC GAC GGC CGG GAA GTT TCT TTC CAC CGG TGC GTT TTG 294 Leu With Leu Ser asp Gly Arg Glu With Ser Phe His Arg Cys With Leu 70 75 80 TCA GCG AGA AGC TCT TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAG 342 Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala Ala Ala Lys Lys 85 90 95 100 GAG AAA GAC TCC AAC AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG 390 Glu Lys asp Ser Asn Asn Thr Ala Ala With Lys Leu Glu Leu Lys Glu 105 110 115 ATT GCC AAG DAM TAC GAA GTC GGT TTC DAM TCG GTT GTG ACT GTT TTG 438 Ile Ala Lys asp Tyr Glu With Gly Phe asp Ser With With Thr With Leu 120 125 130 GCT TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT 486 Ala Tyr With Tyr Ser Ser Arg With Arg Pro Pro Pro Lys Gly With Ser 135 140 145 GAA TGC GCA GAC GAG AAT TGC TGC CAC GTG GCT TGC CGG CCG GCG GTG 534 Glu Cys Ala asp Glu Asn Cys Cys His With Ala Cys Arg Pro Ala With 150 155 160 DAM TTC ATG TTG GAG GTT CTC TAT TTG GCT TTC ATC TTC AAG ATC CCT 582 asp Phe Met Leu Glu With Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro 165 170 175 180 GAA TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA 630 Glu Leu Ile Thr Leu Tyr Gin Arg His Leu Leu asp With With asp Lys 185 190 195 GTT GTT OVER THE GAG GAC ACA TTG GTT OVER THE CTC AAG CTT GCT AAT OVER THE TGT 678 With With Ile Glu asp Thr Leu With Ile Leu Lys Leu Ala Asn Ile Cys 200 205 210 GGT AAA GCT TGT ATG AAG CTA TTG DAM AGA TGT AAA GAG ATT ATT GTC 726 Gly Lys Ala Cys Met Lys Leu Leu asp Arg Cys Lys Glu Ile Ile With 215 220 225 AAG TCT AAT GTA DAM ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA GAG 774 Lys Ser Asn With asp Met With Ser Leu Glu Lys Ser Leu Pro Glu Glu 230 235 240 CTT GTT AAA GAG OVER THE ATT DAM AGA CGT AAA GAG CTT GGT TTG GAG GTA 822 Leu With Lys Glu Ile Ile asp Arg Arg Lys Glu Leu Gly Leu Glu With 245 250 255 260 CCT AAA GTA AAG AAA CAT GTC TCG AAT GTA CAT AAG GCA CTT GAC TCG 870 Pro Lys With Lys Lys His With Ser Asn With His Lys Ala Leu asp Ser 265 270 275

HU 224 480 Β1

DAM asp DAM asp ATT Ile GAG Glu 280 TTA Leu GTC With AAG Lys TTG Leu CTT Leu 285 TTG Leu AAA Lys GAG Glu DAM asp CAC His 290 ACC Thr AAT Asn 918 CTA DAM DAM GCG TGT GCT CTT CAT TTC GCT GTT GCA TAT TGC AAT GTG 966 Leu asp asp below Cys below Leu His Phe below With below Tyr Cys Asn With 295 300 305 AAG ACC GCA ACA DAM CTT TTA AAA CTT DAM CTT GCC DAM GTC AAC CAT 1014 Lys Thr below Thr asp Leu Leu Lys Leu asp Leu below asp With Asn His 310 315 320 AGG AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG AAG 1062 Arg Asn Pro Arg Gly Tyr Thr With Leu His With below below Met Arg Lys 325 330 335 340 GAG CCA CAA TTG OVER THE CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA 1110 Glu Pro Gin Leu Ile Leu Ser Leu Leu Glu Lys Gly below Ser below Ser 345 350 355 GAA GCA ACT TTG GAA GGT AGA ACC GCA CTC ATG ATC GCA AAA CAA GCC 1158 Glu below Thr Leu Glu Gly Arg Thr below Leu Met Ile below Lys Gin below 360 365 370 ACT ATG GCG GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT TCT 1206 Thr Met below With Glu Cys Asn Asn Ile Pro Glu Gin Cys Lys His Ser 375 380 385 CTC AAA GGC CGA CTA TGT GTA GAA OVER THE CTA GAG CAA GAA GAC AAA CGA 1254 Leu Lys Gly Arg Leu Cys With Glu Ile Leu Glu Gin Glu asp Lys Arg 390 395 400 CAA CAA ATT CCT AGA DAM GTT CCT CCC TCT TTT GCA GTG GCG GCC DAM 1302 Glu Gin Ile Pro Arg asp With Pro Pro Ser Phe below With below below asp 405 410 415 420 GAA TTG AAG ATG ACG CTG CTC DAM CTT GAA AAT AGA GTT GCA CTT GCT 1350 Glu Leu Lys Met Thr Leu Leu asp Leu Glu Asn Arg With below Leu below 425 430 435 CAA CGT CTT TTT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC GAA 1398 Gin Arg Leu Phe Pro Thr Glu below Gin below below Met Glu Ile below Glu 440 445 450 ATG AAG GGA ACA TGT GAG TTC OVER THE GTG ACT AGC CTC GAG CCT GAC CGT 1446 Met Lys Gly Thr Cys Glu Phe Ile With Thr Ser Leu Glu Pro asp Arg 455 460 465 CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAG OVER THE GCA CCT TTC 1494 Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly With Lys Ile below Pro Phe 470 475 480 AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA ACC 1542 Arg Ile Leu Glu Glu His Gin Ser Arg Leu Lys below Leu Ser Lys Thr 485 490 495 500 GTG GAA CTC GGG AAA CGA TTC TTC CCG CGC TGT TCG GCA GTG CTC GAC 1590 With Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser below With Leu asp 505 510 515

HU 224 480 Β1

CAG ATT ATG AAC TGT TGA Gin Ile Met Asn Cys *

520 (2) INFORMATION FORUM SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 522 amino acids (Β) ΤΥΡΕ: amino acid (D) TOPOLOGY: linear (ii) MOLECULE ΤΥΡΕ: protein

1608

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Tyr Glu Ile Ser 15 Ser Met 1 asp Thr Thr Ile 5 asp Gly Phe below asp 10 Ser Thr Ser Phe Val below Thr asp Asn Thr asp Ser Ser Ile With Tyr Leu 20 25 30 below below Glu Gin With Leu Thr Gly Pro asp With Ser below Leu Gin Leu 35 40 45 Leu Ser Asn Ser Phe Glu Ser With Phe asp Ser Pro asp asp Phe Tyr 50 55 60 Ser asp Under Lys Leu With Leu Ser asp Gly Arg Glu With Ser Phe His 65 70 75 80 Arg Cys Val Leu Ser below Arg Ser Ser Phe Phe Lys Ser below Leu below 85 90 95 below below Lys Lys Glu Lys asp Ser Asn Asn Thr below below With Lys Leu 100 105 110 Glu Leu Lys Glu Ile below Lys asp Tyr Glu With Gly Phe asp Ser With 115 120 125 With Thr Val Leu below Tyr With Tyr Ser Ser Arg With Arg Pro Pro Pro 130 135 140 Lys Gly Val Ser Glu Cys below asp Glu Asn Cys Cys His With below Cys 145 150 155 160 Arg Pro Ala Val asp Phe Met Leu Glu With Leu Tyr Leu below Phe Ile 165 170 175 Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gin Arg His Leu Leu asp 180 185 190 With With Asp Lys With With Ile Glu asp Thr Leu With Ile Leu Lys Leu 195 200 205 below Asn Ile Cys Gly Lys below Cys Met Lys Leu Leu asp Arg Cys Lys 210 215 220 Glu Ile Ile Val Lys Ser Asn With asp Met With Ser Leu Glu Lys Ser 225 230 235 240 Leu Pro Glu Glu Leu With Lys Glu Ile Ile asp Arg Arg Lys Glu Leu 245 250 255

HU 224 480 Β1

Gly Leu Glu With Pro Lys With Lys Lys His With Ser Asn With His Lys 260 265 270 below Leu Asp Ser asp asp Ile Glu Leu With Lys Leu Leu Leu Lys Glu 275 280 285 asp His Thr Asn Leu asp asp below Cys below Leu His Phe below With below 290 295 300 Tyr Cys Asn With Lys Thr below Thr asp Leu Leu Lys Leu asp Leu below 305 310 315 320 asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr With Leu His With below 325 330 335 below Met Arg Lys Glu Pro Gin Leu Ile Leu Ser Leu Leu Glu Lys Gly 340 345 350 below Ser Ala Ser Glu below Thr Leu Glu Gly Arg Thr below Leu Met Ile 355 360 365 below Lys Gin below Thr Met below With Glu Cys Asn Asn Ile Pro Glu Gin 370 375 380 Cys Lys His Ser Leu Lys Gly Arg Leu Cys With Glu Ile Leu Glu Gin 385 390 395 400 Glu Asp Lys Arg Glu Gin Ile Pro Arg asp With Pro Pro Ser Phe below 405 410 415 With Ala Ala asp Glu Leu Lys Met Thr Leu Leu asp Leu Glu Asn Arg 420 425 430 With Down Leu below Gin Arg Leu Phe Pro Thr Glu below Gin below below Met 435 440 445 Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile With Thr Ser Leu 450 455 460 Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly With Lys 465 470 475 480 Ile Down Pro Phe Arg Ile Leu Glu Glu His Gin Ser Arg Leu Lys below 485 490 495 Leu Ser Lys Thr With Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser 500 505 510 below Val Leu asp Gin Ile Met Asn Cys ★ 515 520 (2) INFORMATION FORUM SEQ ID NO: 13: (I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1194 base pairs (Β) ΤΥΡΕ: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE ΤΥΡΕ: CDNA (ix) FEATURE: (A) NAME / KEY: CDS

HU 224,480 Β1 (B) LOCATION: 1 ... 1194 (D) OTHER INFORMATION: / product = "Altered form of NIM1" / note = "N-terminal / C-terminal chimera."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TAT Tyr 10 GTT With TAC AGC AGC AGA GTG 48 ATG Met 1 DAM asp TCG Ser GTT With GTG ACT GTT With TTG GCT With 5 Thr Leu below Tyr Ser Ser Arg 15 With AGA CCG CCG CCT AAA GGA GTT TCT GAA TGC GCA GAC GAG AAT TGC TGC 96 Arg Pro Pro Pro Lys Gly With Ser Glu Cys below asp Glu Asn Cys Cys 20 25 30 CAC GTG GCT TGC CGG CCG GCG GTG DAM TTC ATG TTG GAG GTT CTC TAT 144 His With below Cys Arg Pro below With asp Phe Met Leu Glu With Leu Tyr 35 40 45 TTG GCT TTC ATC TTC AAG ATC CCT GAA TTA ATT ACT CTC TAT CAG AGG 192 Leu below Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gin Arg 50 55 60 CAC TTA TTG GAC GTT GTA GAC AAA GTT GTT OVER THE GAG GAC ACA TTG GTT 240 His Leu Leu asp With With asp Lys With With Ile Glu asp Thr Leu With 65 70 75 80 OVER THE CTC AAG CTT GCT AAT OVER THE TGT GGT AAA GCT TGT ATG AAG CTA TTG 288 Ile Leu Lys Leu below Asn Ile Cys Gly Lys below Cys Met Lys Leu Leu 85 90 95 DAM AGA TGT AAA GAG ATT ATT GTC AAG TCT AAT GTA DAM ATG GTT AGT 336 asp Arg Cys Lys Glu Ile Ile With Lys Ser Asn With asp Met With Ser 100 105 110 CTT GAA AAG TCA TTG CCG GAA GAG CTT GTT AAA GAG OVER THE ATT DAM AGA 384 Leu Glu Lys Ser Leu Pro Glu Glu Leu With Lys Glu Ile Ile asp Arg 115 120 125 CGT AAA GAG CTT GGT TTG GAG GTA CCT AAA GTA AAG AAA CAT GTC TCG 432 Arg Lys Glu Leu Gly Leu Glu With Pro Lys With Lys Lys His With Ser 130 135 140 AAT GTA CAT AAG GCA CTT GAC TCG DAM DAM ATT GAG TTA GTC AAG TTG 480 Asn With His Lys below Leu asp Ser asp asp Ile Glu Leu With Lys Leu 145 150 155 160 CTT TTG AAA GAG DAM CAC ACC AAT CTA DAM DAM GCG TGT GCT CTT CAT 528 Leu Leu Lys Glu asp His Thr Asn Leu asp asp below Cys below Leu His 165 170 175 TTC GCT GTT GCA TAT TGC AAT GTG AAG ACC GCA ACA DAM CTT TTA AAA 576 Phe below With below Tyr Cys Asn With Lys Thr below Thr asp Leu Leu Lys 180 185 190 CTT DAM CTT GCC DAM GTC AAC CAT AGG AAT CCG AGG GGA TAT ACG GTG 624 Leu asp Leu below asp With Asn His Arg Asn Pro Arg Gly Tyr Thr With 195 200 205 CTT CAT GTT GCT GCG ATG CGG AAG GAG CCA CAA TTG OVER THE CTA TCT CTA 672 Leu His With below below Met Arg Lys Glu Pro Gin Leu Ile Leu Ser Leu

210 215 220

HU 224 480 Β1

TTG Leu 225 GAA AAA GGT Gly GCA below AGT Ser 230 GCA below TCA Ser GAA Glu GCA below ACT Thr 235 TTG Leu GAA Glu GGT Gly AGA Arg ACC Thr 240 720 Glu Lys GCA CTC ATG ATC GCA AAA CAA GCC ACT ATG GCG GTT GAA TGT AAT AAT 768 below Leu Met Ile below Lys Gin below Thr Met below With Glu Cys Asn Asn 245 250 255 ATC CCG GAG CAA TGC AAG CAT TCT CTC AAA GGC CGA CTA TGT GTA GAA 816 Ile Pro Glu Gin Cys Lys His Ser Leu Lys Gly Arg Leu Cys With Glu 260 265 270 OVER THE CTA GAG CAA GAA GAC AAA CGA GAA CAA ATT CCT AGA DAM GTT CCT 864 Ile Leu Glu Gin Glu asp Lys Arg Glu Gin Ile Pro Arg asp With Pro 275 280 285 CCC TCT TTT GCA GTG GCG GCC DAM GAA TTG AAG ATG ACG CTG CTC DAM 912 Pro Ser Phe below With below below asp Glu Leu Lys Met Thr Leu Leu asp 290 295 300 CTT GAA AAT AGA GTT GCA CTT GCT CAA CGT CTT TTT CCA ACG GAA GCA 960 Leu Glu Asn Arg With below Leu below Gin Arg Leu Phe Pro Thr Glu below 305 310 315 320 CAA GCT GCA ATG GAG ATC GCC GAA ATG AAG GGA ACA TGT GAG TTC OVER THE 1008 Gin below below Met Glu Ile below Glu Met Lys Gly Thr Cys Glu Phe Ile 325 330 335 GTG ACT AGC CTC GAG CCT GAC CGT CTC ACT GGT ACG AAG AGA ACA TCA 1056 With Thr Ser Leu Glu Pro asp Arg Leu Thr Gly Thr Lys Arg Thr Ser 340 345 350 CCG GGT GTA AAG OVER THE GCA CCT TTC AGA ATC CTA GAA GAG CAT CAA AGT 1104 Pro Gly With Lys Ile below Pro Phe Arg Ile Leu Glu Glu His Gin Ser 355 360 365 AGA CTA AAA GCG CTT TCT AAA ACC GTG GAA CTC GGG AAA CGA TTC TTC 1152 Arg Leu Lys below Leu Ser Lys Thr With Glu Leu Gly Lys Arg Phe Phe 370 375 380 CCG CGC TGT TCG GCA GTG CTC GAC CAG ATT ATG AAC TGT TGA 1194 Pro Arg Cys Ser below With Leu asp Gin Ile Met Asn Cys k 385 390 395

(2) INFORMATION FORUM SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 398 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Met asp Ser With With Thr With Leu below Tyr With Tyr Ser Ser Arg With 1 5 10 15 Arg Pro Pro Pro Lys Gly With Ser Glu Cys below asp Glu Asn Cys Cys 20 25 30 His With below Cys Arg Pro below With asp Phe Met Leu Glu With Leu Tyr

40 45

HU 224 480 Β1

Leu below 50 Phe Ile Phe Lys Ile 55 Pro Glu Leu Ile Thr 60 Leu Tyr Gin Arg His Leu Leu asp With With asp Lys With With Ile Glu asp Thr Leu With 65 70 75 80 Ile Leu Lys Leu below Asn Ile Cys Gly Lys below Cys Met Lys Leu Leu 85 90 95 asp Arg Cys Lys Glu Ile Ile With Lys Ser Asn With asp Met With Ser 100 105 110 Leu Glu Lys Ser Leu Pro Glu Glu Leu With Lys Glu Ile Ile asp Arg 115 120 125 Arg Lys Glu Leu Gly Leu Glu With Pro Lys With Lys Lys His With Ser 130 135 140 Asn With His Lys below Leu asp Ser asp asp Ile Glu Leu With Lys Leu 145 150 155 160 Leu Leu Lys Glu asp His Thr Asn Leu asp asp below Cys below Leu His 165 170 175 Phe below With below Tyr Cys Asn With Lys Thr below Thr asp Leu Leu Lys 180 185 190 Leu asp Leu below asp With Asn His Arg Asn Pro Arg Gly Tyr Thr With 195 200 205 Leu His With below below Met Arg Lys Glu Pro Gin Leu Ile Leu Ser Leu 210 215 220 Leu Glu Lys Gly below Ser below Ser Glu below Thr Leu Glu Gly Arg Thr 225 230 235 240 below Leu Met Ile below Lys Gin below Thr Met below With Glu Cys Asn Asn 245 250 255 Ile Pro Glu Gin Cys Lys His Ser Leu Lys Gly Arg Leu Cys With Glu 260 265 270 Ile Leu Glu Gin Glu asp Lys Arg Glu Gin Ile Pro Arg asp With Pro 275 280 285 Pro Ser Phe below With below below asp Glu Leu Lys Met Thr Leu Leu asp 290 295 300 Leu Glu Asn Arg With below Leu below Gin Arg Leu Phe Pro Thr Glu below 305 310 315 320 Gin below below Met Glu Ile below Glu Met Lys Gly Thr Cys Glu Phe Ile 325 330 335 With Thr Ser Leu Glu Pro asp Arg Leu Thr Gly Thr Lys Arg Thr Ser 340 345 350 Pro Gly With Lys Ile below Pro Phe Arg Ile Leu Glu Glu His Gin Ser

355 360 365

HU 224 480 Β1

Arg Leu Lys Alá Leu Ser Lys Thr Val Glu Teu Gly Lys Arg Phe Phe 370 375 380

Pro Arg Cys Ser Alá Val Leu Asp Gin Ile Met Asn Cys *

385 390 395 (2) INFORMATION FORUM SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 786 base pairs (Β) ΤΥΡΕ: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE ΤΥΡΕ: cDNA (ix) FEATURE:

(A) NAME / KEY: CDS (B) LOCATION: 1 ... 786 (D) OTHER INFORMATION: / product =, Altered form of NIM1 "/ note =" Ankyrin domains of NIM1. "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GAG Glu CTT AAG GAG Glu 15 ATT Ile 48 ATG Met 1 GAC asp TCC AAC Ser Asn AAC Asn 5 ACC Thr GCC below GCC below GTG AAG CTC With Lys 10 Leu Leu Lys GCC AAG DAM TAC GAA GTC GGT TTC DAM TCG GTT GTG ACT GTT TTG GCT 96 below Lys asp Tyr Glu With Gly Phe asp Ser With With Thr With Leu below 20 25 30 TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT GAA 144 Tyr With Tyr Ser Ser Arg With Arg Pro Pro Pro Lys Gly With Ser Glu 35 40 45 TGC GCA GAC GAG AAT TGC TGC CAC GTG GCT TGC CGG CCG GCG GTG DAM 192 Cys below asp Glu Asn Cys Cys His With below Cys Arg Pro below With asp 50 55 60 TTC ATG TTG GAG GTT CTC TAT TTG GCT TTC ATC TTC AAG ATC CCT GAA 240 Phe Seven Leu Glu With Leu Tyr Leu below Phe Ile Phe Lys Ile Pro Glu 65 70 75 80 TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA GTT 288 Leu Ile Thr Leu Tyr Gin Arg His Leu Leu asp With With asp Lys With 85 90 95 GTT OVER THE GAG GAC ACA TTG GTT OVER THE CTC AAG CTT GCT AAT OVER THE TGT GGT 336 With Ile Glu asp Thr Leu With Ile Leu Lys Leu below Asn Ile Cys Gly 100 105 110 AAA GCT TGT ATG AAG CTA TTG DAM AGA TGT AAA GAG ATT ATT GTC AAG 384 Lys below Cys Met Lys Leu Leu asp Arg Cys Lys Glu Ile Ile With Lys 115 120 125 TCT AAT GTA DAM ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA GAG CTT 432 Ser Asn With asp Met With Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu 130 135 140 GTT AAA GAG OVER THE ATT DAM AGA CGT AAA GAG CTT GGT TTG GAG GTA CCT 480 With Lys Glu Ile Ile asp Arg Arg Lys Glu Leu Gly Leu Glu With Pro 145 150 155 160

HU 224 480 Β1

528

AAA Lys GTA AAG AAA CAT GTC With TCG AAT Ser Asn GTA With CAT His 170 AAG Lys GCA below CTT Leu GAC asp TCG Ser 175 DAM asp With Lys Lys His 165 DAM ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG DAM CAC ACC AAT CTA asp Ile Glu Leu With Lys Leu Leu Leu Lys Glu asp His Thr Asn Leu 180 185 190 DAM DAM GCG TGT GCT CTT CAT TTC GCT GTT GCA TAT TGC AAT GTG AAG asp asp below Cys below Leu His Phe below With below Tyr Cys Asn With Lys 195 200 205 ACC GCA ACA DAM CTT TTA AAA CTT DAM CTT GCC DAM GTC AAC CAT AGG Thr below Thr asp Leu Leu Lys Leu asp Leu below asp With Asn His Arg 210 215 220 AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG AAG GAG Asn Pro Arg Gly Tyr Thr With Leu His With below below Met Arg Lys Glu 225 230 235 240 CCA CAA TTG OVER THE CTA TGT CTA TTG GAA AAA GGT GCA AGT GCA TCA GAA Pro Gin Leu Ile Leu Ser Leu Leu Glu Lys Gly below Ser below Ser Glu 245 250 255 GCA ACT TTG GAA GGT TGA below Thr Leu Glu Gly ★

260

576

624

672

720

768

786 (2) INFORMATION FORUM SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 262 amino acids (Β) ΤΥΡΕ: amino acid (D) TOPOLOGY: linear (ii) MOLECULE ΤΥΡΕ: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

Met Asp Ser Asn Asn Thr Alá Alá Val Lys Leu Glu Leu Lys Glu Ile

10 15

Alá Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Al 20 25 30

Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu 35 40 45

Cys Alá Asp Glu Asn Cys Cys His Val Alá Cys Arg Pro Alá Val Asp 50 55 60

Phe Met Leu Glu Val Leu Tyr Leu Alá Phe Ile Phe Lys Ile Pro Glu

70 75 80

Leu Ile Thr Leu Tyr Gin Arg His Leu Leu Asp Val Val Asp Lys Val

90 95

Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Alá Asn Ile Cys Gly 100 105 110

Lys Alá Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val Lys 115 120 125

HU 224 480 Β1

Ser Asn Val asp Met With Ser 135 Leu Glu Lys Ser Leu 140 Pro Glu Glu 130 With Lys Glu Ile Ile asp Arg Arg Lys Glu Leu Gly Leu Glu With 145 150 155 Lys With Lys Lys His With Ser Asn With His Lys below Leu asp Ser 165 170 175 asp Ile Glu Leu With Lys Leu Leu Leu Lys Glu asp His Thr Asn 180 185 190 asp asp below Cys below Leu His Phe below With below Tyr Cys Asn With 195 200 205 Thr below Thr asp Leu Leu Lys Leu asp Leu below asp With Asn His 210 215 220 Asn Pro Arg Gly Tyr Thr With Leu His With below below Met Arg Lys 225 230 235 Pro Gin Leu Ile Leu Ser Leu Leu Glu Lys Gly below Ser below Ser 245 250 255

Leu

Pro

160

asp

Leu

Lys

Arg

Glu

240

Glu

Alá Thr Leu Glu Gly *

260 (2) INFORMATION FORUM SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 amino acids (Β) ΤΥΡΕ: amino acid (C) STRANDEDNESS: female relevant (D) TOPOLOGY: female relevant (ii) MOLECULE ΤΥΡΕ: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

Ile 1 Arg Arg Met Arg 5 Arg below Leu Asp Alá Alá Asp 10 Ile Glu Leu 15 Lys Leu Met With Met Gly Glu Gly Leu asp Leu asp asp below Leu 20 25 30 With His Tyr below With Gin His Cys Asn

40 (2) INFORMATION FORUM SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 amino acids (Β) ΤΥΡΕ: amino acid (C) STRANDEDNESS: female relevant (D) TOPOLOGY: female relevant (ii) MOLECULE ΤΥΡΕ: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

With

below

Pro 1 Thr Gly Lys Thr 5 below Leu His Leu below 10 below Glu Met With Ser 15 asp Met With Ser With Leu Leu asp His His below asp Xaa Asn Phe 20 25 30 Thr Xaa asp Gly With Thr

Pro

Arg

EN 224 480 Β1 (2) INFORMATION FORUM SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 amino acids (Β) ΤΥΡΕ: amino acid (C) STRANDEDNESS: female relevant (D) TOPOLOGY: female relevant (ii) MOLECULE ΤΥΡΕ: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

Ile Arg Arg Met Arg Arg below Leu asp below below asp Ile Glu Leu With 1 5 10 15 Lys Leu Met With Mec Gly Glu Gly Leu asp Leu asp asp below Leu below 20 25 30 With His Tyr below With Gin His Cys Asn

40 (2) INFORMATION FORUM SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 amino acids (Β) ΤΥΡΕ: amino acid (C) STRANDEDNESS: female relevant (D) TOPOLOGY: female relevant (ii) MOLECULE ΤΥΡΕ: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: His 10 Leu Alá Alá Glu Met Val 15 Arg Arg 1 Pro Asp Ser 5 Lys Thr below Leu Ser Pro Asp Met With Ser With Leu Leu asp Gin 20 25

(2) INFORMATION FORUM SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 amino acids (Β) ΤΥΡΕ: amino acid (C) STRANDEDNESS: female relevant (D) TOPOLOGY: female relevant (ii) MOLECULE ΤΥΡΕ: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

Ile 1 Arg Arg Met Arg Arg 5 below Leu Asp Alá 10 below asp Ile Glu Leu 15 With Lys Leu Met With Met Gly Glu Gly Leu asp Leu asp asp below Leu below 20 25 30 With His Tyr below With Gin His Cys Asn

40 (2) INFORMATION FORUM SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 amino acids (Β) ΤΥΡΕ: amino acid (C) STRANDEDNESS: female relevant (D) TOPOLOGY: female relevant (ii) MOLECULE ΤΥΡΕ: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

Arg Arg Pro Asp Ser Lys Thr Alá Leu His Leu Alá Alá Glu Met Val

10 15

HU 224 480 Β1

Ser Pro Asp Met Val Ser Val Leu Leu Asp Gin 20 25 (2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 amino acids (B) TYPE: anino acid (C) STRANDEDNESS: female relevant (D) TOPOLOGY: female relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

Ile Arg Arg Met Arg Arg below Leu asp below below asp Ile Glu Leu With 1 5 10 15 Lys Leu Met With Met Gly Glu Gly Leu asp Leu asp asp below Leu below 20 25 30 With His Tyr below With Gin His Cys Asn

40 (2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: female relevant (D) TOPOLOGY: female relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

Pro Thr Gly Lys Thr Alá Leu His Leu Alá Alá Glu Met Val Ser Pro

10 15

Asp Met Val (2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(x) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

CAACAGCTTC GAAGCCGTCT TTGACGCGCC GGATG 35 (2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

CATCCGGCGC GTCAAAGACG GCTTCGAAGC TGTTG 35 (2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single

HU 224 480 Β1 (D) TOPOLOGY: linear (ii) MOLECULE ΤΥΡΕ: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

GGAATTCAAT GGATTCGGTT GTGACTGTTT TG 32 (2) INFORMATION FORUM SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs (Β) ΤΥΡΕ: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE ΤΥΡΕ: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

GGAATTCTAC AAATCTGTAT ACCATTGG 28 (2) INFORMATION FORUM SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs (Β) ΤΥΡΕ: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE ΤΥΡΕ: other nucleic acid (A) DESCRIPTION: / desc =, oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 29:

CGGAATTCGA TCTCTTTAAT TTGTGAATTT C 31 (2) INFORMATION FORUM SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs (Β) ΤΥΡΕ: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE ΤΥΡΕ: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

GGAATTCTCA ACAGTTCATA ATCTGGTCG 29 (2) INFORMATION FORUM SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs (Β) ΤΥΡΕ: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE ΤΥΡΕ: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

GGAATTCAAT GGACTCCAAC AACACCGCCG C 31 (2) INFORMATION FORUM SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs (Β) ΤΥΡΕ: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE ΤΥΡΕ: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

GGAATTCTCA ACCTTCCAAA GTTGCTTCTG ATG 33

Claims (25)

PATENT CLAIMS A method of protecting plants against pathogen attack, comprising the steps of: (a) providing a plant transformed with a chimeric gene, said gene comprising a promoter active in plants operably linked to: (i) a nucleotide sequence encoding a wild-type NIM1 protein that participates in a signal transduction cascade leading to systemic acquired resistance (SAR) in plants; as a regulator, wherein the resulting plant has a first-degree disease resistance; and (b) applying to said plant at least one microbicide that imparts to the plant a second level of disease resistance; wherein the application of the microbicide to said plant provides a synergistically increased level of disease resistance greater than the sum of the first and second levels of disease resistance. The method of claim 1, wherein the wild-type NIM1 protein comprises the amino acid sequence set forth in SEQ ID NO: 2. The method of claim 1, wherein the nucleotide sequence encoding the wild-type NIM1 protein comprises the coding sequence set forth in SEQ ID NO: 1. 4. The method of claim 1, wherein the modified form of the NIM1 protein comprises alanine instead of serine at amino acid positions 55 and 59 of SEQ ID NO: 2. The method of claim 4, wherein the modified form of the NIM1 protein is SEQ ID NO: 8 contains the amino acid sequence. The method of claim 4, wherein the modified form of the NIM1 protein is encoded by the nucleotide sequence shown in SEQ ID NO: 7. The method of claim 1, wherein the modified form of the NIM1 protein is about 1-125 of SEQ ID NO: 2. has a truncated N-terminal amino acid sequence corresponding to its amino acid positions. 8. The method of claim 7, wherein the modified form of the NIM1 protein comprises the amino acid sequence set forth in SEQ ID NO: 10. The method of claim 7, wherein the modified form of the NIM1 protein is as defined in SEQ ID NO: 9 is encoded by the nucleotide sequence. 10. The method of claim 1, wherein the modified form of the NIM1 protein is SEQ ID NO: 2 is about 522-593. has a truncated C-terminal amino acid sequence corresponding to its amino acid position. 11. The method of claim 10, wherein the modified form of the NIM1 protein is SEQ ID NO. 12 contains the amino acid sequence. The method of claim 10, wherein the modified form of the NIM1 protein is encoded by the nucleotide sequence shown in SEQ ID NO: 11. 13. The method of claim 1, wherein the modified form of the NIM1 protein is about 1-125 of SEQ ID NO: 2. truncated N-terminal corresponding to the amino acid positions of SEQ ID NO: 2 and about 522-593 of SEQ ID NO: 2. has a truncated C-terminal amino acid sequence corresponding to its amino acid positions. The method of claim 13, wherein the modified form of the NIM1 protein comprises the amino acid sequence set forth in SEQ ID NO: 14. 15. The method of claim 13, wherein the modified form of the NIM1 protein is encoded by the nucleotide sequence shown in SEQ ID NO: 13. 16. The method of claim 1, wherein the modified form of the NIM1 protein is substantially about 103-362 of SEQ ID NO: 2. consists of ankyrin motifs corresponding to its amino acid positions. 17. The method of claim 16, wherein the modified form of the NIM1 protein comprises the amino acid sequence set forth in SEQ ID NO: 16. 18. The method of claim 16, wherein the modified form of the NIM1 protein is encoded by the nucleotide sequence shown in SEQ ID NO: 15. 19. The method of any one of claims 1, 4, 7, 10, 13 or 16, wherein the NIM1 protein is encoded by a nucleotide sequence which, under the following conditions, is SEQ ID NO: 1,7. , 9, 11, 13 or 15: hybridization in 1% BSA; 520 mM NaPO ₄ , pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours and wash in 6 * SSC for 15 minutes (3 *), 3 * SSC for 15 minutes (1 *) at 55 ° C. 20. The process according to any one of claims 1 to 3, wherein the microbicide is a fungicide as follows: 4- [3- (4-chlorophenyl) -3- (3,4-dimethoxyphenyl) acryloyl] morpholine ("dimethomorph"); 5-methyl-1,2,4-triazolo [3,4-b] [1,3] benzothiazole ("tricyclazole"); 3-Allyloxy-1,2-benzothiazole-1,1-dioxide ("probonazole"); α- [2- (4-Chloro-phenyl) -ethyl] -α- (1,1-dimethylethyl) -1H-1,2,4-triazole-1-ethanol ("tebuconazole"); 1 - [[3- (2-chlorophenyl) -2- (4-fluorophenyl) oxiran-2-yl] methyl] -1H-1,2,4-triazole ("epoxyconazole"); α- (4-chlorophenyl) -α- (1-cyclopropylethyl) -1H-1,2,4-triazole-1-ethanol ("cyproconazole"); HU 224 480 Β1 5- (4-chlorobenzyl) -2,2-dimethyl-1- (1H-1,2,4-triazol-1-ylmethyl) cyclopentanol ("metconazole"); 2- (2,4-Dichlorophenyl) -3- (1H-1,2,4-triazol-1-yl) propyl-1,1,2,2-tetrafluoroethyl ether ("tetraconazole") '); methyl (E) -2- {2- [6- (2-cyanophenoxy) pyrimidin-4-yloxy] phenyl} -3-methoxyacrylate ("ICIA 5504", "azoxystrobin"); methyl (E) -2-methoxyimino-2- [α- (o-tolyloxy) -o-tolyl] acetate ("BAS 490 F", cresoxime methyl); 2- (2-phenoxyphenyl) - (E) -2-methoxyimino-N-methyl-acetamide; [2- (2,5-dimethylphenoxy) phenyl] - (E) -2-methoxyimino-N-methylacetamide; (1R, 3S / 1S, 3R) -2,2-Dichloro-N - [(R) -1- (4-chloro-phenyl) -ethyl] -ethyl-3-methyl-cyclopropanecarboxamide ("KTU 3616" ); ethylene bis (dithiocarbamate) polymer manganese zinc complex ("mancozeb"); 1- [2- (2,4-Dichlorophenyl) -4-propyl-1,3-dioxolan-2-ylmethyl] -1H-1,2,4-triazole ("propiconazole"); 1- {2- [2-Chloro-4- (4-chloro-phenoxy) -phenyl] -4-methyl-1,3-dioxolan-2-ylmethyl} -1H-1,2,4-triazole ( "difenoconazole"); 1- (2- (2,4-dichlorophenyl) phenyl] -1H-1,2,4-triazole ("penconazole"); cis-4- [3- (4-tert-butyl-phenyl) -2-methyl-propyl] -2,6-dimethyl-morpholine ("fenpropimorph"); 1- [3- (4-tert-Butyl-phenyl) -2-methyl-propyl] -piperidine ("fenpropidin"); 4-cyclopropyl-6-methyl-N-phenyl-2-pyrimidinamine ("cyprodinyl"); (RS) -N- (2,6-dimethylphenyl) -N- (methoxyacetyl) alanine methyl ester ("metalaxyl"); (R) -N- (2,6-Dimethylphenyl) -N- (methoxyacetyl) alanine methyl ester ("R-metalaxyl"); 1,2,5,6-Tetrahydro-4H-pyrrolo [3,2,1-ij] quinolin-4-one ("pyroquilone") or ethyl hydrogen phosphonate ("fosetyl"). 21. The method of claim 20, wherein the fungicide is metalaxyl. 22. A process according to any one of claims 1 to 5, characterized in that the microbicide is a benzothiadiazole, an isonicotinic acid or a salicylic acid compound. 23. A process according to any one of claims 1 to 3, characterized in that two microbicides are applied simultaneously to the plant. 24. The process of claim 23 wherein one of the microbicides is a fungicide: 4- [3- (4-chlorophenyl) -3- (3,4-dimethoxyphenyl) acryloyl] morpholine ( "dimethomorph"); 5-methyl-1,2,4-triazolo [3,4-b] [1,3] benzothiazole ("tricyclazole"); 3-Allyloxy-1,2-benzothiazole-1,1-dioxide ("probonazole"); α- [2- (4-chlorophenyl) ethyl] -α- (1,1-dimethylethyl) -1H-1,2,4-triazole-1-ethanol ("tebuconazole"); 1 - [[3- (2-chlorophenyl) -2- (4-fluorophenyl) oxiran-2-yl] methyl] -1H-1,2,4-triazole ("epoxyconazole"); α- (4-chlorophenyl) -α- (1-cyclopropylethyl) -1H-1,2,4-triazole-1-ethanol ("cyproconazole"); 5- (4-chlorobenzyl) -2,2-dimethyl-1- (1H-1,2,4-triazol-1-ylmethyl) cyclopentanol ("metconazole"); 2- (2,4-Dichlorophenyl) -3- (1H-1,2,4-triazol-1-yl) -propyl-1,1,2,2-tetrafluoroethyl ether ("tetraconazole") '); methyl (E) -2- {2- [6- (2-cyanophenoxy) pyrimidin-4-yloxy] phenyl} -3-methoxyacrylate ("ICI A 5504", "azoxystrobin"); methyl (E) -2-methoxyimino-2- [α- (o-tolyloxy) -o-tolyl] acetate ("BAS 490 F", cresoxime methyl); 2- (2-phenoxyphenyl) - (E) -2-methoxyimino-N-methylacetamide; [2- (2,5-dimethylphenoxy) phenyl] - (E) -2-methoxyimino-N-methylacetamide; (1R, 3S / 1S, 3R) -2,2-Dichloro-N - [(R) -1- (4-chloro-phenyl) -ethyl] -ethyl-3-methyl-cyclopropanecarboxamide ("KTU 3616") ; ethylene bis (dithiocarbamate) polymer manganese zinc complex ("mancozeb"); 1- (2- (2,4-dichloro-phenyl) -4-propyl-1,3-dioxolan-2-ylmethyl) -1H-1,2,4-triazole ("propiconazole"); 1- {2- [2-Chloro-4- (4-chloro-phenoxy) -phenyl] -4-methyl-1,3-dioxolan-2-ylmethyl} -1H-1,2,4 -triazole ("difenoconazole"); 1- (2- (2,4-dichlorophenyl) pentyl] -1H-1,2,4-triazole ("penconazole"); cis-4- [3- (4-tert-butyl-phenyl) -2-methyl-propyl] -2,6-dimethyl-morpholine ("fenpropimorph"); 1- [3- (4-tert-Butyl-phenyl) -2-methyl-propyl] -piperidine ("fenpropidin"); 4-cyclopropyl-6-methyl-N-phenyl-2-pyrimidinamine ("cyprodinyl"); (RS) -N- (2,6-Dimethylphenyl) -N- (methoxyacetyl) alanine methyl ester ("metalaxyl"); (R) -N- (2,6-Dimethylphenyl) -N- (methoxyacetyl) alanine methyl ester ("R-metalaxyl"); 1,2,5,6-tetrahydro-4H-pyrrolo [3,2,1-ijquinolin-4-one ("pyroquilone") or ethyl hydrogen phosphonate ("fosetyl"). 25. The method of claim 24, wherein the fungicide is metalaxyl and the other microbicide is a benzothiadiazole derivative.