FR2757875A1 - METHODS OF USING THE NIM1 GENE TO PROVIDE PLANT RESISTANCE TO VEGETABLES - Google Patents

Abstract

The invention concerns the location and characterization of a gene (designated NIM1) that is a key component of the SAR pathway and that in connection with chemical and biological inducers enables induction of SAR gene expression and broad spectrum disease resistance in plants. The NIM1 gene product is a structural homologue of the mammalian signal transduction factor I kappa B subclass alpha . The present invention exploits this discovery to provide altered forms of NIM1 that act as dominant-negative regulators of the systemic acquired resistance (SAR) signal transduction pathway. These altered forms of NIM1 confer the opposite phenotype as the nim1 mutant in plants transformed with the altered forms of NIM1, i.e. the transgenic plants exhibit constitutive SAR gene expression and a constitutive immunity (CIM) phenotype. The invention further concerns transformation vectors and processes for overexpressing the NIM1 gene in plants. The transgenic plants thus created have broad spectrum disease resistance. The present invention further concerns DNA molecules encoding altered forms of the NIM1 gene, expression vectors containing such DNA molecules, and plants and plant cells transformed therewith. The invention further concerns transformation vectors and processes for overexpressing the NIM1 gene in plants. Disclosed are vectors and processes for producing overexpression of the NIM1 gene in plants. The invention also concerns methods of activating SAR in plants and conferring to plants a CIM phenotype and broad spectrum disease resistance by transforming the plants with DNA molecules encoding altered forms of the NIM1 gene product.

Description

METHODS OF USING THE NIMi GENE TO CONFER
VEGETABLE DISEASE RESISTANCE
The present invention generally relates to the resistance of plants to a broad spectrum of diseases, including the phenomenon of systemic acquired resistance (SAR).

More particularly, the present invention relates to the recombinant expression of wild and modified forms of the NIAI gene, which is involved in the signal transduction cascade leading to SAR to create transgenic plants with resistance to a broad spectrum of diseases. . The present invention also relates to the high level expression of the NIMi gene cloned in transgenic plants having resistance to a broad spectrum of diseases.

 Plants and plants are constantly attacked by a wide range of pathogenic organisms, including viruses, bacteria, fungi and nematodes. Cultivated plants are particularly vulnerable because they generally grow as genetically uniform monocultures; if an illness appears, the losses can be severe. However, most plants have their own innate defense mechanisms against pathogenic organisms. A natural variant, providing resistance to plant pests, has been identified by breeders and has been introduced into many crop plants.

These natural disease resistance genes often provide a high level of resistance to or immunity against pathogenic microorganisms.

Systemic acquired resistance (SAR) is a component of plants with complex systems used to defend them against pathogenic microorganisms (Hunt and Ryals, Crit Rev. in Plant, Sci 15, 583-606 (1996), incorporated here as a reference in its entirety
Ryals et al., Plant Cell 8, 1809-1819 (1996), incorporated herein by reference in its entirety. See also U.S. Patent No. 5,614,395, incorporated herein by reference in its entirety.) SAR is a particularly important aspect of plant-pathogenic microorganism responses because it is a systemic resistance that can be induced. by a pathogenic microorganism, against a broad spectrum of infectious agents, including viruses, bacteria and pathogenic fungi.When the SAR signal transduction pathway is blocked, plants become more susceptible to pathogenic microorganisms that normally create a disease, and likewise become susceptible to certain infectious agents that normally should not cause disease (Gaffney et al., Science 261, 754-756 (1993), incorporated herein by reference in its entirety; Delaney et al. Science 266, 1247-1250 (1994), incorporated herein by reference in its entirety, Delaney et al.
Proc. Nati. Acad. Sci. USA 92, 6602-6606 (1995), incorporated herein by reference in its entirety; Delaney,
Plant. Phys. 113, 5-12 (1997), incorporated herein by reference in its entirety; Bi et al., Plant. J. 8, 235245 (1995), incorporated herein by reference in its entirety; Mauch-Mani and Slusarenko, Plant Cell 8, 203212 (1996), incorporated herein by reference in its entirety). These observations indicate that the SAR signal transduction pathway is critical for maintaining plant health.

In theory, the SAR response can be subdivided into two phases. In the initiation phase, an infection is recognized by a pathogenic organism, and a signal is released, which crosses the phloem to arrive at the distant tissues. This systemic signal is perceived by the target cells, which react by the expression of the genes of the
SAR and disease resistance. The maintenance phase of the SAR is the time between a few weeks and the entire life of the plant, during which the plant is in a quasi-permanent state, with maintenance of disease resistance (Ryals et al. 1996).

Accumulation of salicylic acid (SA) appears to be necessary for SAR signal transduction. Plants that can not accumulate AS due to treatment with specific inhibitors, epigenetic phenylalanine-ammonia-lyase repression, or transgene expression of salicylate-hydroxylase, which specifically degrades the SA, can not induce either SAR gene expression or disease resistance (Gaffney et al., 1993, Delaney et al., 1994, Mauch-Mani and Slusarenko 1996, Maher et al., Proc Nat. Acad Sci USA 91, 7802-7806 (1994), incorporated herein by reference in its entirety.
Pallas et al., Plant J. 10, 281-293 (1996), incorporated herein by reference). Although it has been proposed that the
SA can serve as a systemic signal, this point is currently the subject of controversy, and, to date, all that is known of certain is that, if the SA does not accumulate, then the transduction SAR signal is blocked (Pallas et al., 1996, Shulaev et al., 1995,
Plant Cell 7, 1691-1701 (1995), incorporated herein by reference in its entirety; Vernooij et al., Plant Cell 6, 959-965 (1994), incorporated herein by reference in its entirety).

Recently, Arabidopsis has been shown to be a model system for studying SAR (Uknes et al., Plant Cell 4, 645-656 (1992), incorporated herein by reference in its entirety; Uknes et al. ., Plant Mol.
Microbe Interact 6, 692-698 (1993), incorporated herein by reference in its entirety; Cameron et al., Plant J 5, 715-725 (1994), incorporated herein by reference in its entirety; Mauch-Mani and Slusarenko, Mol. seedling
Microbe Interact. 7, 378-383 (1994), incorporated herein by reference in its entirety; Dempsey and Klessig,
Bulletin of The Paster Institute 93, 167-186 (1995), incorporated herein by reference in its entirety). It has been shown that SAR can be activated in Arabidopsis by pathogenic microorganisms as well as by chemical agents 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), incorporated herein by reference in its entirety; Lawton et al., Plant J. 10, 71-82 (1996), incorporated herein by reference in its entirety by reference in its entirety). After treatment with INA or BTH, or after infection with a pathogenic microorganism, at least three pathogenesis-related protein genes,
PR), namely PR-1, PR-2 and PR-5, are induced in a coordinated manner, simultaneously with the onset of resistance (Uknes et al., 1992, 1993). In tobacco, which is the best characterized species, treatment with a pathogenic microorganism or immunizing compound causes the expression of at least 9 sets of genes (Ward et al., Plant Cell, 1085- 1094 (1991), incorporated herein by reference in its entirety). Disease resistant transgenic plants have been created by transforming plants using different SAR genes (US Patent No. 5,614,395).

A number of Arabidopsis mutants have been isolated, which have altered signal transduction
SAR (Delaney, 1997). The first of these mutants are so-called mutants lsd (in English lesions simulating features, disease simulating lesions), and acd2 (in English: accelerated cell death, accelerated death of cells) (Dietrich et al., Cell77 , 551-563 (1994), incorporated herein by reference in its entirety;
Greenberg et al., Cell 77, 551-563 (1994), incorporated herein by reference in its entirety). All these mutants show some degree of spontaneous formation of necrotic lesions on their leaves, high levels of SA, mRNA accumulation for the genes of the
SAR, and a significantly enhanced resistance to diseases. At least 7 different lsd mutants have been isolated and characterized (Dietrich et al., 1994, Weymann et al., Plant Cell 7, 2013-2022 (1995), incorporated herein by reference in its entirety). Another interesting class of mutants is represented by the kin mutants: (constitutive immunity, constitutive immunity) (Lawton et al., 1993 "The Molecular Biology of Systemic Acquired Resistance", in Mechanisms of Defense
Responses in Plants, B. Fritig and M. Legrand, eds (Dordrecht, The Netherlands, Academic Editions Kluwer), pp. 422-432 (1993), incorporated herein by reference in its entirety). See also PCT International Application WO 94/16077, these two references being incorporated herein in their entirety. Like the lsd and acd2 mutants, the cim mutants show a high accumulation of SA and a high level of expression and resistance of the SAR gene, but unlike lsd or acd2, they do not exhibit detectable lesions on their leaves.

Cprl (constitutive expresser of PR genes, constitutive expression of PR genes) may be a type of cim mutant; however, since the presence of microscopic lesions on the leaves of the cpr1 is not excluded, the cpr1 may be a type of lsd mutant (Bowling et al.,
Plant Cell, 1845-1857 (1994), incorporated herein by reference in its entirety).

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

Obviously, this mutant has an early blockade in SAR signaling. Nprl (nonexpresser of PR genes) is a mutant that can not cause the expression of the SAR signaling pathway after INA treatment (Cao et al., Plant Cell 6, 1583-1592 (1994), incorporated herein by reference in its entirety). Eds (enhanced disease susceptibility) mutants have been isolated based on their ability to support a bacterial infection after inoculation of a low bacterial concentration (Glazebrook et al., Genetics 143, 973-982). (1996), incorporated herein by reference in its entirety, Parker et al., Plant Cell 8, 2033-2046 (1996), incorporated herein by reference in its entirety).

Some eds mutants are phenotypically very similar to nprl, and recently eds5 and eds53 have been shown to be allelic against nprl (Glazebrook et al., 1996). NimI (in English: noninducible immunity, non-induble immunity) is a mutant that supports the growth of P. parasitica (that is, the agent that causes late blight) after treatment with 1 ' INA (Delaney et al., 1995, international application
PCT WO 94/16077). Although NIML can accumulate AS after infection with a pathogenic microorganism, it can not cause SAR gene expression or disease resistance, suggesting that the mutation is blocking the downstream pathway of AS. . Niml also has a reduced ability to respond to INA or BTH, suggesting that blockage exists downstream of the action of these chemical agents (Delaney et al., 1995).
Lawton et al., 1996).

 Recently, two Arabidopsis allelic genes have been isolated and characterized, the mutants of which are responsible for the nimi and nprl phenotypes respectively (Ryals et al., Plant Cell 9, 425-439 (1997), incorporated herein by reference in its Cao et al., Cell 88, 57-63 (1997), incorporated herein by reference in its entirety). The wild-type NIMi gene product is involved in the signal transduction cascade leading to both SAR and gene-for-gene disease resistance in Arabidopsis (Ryals et al., 1997).

Ryals et al. (1997) also describe the isolation of 5 additional nimI alleles, which show a range of phenotypes ranging from low degradation of chemically induced PR-1 gene expression and fungal resistance to mutants. strongly blocked. Transformation of the wild-type NPR1 gene into nprl mutants not only completes the mutations, restores the sensitivity of SAR induction in terms of PR gene expression and disease resistance, but also makes the transgenic plants more resistant to P. syringae infection in the absence of induction of SAR (Cao et al., 1997).

NF-KB / IKB signal transduction pathways
NF-KB / IKB signaling pathways have been implicated in disease resistance responses across a range of organisms from Drosophila to mammals. In mammals, NF-KB / IKB signal transduction may be mediated by a number of different stimuli, which include exposure of cells to lipopolysaccharide, tumor necrosis factor, interleukin-1 (IL-1) or viral infection (Baeuerle and Baltimore, Cell 87, 13-20 (1996), Baldwin, Annu Rev. Immunol 14, 649-681 (1996)). The activated pathway leads to the synthesis of a number of factors involved in inflammation and immune responses, including IL-2, IL-6, IL-8, and granulocyte / macrophage colonies (deMartin et al., Gene 152, 253-255 (1995)).

In transgenic mouse studies, the elimination of NF-KB / IKB signal transduction leads to a defective immune response, which includes increased sensitivity to bacterial and viral pathogenic microorganisms (Beg and Baltimore, Science 274, 782784 (1996) Van Antwerp et al., Science 274, 787-789 (1996) Wang et al., Science 274, 784-787 (1996)
Baeuerle and Baltimore (1996)). In Arabidopsis, SAR is functionally analogous to inflammation, in that normal resistance processes are enhanced after activation of SAR, leading to increased resistance to disease (Bi et al., 1995, Cao et al., 1994). Delaney et al., 1995, Delaney et al., 1994
Gaffney et al., 1993; Mauch-Mani and Slusarenko 1996
Delaney, 1997). In addition, inactivation of the pathway leads to increased susceptibility to bacterial, viral and fungal pathogenic microorganisms. It is interesting to note that SA has been reported to block the activation of NF-κB in mammalian cells (Kopp et al.
Ghosh, Science 265, 956-959 (1994)), while SA activates signal transduction in Arabidopsis.

The bacterial infection of Drosophila activates a cascade of signal transductions, which leads to the synthesis of a number of antifungal proteins such as cercropin B, defensin, diperericin and drosomycin (lp et al., Cell 75, 753-763 (1993) Lemaitre et al., Cell 86, 973-983 (1996)). This induction is dependent on the dorsal and dif gene product, two homologs of NF-KB, and is repressed by cactus, a homologue of IKB, in the fly. Mutants that exhibit reduced synthesis of antifungal and antibacterial proteins exhibit greatly reduced resistance to infection.

 Despite extensive research, and despite the use of sophisticated and intensive crop protection measures, including plant genetic transformation, disease losses continue to be billions of dollars each year. As a result, there is a continuing need for the development of new crop protection measures, based on an ever greater understanding of the genetic basis of plant resistance to disease.

 The definitions below will facilitate the understanding of the present invention.

Plant cell
It is the structural and physiological unit of plants, including a protoplast and the cell wall. The term "plant cell" refers to any cell that is part of or derived from a plant. Examples of cells are differentiated cells that are part of a living plant; differentiated cells in culture; undifferentiated cells in culture; undifferentiated tissue cells such as callus or tumors; differentiated cells of seeds, embryos, propagules and pollen.

Vegetable fabric
It is a group of plant cells organized in a structural and functional unit. Plant tissues include all plant tissues, during planting or in cultivation. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures and all groups of plant cells organized in structural and / or functional units. The use of this expression, together with any specific type of plant tissue, or in the absence of any specific type of plant tissue from the above list, or otherwise encompassed by this definition, does not exclude no other type of plant tissue.

Protoplast
It is a plant cell without a cell wall.

Descending plant
It is a plant of a future generation, obtained sexually or asexually, which includes, but is not limited to, offspring.

Transgenic plant
It is a plant in whose genome a recombinant DNA is stably incorporated.

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

Recombinant DNA technology (or recombinant DNA technology):
It is the technology that produces the recombinant DNA in vitro and transfers the recombinant DNA into cells where it can be expressed or multiplied (see
Concise Dictionary of Biomedicine and Molecular Biology,
Ed. Juo, CRC Press, Boca Raton (1996)), for example the transfer of DNA into one or more protoplasts or cells in different forms, and in particular for example (1) naked DNA in circular, linear forms or supertorsed, (2) DNA contained in nucleosomes or chromosomes or nuclei or parts thereof, (3) DNA complexed or associated with other molecules, (4) DNA enclosed in liposomes, spheroplasts, cells or protoplasts, or (5) DNA transferred from organisms other than the host organism (eg
Agrobacterium tumefaciens). These methods, as well as other methods for introducing recombinant DNA into cells, are known in the art and can be used to produce the transgenic cells or transgenic plants of the present invention.

The recombinant DNA technology also includes the homologous recombination methods described in
Treco et al., WO 94/12650, and Treco et al., WO 95/31560, which can apply to increasing peroxidase activity in a monocotyledon. This should result in an increase in the number of copies of the sequences encoding peroxidase within the plant.

 The initial insertion of the recombinant DNA into the genome of the RO plant is not defined as being performed by the traditional methods of plant breeding, but rather by the technical methods described in the invention. After initial insertion, transgenic offspring can be propagated using essentially traditional breeding techniques.

Chimeric gene
It is a DNA molecule containing at least two heterologous parts, for example parts which derive from pre-existing DNA sequences which are not associated in their pre-existing states, these sequences having preferably been generated using recombinant DNA technology.

Expression cassette
It is a DNA molecule comprising a promoter and a terminator between which can be inserted a coding sequence.

Coding sequence
It is a DNA molecule that, when transcribed and translated, leads to the formation of a polypeptide or protein.

Gene:
A discrete chromosomal region comprising a regulatory DNA sequence responsible for the regulation of expression, i.e., transcription and transduction, and a coding sequence that is transcribed and translated to give a polypeptide or distinct protein.

 The present invention describes the identification, isolation and characterization of the NIAI gene, which encodes a protein involved in the signal transduction cascade sensitive to biological and chemical inducers, which leads to systemic resistance acquired in plants.

Accordingly, the present invention discloses an isolated DNA molecule (NIT1 gene), which encodes a protein
NIM1 involved in the signal transduction cascade leading to systemic resistance acquired in plants.

In the context of the present invention, there is disclosed a DNA molecule which encodes the NIM1 protein which hybridises under the following conditions to give the
BAC-04, ATCC deposit No. 97543: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; 7% sodium lauryl sulphate
EDTA 1 mM; sodium chloride 250 mM at 550C for 18 to 24 hours, and wash in 6XSSC for 15 minutes (X3), 3XSSC for 15 minutes (X1) at 550C. In a particularly preferred embodiment, the NIM1 gene is located inside the cosmid D7, ATCC deposit NO 97736.

 The NIMi gene described in the invention can be isolated from a dicotyledon such as Arabidopsis, tobacco, cucumber or tomato. Alternatively, the NIM1 gene can be isolated from a monocotyledonous plant such as corn, wheat or barley.

In addition, an encoded NIM1 protein comprising the amino acid sequence SEQ ID NO: 3 is described. The invention furthermore describes the sequence coding for the NIM1 gene, which hybridises under the following conditions to the coding sequence SEQ ID NO: 2
Hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; sodium lauryl sulphate 7%; EDTA 1 mM; sodium chloride 250 mM at 550C for 18 to 24 hours, and wash in 6XSSC for 15 minutes (X3), 3XSSC for 15 minutes (X1) at 550C. In a particularly preferred embodiment, the sequence coding for the NIMi gene comprises the coding sequence SEQ ID NO: 2.

The present invention also discloses a chimeric gene comprising a plant-active promoter operably linked to a coding sequence of the gene
NIM1, a recombinant vector comprising such a chimeric gene, wherein the vector is capable of stable transformation in a host, and a host transformed under stable conditions using such a vector. Preferably, the host is a plant such as one of the following agronomically important crops; rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic eggplant pepper celery carrot squash pumpkin zucchini cucumber apple pear quince melon plum cherry peach nectarine apricot strawberry grape raspberry ripe pineapple avocado papaya mango banana, soya, tobacco, tomato, sorghum and sugar cane.

 In a particularly preferred embodiment, the NIM1 protein is expressed in a transformed plant at higher levels than in a wild-type plant.

 The present invention also relates to a method for conferring a CIM phenotype to a plant, by transformation of the plant using a recombinant vector comprising a chimeric gene which in itself comprises a promoter active in plants and linked by operational link to a sequence encoding the NIM1 gene, wherein the encoded NIM1 protein is expressed in the transformed plant in larger amounts than in a wild type plant.

 In addition, the present invention relates to a method for activating systemic resistance acquired in a plant, by transforming the plant with a recombinant vector comprising a chimeric gene, which itself comprises a promoter active in plants, bound by operably linking to a sequence encoding the NIAI gene, wherein the encoded NIM1 protein is expressed in the plant transformed in larger amounts than in a wild type plant.

 In addition, the present invention relates to a method for conferring resistance to a broad spectrum of diseases to a plant by transformation of the plant with a recombinant vector comprising a chimeric gene which itself comprises an active promoter in a plant. the plants, operably linked to a sequence encoding the NIAI gene, wherein the encoded NIM1 protein is expressed in the transformed plant in larger amounts than in a wild-type plant.

 Another aspect of the present invention exploits the fact that the SAR pathway in plants has functional parallels with the regulatory scheme of NF-KB / IKB in mammals and flies, as well as the discovery of which the NIMi gene produced is a structural homolog of the subclass a of the signal transduction factor IKB in a mammal. IUK mutations have been described which act as super-repressors or dominant-negatives of the NF-KB / IaB regulatory scheme. The present invention encompasses modified forms of the wild-type NIM1 gene (SEQ NO: 2), which act as dominant-negative regulators of the SAR signal transduction pathway. These modified forms of NIM1 confer the opposite phenotype on plants transformed with its aid, as niml mutant; plants, for example those transformed with modified forms of NIAI, exhibit an expression of the constitutive gene for SAR and a CIM phenotype.

 The present invention also relates to DNA molecules that hybridize to a DNA molecule according to the present invention as defined above, but preferably to an oligonucleotide probe obtainable from this DNA molecule. , comprising a contiguous portion of the coding sequence for said modified forms of NiMi and having a length of at least 10 nucleotides, under moderately stringent conditions.

Factors that affect the stability of hybrids determine the stringency of hybridization. One of its factors is the melting temperature T ,,,,, which can easily be calculated by the formula given in DNA
PROBES, George H. Keller and Mark M. Manak, Macmillan
Publishers Ltd, 1993, Section one: Molecular Hybridization Technology, page 8 and following.

 The preferred hybridization temperature is of the order of about 25 ° C lower than the calculated melting temperature Tm, and is preferably between about 12 and 150 ° C below the calculated melting temperature T ,,,, and, in the case of oligonucleotides, it is in the range of about 5 to 100C below the melting temperature T 1.

In one embodiment of the present invention, the NIM1 gene is modified so that the encoded product has alanines instead of serines in the amino acid positions corresponding to positions 5559 of the amino acid sequence of the NIM1 of wild-type Arabidopsis (SEQ ID NO: 3). An example of a preferred embodiment of this modified form of the NIM1 gene, which leads to changes of these serine residues to alanine residues, is presented in the sequence SEQ ID
NO: 22. An exemplary, dominant-negative form of the NIM1 protein, with alanines instead of serines at the amino acid positions exemplified by a preferred embodiment of this modified form of the NIM1 gene, which encodes a gene product having a N-terminal deletion, is presented in the sequence SEQ ID NO: 24. An exemplary dominant negative form of the NIM1 protein comprising an Nterminal deletion is presented in the sequence SEQ ID NO: 25. The present invention also relates to modified forms of NIAI alleles, wherein the coding sequence of such an allele hybridizes under moderately stringent conditions to the coding sequence SEQ ID NO: 24; the following conditions are particularly preferred: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; 7% sodium lauryl sulphate
EDTA 1 mM; 250mM sodium chloride at 55 ° C for 18-24 hours, and wash in 6XSSC for 15 minutes (X3), 3XSSC for 15 minutes (X1) at 550 ° C. In these embodiments, NIM1 alleles that hybridize SEQ ID NO: 24 under the above conditions are modified such that the encoded product has an N-terminal deletion which eliminates lysine residues that may serve as potential ubiquitinization sites, in addition to serines at the amino acid positions corresponding to positions 55 and 59 of the wild-type gene product.

In yet another embodiment of the present invention, the NIM1 gene is modified so that the encoded product has a C-terminal truncation, which is believed to lead to improved intrinsic stability, by blocking constitutive phosphorylation. serine and threonine residues at the Cterminal site of the wild-type gene product. An example of a preferred embodiment of this modified form of the NIAI gene, which encodes a gene product having a C-terminal deletion, is presented in the SEQ sequence
ID NO: 26. An exemplary dominant-negative form of the NIM1 protein having a C-terminal deletion is presented in the sequence SEQ ID NO: 27. The present invention also relates to modified forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under moderately stringent conditions to the coding sequence SEQ ID NO: 26; the following conditions are particularly preferred: hybridization in 1% BSA; NaPO4 520 mM, pH 7.2; sodium lauryl sulphate 7%; EDTA 1 mM; 250mM sodium chloride at 550C for 18-24 hours, and wash in 6XSSC for 15 minutes (X3), 3XSSC for 15 minutes (X1) at 55 ° C. In these embodiments, the NIM1 alleles that hybridize SEQ ID NO: 26 under the above conditions are modified such that the coded product has a C-terminal deletion which removes serine and threolin residues.

 In yet another embodiment of the present invention, the NIM1 gene is modified so that the encoded product has both an N-terminal deletion and a C-terminal truncation, leading to the advantages of the two embodiments described above. of the invention.

A preferred embodiment of the invention is a modified form of the NIM1 protein, which has an N-terminal amino acid truncation corresponding approximately to amino acid positions 1-125 of the sequence SEQ ID NO: 2 and a Cterminal truncation of amino acids corresponding approximately to amino acid positions 522-593 of SEQ sequence
ID NO: 3.

The sequence SEQ ID NO: 28 shows an example of a preferred embodiment of this modified form of the NIM1 gene, which encodes a gene product comprising both an N-terminal deletion and a C-terminal deletion. The sequence SEQ ID NO: 29 has a dominant-negative exemplary form of the NIM1 protein comprising a deletion
C-terminus. The present invention also relates to modified forms of NIM1 alleles, wherein the coding sequence of such an allele hybridizes under moderately stringent conditions to the SEQ ID coding sequence.
NO: 28; The following conditions are particularly preferred: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; sodium lauryl sulphate 7%; EDTA 1 mM; sodium chloride 250 mM at 550C for 18 to 24 hours, and wash in 6XSSC for 15 minutes (X3), 3XSSC for 15 minutes (X1) at 550C. In these embodiments, NIM1 alleles that hybridize to SEQ ID sequence
NO: 28 under the above conditions are modified so that the coded product has both an Nterminal deletion, which eliminates lysine residues that may serve as potential sites for ubiquitinization in addition to serines on the corresponding amino acid positions. at positions 55 and 59 of the wild-type gene product, a C-terminal deletion, which removes serine and threonine residues.

 In yet another embodiment of the present invention, the NiMi gene is modified such that the encoded product consists essentially of only ankyrin domains of the wild-type gene product.

An isolated DNA molecule is preferred, wherein said modified form of the NIM1 protein consists essentially of ankyrin units corresponding approximately to the amino acid positions 103-362 of the sequence SEQ ID NO: 3. An example of a preferred embodiment of this modified form of the NDM1 gene, which encodes the ankyrin domains, is presented in the sequence
SEQ ID NO: 30. The sequence SEQ ID NO: 31 has an exemplary, dominant-negative form of the NIM1 protein, consisting essentially and only of ankyrin domains. The present invention also relates to modified forms of MIMI alleles, wherein the coding sequence of such an allele hybridizes under moderately stringent conditions to the coding sequence SEQ ID NO: 30; the following conditions are particularly preferred: hybridization in BSA at 18; 520 mM NaPO4, pH 7.2; sodium lauryl sulphate 7%; EDTA 1 mM; sodium chloride 250 mM at 550C for 18 to 24 hours, and wash in 6XSSC for 15 minutes (X3), 3XSSC for 15 minutes (X1) at 550C. In these embodiments, the NIM1 alleles that hybridize to SEQ ID NO: 30 under the above conditions are modified such that the encoded product consists essentially of ankyrin domains of the wild-type gene product.

 Thus, the present invention relates to DNA molecules encoding modified forms of the NIM1 gene, such as those described above, and all DNA molecules that hybridize therewith using moderately stringent conditions.

 The present invention also relates to a chimeric gene comprising a promoter active in plants, linked by operational binding to one of the modified forms described above of the NIM1 gene, a recombinant vector comprising this chimeric gene, where the vector can be transformed into stable conditions in a host cell, as well as a host cell transformed under stable conditions by such a vector. Preferably, the host is a plant such as one of the following agronomically important crops; rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic eggplant pepper celery carrot squash pumpkin zucchini cucumber apple pear quince melon plum cherry peach nectarine apricot strawberry grape raspberry ripe pineapple avocado papaya mango banana, soya, tobacco, tomato, sorghum and sugar cane.

 The present invention also relates to a method for conferring a CIM phenotype to a plant, by transformation of the plant with a recombinant vector comprising a chimeric gene which itself comprises a promoter active in plants and linked by operational binding to one of the forms The above-described modified modifications of the NIM1 gene, wherein the coded, dominant-negative form of the NIM1 protein is expressed in the transformed plant and confers a CIM phenotype on the plant.

 In addition, the present invention relates to a method for activating systemic resistance acquired in a plant, by transforming the plant with a recombinant vector comprising a chimeric gene, which itself comprises a promoter active in plants and operably linked to the plant. one of the modified forms described above of the NIM1 gene, wherein the coded, dominant-negative form of the NIM1 protein is expressed in the transformed plant and activates the systemic resistance acquired in the plant.

 In addition, the present invention relates to a method for conferring resistance to a broad spectrum of diseases to a plant by transformation of the plant with a recombinant vector comprising a chimeric gene which itself comprises a promoter active in plants and operatively linked to one of the modified forms described above of the NIM1 gene, where the coded, dominant-negative form of the NIM1 protein is expressed in the transformed plant and confers on the plant a resistance to a broad spectrum of diseases.

 In yet another aspect, the present invention relates to a method of screening a NIM1 gene involved in the signal transduction cascade leading to systemic resistance acquired in a plant, which involves probing a library or library of CDNA from said plant with a sequence encoding NIM1 which hybridizes in the following set of conditions to the coding sequence SEQ ID NO: 2: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; sodium lauryl sulphate 7%; EDTA 1 mM; 250 mM sodium chloride at 55 ° C for 18 to 24 hours, and wash in 6XSSC for 15 minutes (X3), 3XSSC for 15 minutes (X1) at 550 ° C.

The invention also relates to the following objects
An isolated DNA molecule according to the invention, wherein said modified form of the NIM1 protein has alanines instead of serines at the amino acid positions corresponding to positiosn 55 and 59 of the sequence SEQ ID NO: 3, where said DNA molecule hybridizes under the following conditions to the nucleotide sequence SEQ ID NO: 22: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; sodium lauryl sulphate 7%; EDTA 1 mM; 250mM sodium chloride at 550C for 18-24 hours, and wash in 6XSSC for 15 minutes (X3), 3XSSC for 15 minutes (X1) at 550C.

An isolated DNA molecule according to the invention, wherein said modified form of the NIM1 protein has an N-terminal amino acid truncation corresponding approximately to amino acid positions 1-125 of the sequence SEQ ID NO: 3, wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence SEQ ID NO: 24: hybridization in
BSA at 1%; 520 mM NaPO4, pH 7.2; sodium lauryl sulphate 7%; EDTA 1 mM; 250mM sodium chloride at 550C for 18-24 hours, and wash in 6XSSC for 15 minutes (X3), 3XSSC for 15 minutes (X1) at 55 ° C.

An isolated DNA molecule according to the invention, wherein said modified form of the NIM1 protein has a C-terminal amino acid truncation corresponding approximately to the amino acid positions 522-593 of the sequence SEQ ID NO: 3, wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence SEQ ID NO: 26: Hybridization in
BSA at 1%; 520 mM NaPO4, pH 7.2; sodium lauryl sulphate 7%; EDTA 1 mM; sodium chloride 250 mM at 550C for 18 to 24 hours, and wash in 6XSSC for 15 minutes (X3), 3XSSC for 15 minutes (X1) at 550C.

 An isolated DNA molecule according to the invention, wherein said modified form of the NIM1 protein comprises the amino acid sequence SEQ ID NO: 28, wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence SEQ ID NO: 28: Hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; sodium lauryl sulphate 7%; EDTA 1 mM; 250 mM sodium chloride at 55 ° C for 18 to 24 hours, and wash in 6XSSC for 15 minutes (X3), 3XSSC for 15 minutes (X1) at 55 ° C.

An isolated DNA molecule according to the invention, wherein said modified form of the NIM1 protein consists essentially of ankyrin units corresponding approximately to the amino acid positions 103362 of the sequence SEQ ID NO: 3, wherein said DNA molecule hybridises under the following conditions to the nucleotide sequence SEQ ID NO: 30: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; 7% sodium lauryl sulphate
EDTA 1 mM; sodium chloride 250 mM at 550C for 18 to 24 hours, and wash in 6XSSC for 15 minutes (X3), 3XSSC for 15 minutes (X1) at 550C.

 A modified form of the NIM1 gene according to the invention, which has been synthesized by mutagenization.

 Use of an isolated DNA molecule according to the invention for activating systemic resistance acquired in a plant cell, a plant and their descendants.

 Use of an isolated DNA molecule according to the invention to confer resistance to a broad spectrum of diseases to a plant cell, a plant and their descendants.

 Use of an isolated DNA molecule according to the invention for conferring a CIM phenotype on a plant cell, a plant and their descendants.

 Use of resistant plants and their descendants according to the invention for incorporating disease resistance character into plant lines by selection.

 Use of NiMi gene variants to confer disease resistance and activate SAR gene expression in plants transformed with this gene.

 A method for producing a modified form of a NiMi gene.

 A method for producing transgenic offspring of a transgenic parent plant comprising an isolated DNA molecule encoding a modified form of a NIM1 protein according to the invention, which comprises transforming said parent plant with a dimer molecule a recombinant vector according to the invention, and to transfer the character to the descendants of said transgenic parent plant, using known techniques of plant breeding.

A method of producing a DNA molecule comprising a portion of DNA containing a portion of DNA encoding a modified form of a NIM1 protein, which comprises
a) preparing a nucleotide probe capable of hybridizing specifically to a modified form of an NIjM1 gene or mRNA, wherein said probe comprises a contiguous portion of the coding sequence for a modified form of a NIjMl having a length of at least 10 nucleotides.
b) probing for other modified forms of a NIjM1 coding sequence into populations of cloned genomic DNA fragments or cDNA fragments from a selected organism, by use of the nucleotide probe prepared in step a); and
c) isolating and multiplying a DNA molecule comprising a portion of DNA containing a portion of DNA encoding a modified form of a NIM1 protein.

A method for isolating a DNA molecule comprising a DNA portion containing a modified form of an NLM1 sequence, which consists of
a) preparing a nucleotide probe capable of hybridizing in a specific manner to a modified form of a NIM1 gene or an mRNA, wherein said probe comprises a contiguous portion of the coding sequence for a modified form of a NiMi having a length of at least 10 nucleotides.
b) probing for other modified forms of a coding sequence of the NIM1 in populations of cloned genomic DNA fragments or cDNA fragments from a selected organism, using the probe nucleotide prepared in step a); and
c) isolating a DNA molecule comprising a portion of DNA containing a modified form of a NIM1 gene.

 A method of producing transgenic plants that express higher levels of the NIM1 gene than the wild type, or their functional and mutant variants.

 A method for producing transgenic plants that express higher than wild-type NiMi gene levels, or functional and mutant variants thereof, where NIMl gene expression is at a level that is at least two-fold higher than the wild type of expression of the NiMi gene in wild type plants.

 A process for producing transgenic plants that express higher levels of the NIM1 gene than the wild type, or their functional and mutant variants, where the expression of the NIM1 gene is at a level that is at least ten times higher than the level expression of the NIM1 gene in wild-type plants.

The phenotype of the mutant nim
The present invention relates to mutant plants, as well as genes isolated therefrom, that exhibit a defect in their normal response to infection by pathogenic microorganisms, in that they do not express the associated genes. to the SAR. These mutants are called nim mutants (in English: non-inducible immunity, non-inducible immunity), and exhibit a "universal susceptibility to diseases" (in English: universal disposable susceptible, UDS), since they are susceptible to many strains. and many pathotypes of pathogenic microorganisms of the host plant, as well as pathogenic microorganisms that do not normally affect the host plant but normally affect other hosts. These mutants can be selected by treating seed or other biological material with mutagenic agents and then selecting downstream plants for the UDS phenotype by treating the offspring with known chemical inducers (eg INA) of SAR, then plant infection using a known pathogenic microorganism. Non-inducible mutants develop severe pathological symptoms under these circumstances, while wild-type plants acquire systemic resistance acquired through the chemical compound. The nim mutants can also be selected from mutant populations produced by chemical and irradiation mutagenesis, as well as in populations produced by T-DNA insertion and transposon-induced mutagenesis. The techniques for producing mutant plant lines are well known.

 The nim mutants represent useful indicators of the assessment of disease pressure in field pathogenesis assays, where the phenotype of the natural resistance of so-called wild-type plants (i.e. non-mutants) may vary, so that a reliable standard of sensitivity is not obtained. In addition, nim plants have additional utility for testing transgenic candidates for disease resistance. If a nim parent line is used as the recipient of transgenes, the contribution of the transgene to disease resistance can be directly assessed against a baseline level of sensitivity. In addition, the plants can be used as a tool to understand pathogenic plant-microorganism interactions. Host plants do not establish a systemic response to an attack by a pathogenic microorganism, and the permanent development of the pathogenic microorganism represents an ideal system in which one can study its biological interaction with the host.

 Since nim host plants may also be susceptible to pathogenic microorganisms outside the host range in which they normally enter, these plants also have great utility in the molecular, genetic and biological study of hate-microorganism interactions. pathogens. In addition, the UDS phenotype of nim plants makes them even useful for the selection of fungicides. The selected nim mutants in a particular host have considerable utility in the selection of fungicides, if this host and the pathogenic microorganisms of the host are used. The advantage lies in the mutant UDS phenotype, which eliminates the problems encountered by hosts with different sensitivities to different pathogenic and pathotypic microorganisms, or even resistant to certain pathogenic microorganisms or pathotypes.

 The nim mutants can additionally be used to select fungicides against a range of pathogenic microorganisms and pathotypes, using a heterologous host, i.e. a host that normally can not be in the range of species. hosts of a particular pathogenic microorganism. Thus, the susceptibility of Arabidopsis nim mutants to pathogenic microorganisms of other species (eg, crop plant species) facilitates efficient fungicide selection operations, in terms of compounds against important pathogenic microorganisms of the crop plants.

The niml mutant of Arabidopsis thaliana
A mutant of Arabidopsis thaliana, called niml (in English: noninducible immunity, non-inducible immunity), which supports the proliferation of P. parasitica (which is the causal agent of late blight) after treatment with INA is described in Delaney et al. Although NIML can accumulate AS after infection with pathogenic microorganisms, neither SAR gene expression nor disease resistance can be induced, suggesting that the mutation is blocking the downstream pathway. due to. Niml also decreases its ability to respond to INA or BTH, suggesting that blockage exists downstream of the action of these chemicals (Delaney et al., 1995, Lawton et al., 1996). ). The first nimI mutant of Arabidopsis (hereinafter referred to as nimI-1) was isolated from 80,000 plants of a population of Issilewskija, T-DNA-labeled (Ws-0) -decidated Arabidopsis ecotype, by sputtering, on two-week-old plants, 0.33 mM INA, followed by P. parasitica inoculation (Delaney et al., 1995). Plants that support fungal growth after INA treatment are selected as putative mutants. Five additional mutants (referred to in the invention as nim1-2, nim1-3, n7m1-4, nim1-5 and nim1-6) are isolated from 280,000 M2 plants, from a population of Ws-0 mutagenized by the ethyl methanesulfonate (EMS).

To determine whether the mutants are dominant or recessive, Ws-0 plants are used as pollen donors to cross with each of these mutants. F1 plants are then noted for their ability to support fungal overgrowth after INA treatment. as we see on the
As shown in Table 3, all F1 nim1-1, nim1-2, nim1-3, nim1-4 and nim1-6 plants are wild-type phenotypically, indicating a recessive mutation in each line. nim1-5 shows the nim phenotype in F1 plants, indicating that this particular mutant is dominant. For verification, the reciprocal cross is performed by using nim1-5 as a pollen donor to fertilize the Ws-0 plants.

In this case, the 18 F1 plants are nim from the phenotypic point of view, which confirms the dominance of the nim1-5 mutation.

 To determine whether mutations ranging from nim1-2 to nim1-6 are allelic to the previously characterized nim1-1 mutation, nim1-1 pollen is used to fertilize nim1-2 to nimi-6 mutations. Because kamycin resistance is apparent, F1 progeny are identified by their resistance to antibiotics. In all cases, kanamycin-resistant F1 plants are nim, indicating that they are allelic to the nimI-1 mutant. Since the nmi-5 mutant is dominant and obviously homozygous for the mutation, it is necessary to analyze the nimI-1 complementation in the F2 generation. If nim1-1 and nim1-5 were allelic, then all F2 plants should be expected to have a nim phenotype. If this is not the case then 13 of the 16 F2 plants are likely to have a nim phenotype. Of 94 plants, 88 clearly support fungal growth after INA treatment. Six plants have an associated phenotype of black leaf spots, which is reminiscent of a lesion mimicry phenotype, and support a low fungal proliferation after lina treatment.

Since nimI-5 carries a point mutation in the NIM1 gene (see above), it is considered to be a nimI allele.

To determine the relative strength of the different nimI alleles, each mutant was subjected to an analysis of the proliferation of P. parasitica under normal proliferation conditions and after prior treatment with SA, INA or BTH. As we see on the
Table 1, in the course of normal proliferation, the nimI-1, nimI-2, nimI-3, nimi-4 and nimI-6 mutants all support approximately the same fungal proliferation rate, which is somewhat greater than that of the control.
Ws-O. The exception is nimI-5 plants in which fungal growth is delayed by several days compared to other nimI mutants and Ws-0 controls, but eventually all nimI-5 plants succumb to the pathogenic fungus. After SA treatment, the mutants can be grouped into three classes: nim1-4 and nim1-6 show relatively fast fungal growth; the plants nim1-1, nim1-2, nim1-3 have a relatively lower rate of fungal growth; and the fungal proliferation of nimI-5 plants is even slower than in the controls
Ws-0 untreated. After treatment with INA or BTH, the mutants also appear to fall into three classes, where nim1-4 is the most severely compromised in its ability to restrict fungal overgrowth after chemical treatment; niml-l, niml-2, niml-3 and niml-4 are all moderately compromised; and niml-5 is only slightly compromised. In these experiments, Ws-0 does not support fungal growth after treatment with INA or BTH. Thus, with respect to the inhibition of fungal proliferation after chemical treatment, the mutants fall into three classes, with nimi-4 being the most highly compromised, nimI-1, nimI-2, nimi-3, and nimI-6 exhibiting intermediate inhibition of the pathogenic fungus, niml-5 having only a slightly degraded resistance to fungi.

 The accumulation of PR-1 mRNA is also used as a criterion for characterizing the different niml alleles. there might be particularly strong alleles. It is interesting to observe that the accumulation of PR-1 mRNA is high in the nimI-5 mutant, but is only moderately induced after chemical treatment or after infection with P. parasitica. Based on the accumulation of PR-1 mRNA and fungal infection, mutants fall into 3 categories: highly compromised alleles (niml-4 and niml-6); moderately compromised alleles (nimI-1, nimI-2 and nimI-3) and a weakly compromised allele (nimI-5).

Mapping the fine structure of the niml mutation
To determine a coarse map position of the NIM1, 74 plants of the nim F2 phenotype were identified from a cross between nimI-1 (Ws-O) and
Landsberg erecta (Ler) for susceptibility to P. parasitica and lack of PR-1 mRNA accumulation after INA treatment. After testing a number of simple sequence length polymorphism (SSLP) markers (Bell and Ecker, 1994), niml was found to be about 8.2 cmorgans (cM) of nga 128 and 8.2 cM nga 111, on the lower arm of chromosome 1. In a subsequent analysis, it was found that nimI-1 is between nga 111 and about 4 cM of the SSLP marker ATHGENEA.

 For the mapping of the fine structure, 1138 nim plants from an F2 population, obtained by crossing between niml-1 and Ler DP23, were identified on the basis of both their inability to accumulate PR-1 mRNA and their ability to support fungal growth after treatment with INA. The DNA is extracted from these plants, and their zygotic character is noted both at ATHGENEA and at nga 111. As seen in FIGS. 5A-5D, 93 recombinant chromosomes are identified between ATHGENEA and nimI, which gives a genetic distance of about 4.1 cM (93 of 2276), and 239 recombinant chromosomes are identified between nga 111 and nim1, indicating a genetic distance of about 10.5 cM (239 of 2276). Further informative recombinants in the range from ATHGENEA to nga 111 are further analyzed using amplified fragment length polymorphism (AFLP) analysis (Vos et al., 1995).

Initially, we identify 10 markers of AFLP between
ATHGENEA and nga 111, and used to synthesize a low resolution map of the region (Figure 5A).

The markers of AFLP W84.2 (located at 1 cM of niml) and
W85.1 (located at 0.6 cM nimI) are used to isolate yeast artificial chromosome (YAC) clones from the CIC bank (which is the Center for the Study of Yeast Artificial Chromosome, YAC). Human Polymorphism, INRA and CNRS) (Creusot et al., 1995). Two clones of YAC, CIC12H07 and CIC12F04, are identified with
W84.2, and two clones of YAC, CIC7E03 and CIC10G07 (data not shown) are identified with the marker
W85.1. However, it is found that there is a gap between the two sets of lateral YAC clones. From this point on, clones of the bacterial artificial chromosome (BAC) and P1 clones that overlap the CIC 12H07 and the
CIC 12F04 are isolated and mapped, followed by three sequential steps by extending the contiguous BAC / P1 to NM1 (Liu et al., 1995, Chio et al., 1995). At different times during walking, new AFLP polymorphisms are developed which are specific for BAC or P1 clones and are used to determine if the NIM1 gene has been crossed. It is found that NIM1 was crossed when clones of BAC and P1 are isolated, which give rise to the two markers of AFLP polymorphism L84.6a and L84.8. The AFLP marker L84.6a, which is found on the P1 clones, namely P1-18, P1-17 and P1-21, identifies three recombinants, and the L84.8 marker found on the clones P1 which are the clones P1-20, P1-22, P1-23 and P1-24, and the BAC clones that are BAC-04, BAC-05 and BAC-06 identify a recombinant. Since these clones overlap to form a large contiguous structure (2100 kb) and include AFLP markers that are lateral to niai, the gene is located on the contiguous structure. The BAC and P1 clones that comprise the contiguous structure are used to produce 8 additional AFLP markers, showing that nimI is located between the L84.Y1 and L84.8 markers, representing a gap of about 0.09. cM.

 A cosmid library was synthesized in the Agrobacterium-compatible T-DNA cosmid vector pCLD04541 using DNA from BAC-06, BAC-04 and P1-18. A contiguous cosmid structure is put using AFLP markers derived from these clones. Physical mapping shows that the physical distance between L84.Y1 and L84.8 is greater than 98 kb, which gives a ratio of the genetic distance to the physical distance of about 1 megabase per cM. To facilitate the subsequent identification of the NIM1 gene, the sequence of the BAC04 DNA is determined.

Isolation of the NIMl gene
To identify the cosmids that contain the NIM1 gene, the 12 cosmids of Table 4 are transformed into nimI-1 examples, and the ability of the transformants to complete the mutant phenotype is evaluated. The cosmids
D5, E1 and D7 all appear to be complete, which is determined by the ability of the transformants to accumulate PR-1 mRNA after treatment with INA. The ends of these cosmids are sequenced, and appear to be located on the BAC-04 DNA sequence. There are 9918 base pairs in the DNA region shared by the
D7 and D5, which contain the NIM1 gene. As seen in Figure 5D, four putative gene regions are identified in this 10 kb sequence. Region 1 could potentially encode a protein of 19,105
D, region 3 could encode a protein of 44,554 D, and region 4 could encode a region of 52,797 D. Region 2 has four open reading frames of different sizes, located close to each other, which gives to think that we have a gene with three introns.

An analysis using the NetPlantGene program (Hebsgaard et al., 1996) indicates a high probability that open reading frames can be spliced together to form a large open reading frame encoding a 66 039 D protein.

 To determine the region of the gene that contains the NIM1 gene, gel blots, containing RNA isolated from leaf tissue of Ws-O, and the different niml mutants, after treatment with water or chemical agent, are probed with DNA that derives from each of the four regions of the gene. In these experiments, care is taken to mark the probes with a large specific activity, and the autoradiograms are exposed for more than one week. In our past experience, these conditions should identify RNA at concentrations of about one copy per cell. The only region of the gene that produces detectable RNA is region 2 of the gene. As seen in Figure 7, the mRNA identified by the region 2 probe of the gene is induced by the BTH treatment of wild-type plants, but not in any of the mutants. In addition, RNA accumulation is high in all plants after infection with P. parasitica, indicating that this particular gene is induced after infection with a pathogenic microorganism.

 To establish in more detail the region of the gene that encodes NIM1, the DNA sequence from each of the four regions of the gene is determined for each of the nimI alleles and compared to the corresponding region of the gene from Ws. -0. No mutations were detected between Ws-0 and mutant alleles in any of the 3 or 4 regions of the gene, and only a single change was observed in region 1 of the nimI-6 mutant gene. However, there is a mutation consisting of a single base pair in each of the alleles, relative to Ws-0 for region 2. The DNA sequence of the gene 2 region is shown in Figure 6.

As shown in Table 5 and in Figure 6, a single adenosine is inserted in nimI-1 at position 3579, which causes a shift in the reading frame with a consequent modification of seven amino acids and the deletion of 349 amino acids. In nimI-2, there is a cytidine-thymidine transition at position 3763, which replaces a histidine with a tyrosine. In nimI-3, a single adenosine is deleted at position 3301, which causes a frame shift, which modifies 10 amino acids and removes 412 amino acids from the predicted protein. It is interesting to observe that both nimi-4 and nim1-5 have a guanosine-adenosine transition at position 4160, replacing arginine with lysine, and in nim1-6 there is a cytosine-thymidine transition that leads to to a stop codon, which deletes 255 amino acids from the predicted protein. Although the mutation in nimi-4 and nimI-5 alters the mRNA consensus donor splice site, RT-PCR polymerase chain reaction analysis indicates that this mutation does not lead to a change in splicing mRNA (data not shown).

NDMl counterparts
The DNA sequence of region 2 of the gene is used in a Blast search (Altschul et al., 1990), and identifies a match between the expressed Arabidopsis (EST) sequence marker T22612 and significant matches with the markers EST rice S2556, S2861,
S3060 and S3481 (see Figure 8). A probe is used
DNA covering base pairs 2081 to 3266 to screen an Arabidopsis cDNA library, and 14 clones corresponding to region 2 of the gene are isolated. From the cDNA sequence, we can confirm the establishment of the exon / intron boundaries that we see on the
Figure 6. Rapid amplification of the cDNA ends (RACE) by polymerase chain reaction is performed using RNA from INA-treated Ws-0 plants, and the likely starting site is determined. transcription, which turns out to be the A at position 2588 in Figure 6.

Using the NIM1 cDNA as a probe, Arabidopsis NiMi homologs can be identified and isolated by screening genograms or cDNA libraries from different plants, including, but not limited to, culture plants. rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, aubergine, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, ripe, pineapple, avocado, papaya, mango, banana, soy, tobacco, tomato, sorghum and sugar cane. Among the standard techniques for carrying out this operation, mention may be made of the hybridization screening of plaque-based DNA libraries (plaques or colonies, see, for example, Sambrook et al., Molecular
Cloning, eds., Cold Spring Harbor Laboratory Press.

(1989)), and polymerase chain reaction (PCR) using oligonucleotide primers (see, for example, Innis et al., PCR Protocols, a Guide to
Methods and Applications, eds., Academic Press (1990)).

The identified homologs undergo a genetic engineering operation in the expression vectors mentioned below, and are transformed in the above cultures. Transformants are assessed for improved disease resistance by using appropriate pathogenic microorganisms of the test crop plant.

For example, homologues of the NIM1 in the genomes of cucumber, tomato, tobacco, maize, wheat and barley were detected by DNA transfer analysis. The genomic DNA is isolated from cucumber, tomato, tobacco, maize, wheat and barley, digested with BamHI, HindIII, XbaI or SalI restriction enzymes, electrophoretically separated on 0.8% agarose gels and transferred to a nylon membrane by capillary transfer. After cross-linking to W to fix the DNA, the membrane is hybridized under low stringency conditions (1% BSA, 520 mM NaPO 4, pH 7.2, 7% sodium lauryl sulfate).
EDTA 1 mM; 250 mM Sodium Chloride) at 55 ° C for 18 to 24 hours] with 32 P-radiolabelled Arabidopsis thaliana NIM1 cDNA After hybridization, the blots are washed under low stringency conditions [6XSSC for 15 minutes (X3) 3XSSC for 15 minutes, (X1) at 55 ° C; 1XSSC corresponding to 0.15 M NaCl, 15 mM Na citrate, pH 7.0)], and exposed to X-ray film to visualize the bands corresponding to the NIM1.

 In addition, homologs of the expressed sequence markers (ESTs) identified by analogy with the NIM1 gene, such as the rice EST markers described above, can also be used to isolate the homologs. EST markers may be particularly useful for isolating homologues of NIM1 from other monocotyledons.

The homologues can also be obtained by PCR polymerase chain reaction. In this method, comparisons are made between known homologs (eg rice and Arabidopsis). It is then used to manufacture primers for amplification
PCR regions with high similarity or identity between amino acids and DNA. Once an appropriate region is identified, primers are prepared for that region, with different substitutions at the third position of the codon. The PCR reaction is carried out from cDNA or genomic DNA under different standard conditions. When a band appears, it is cloned, and / or sequenced to determine if it is a homologue of NDM1.

A high level of expression of NIMl confers on plants
disease resistance
The present invention also relates to the production of transgenic plants which express higher levels of the NIM1 gene than those of the wild type, or their functional and mutant variants, so that they exhibit resistance to a broad spectrum of disease.

In a preferred embodiment of the invention, the expression of the NIM1 gene corresponds to a level which is at least twice as high as the level of expression of the NIM1 gene of wild type plants, and which is preferably 10 times higher. at the expression level of the wild type. The high level of expression of the NIM1 gene simulates the effects of the inducing compounds, in that it gives rise to plants having a constitutive immunity phenotype (CIM).

 Several methods are described for producing plants that overexpress the NIM1 gene and therefore have a CIM phenotype. A first method is to select transformed plants that have a high level of NSM1 expression, and thus a CIM phenotype due to the effect of the insertion site. Table 6 shows the results of testing different transformants for their resistance to fungal infection.

As can be seen from the table, a number of transformants have a lower than normal fungal growth, and many of them have no visible fungal growth at all. The RNA is prepared from samples taken and analyzed as described in Delaney et al., 1995. The blots are hybridized to the Arabidopsis PR-1 gene probe (Uknes et al., 1992). . Three lines have an early induction of PR-1 gene expression, in that the PR-1 mRNA is evident 24 or 48 hours after the fungal treatment. These three lines also show resistance to fungal infection.

In addition, methods are disclosed for synthesizing plant transformation vectors comprising a plant-activity constitutive promoter, for example the 35S promoter of CaMV, operably linked to a coding region that encodes an active NIM1 protein. High levels of protein
NIM1 active produce the same effect of disease resistance as chemical induction with inducing chemicals such as BTH, INA and SA.

The NlM1 gene is a homologue of IKBα
The NIM1 gene is a key component of the systemic acquired resistance (SAR) pathway of plants (Ryals et al., 1996). The NMI gene is associated with activation of SAR by chemical and biological inducers, and, along with these inducers, is necessary for SAR and SAR gene expression. The location of the NIM1 gene is determined by a molecular biology analysis of the genome of mutant plants known to carry the mutant NIM1 gene, which gives the host plants extreme sensitivity to a wide range of pathogenic microorganisms and makes them unable to respond to pathogenic microorganisms and chemical inducers of SAR. The wild type NIM1 gene of Arabidopsis was mapped and sequenced (SEQ ID NO: 2). The wild type NIM1 gene produced (SEQ ID NO: 3) is involved in the signal transduction cascade leading to both SAR and gene -gene resistance in Arabidopsis (Ryals et al. 1997). Recombinant overexpression of the wild form of NIM1 gives rise to plants with a constitutive immunity (CIM) phenotype, and thus confers disease resistance to transgenic plants. Increased amounts of the active NIM1 protein produce the same effect of disease resistance as chemical induction with inducing chemicals such as BTH, INA and SA.

The sequence of the NIM1 gene (SEQ ID NO: 2) is used in BLAST searches, and the matches are identified on the basis of homology of a relatively well conserved domain in the NIM1 gene sequence, relative to the ankyrin domains. found in a number of proteins such as spectrins, ankyrins, NF-KB and IKB (Michaely and Bennett,
Trends Cell Biol. 2, 127-129 (1992)). However, beyond the ankyrin pattern, a conventional computer analysis does not detect other strong homologies, including homology to IKBa. Despite the negative results of the computer programs, we carry out visual checks in pairs between the protein NIM1 (SEQ ID
NO: 3) and 70 known ankyrin-containing proteins, and there is marked similarity to the IKBa class of transcriptional regulators (Baeuerle et al.
Baltimore 1996; Balwin 1996). As we see on the
9, the NIM1 protein (SEQ ID NO: 3) shares a significant homology with the IKBa proteins of the mouse, rat and pig (respectively SEQ ID NO: 18, 19 and 20).

 NIM1 contains many important structural domains of IKBa throughout the length of the protein, including ankyrin domains (indicated by the underscore in Figure 9), 2 aminoterminal serines (amino acids 55 and 59 of NIM1 ), a pair of lysines (amino acids 99 and 100 of NIM1) and an acidic C-terminal site. Overall, NIM1 and IKBa share an identity at 30% of residues, and conservative replacements at 50% of residues. Thus, there is homology between IKB and NIM1 on all proteins, with an overall similarity of 80%.

One of the functioning pathways of the protein
IKBa in signal transduction involves attaching it to the cytosolic NF-κB transcription factor and preventing it from entering the nucleus and modifying target gene transcription (Baeuerle and Baltimore 1996, Baldwin 1996). The NF-κB target genes regulate (activate or inhibit) several cellular processes, including antiviral, antimicrobial and cell death responses (Baeuerle and Baltimore, 1996). When the signal transduction pathway is activated, IKBα is phosphorylated at two serine residues (amino acids 32 and 36 of mouse IKBα). This programs the ubiquitization at a double lysine (amino acids 21 and 22) of mouse IKBa). After ubiquitization, the NF-KB / IKB complex is sent into the proteosome, where the IKBa degrades, and the NF-KB is released to the nucleus.

The phosphorylated serine residues, important in the function of the I'cBa protein, are conserved in the
NIM1, within a large contiguous block of conserved sequences, between amino acids 35 and 84 (Figure 9).

In contrast to IKBα, where the double lysine is located at about 15 amino acids in the direction of the N-terminal site of the protein, there is in NIM1 a double lysine located at about 40 amino acids towards the end.
C-terminus. In addition, there is a high degree of homology between NIM1 and IicBa in the high serine / threonine carboxyterminal region, which has been found to be important in the basal turnover rate (Sun et al. Mol Cell Biol 16, 1058-1065 (1996)). According to the present invention, and based on the analysis of structural homology and the presence of elements known to be important for the function of IicBa, NIM1 is expected. works like IKBa, since it has similar effects on the regulation of plant genes.

Plants containing the wild-type NIM1 gene, after treatment with inducing chemicals, should comprise a larger amount of the NIM1 gene product (IKB homolog), or less phosphorylation of the NiMi gene product (homologue of the IKB). According to this model, the result is that the counterpart
Plant NF-κB is maintained outside the nucleus, and responses in SAR gene expression and resistance may occur. In mutant nimI plants, a non-functional NIM1 gene product is present. Therefore, according to this model, the NF-κB homolog is free to go to the nucleus and suppress the resistance and expression of the SAR gene.

Consistent with this idea, salicylic acid-treated animal cells exhibit increased stability / abundance of IKB and a reduction of active NF-KB in the nucleus (Kopp and Ghosh, 1994). IKB mutations that act as super-repressors or dominant-negatives are known (Britta-Mareen
Traenckner et al., EMBO 14: 2876-2883 (1995); Brown et al., Science 267: 1485-1488 (1996); Brockman et al.
Molecular and Cellular Biology 15: 2809-2818 (1995); Wang et al., Science 274: 784-787 (1996)). These mutant forms of IKB bind to NF-KB but are not phosphorylated or ubiquitinated, so they are not degraded. NF-KB remains attached to IKB and can not move to penetrate the nucleus.

Modified forms of the NIMl gene
In view of the above, the present invention relates to modified forms of NIM1, which act as dominant-negative regulators of the SAR signal transduction pathway. Plants transformed by these dominant-negative forms of NIM1 have the opposite phenotype as niml mutant plants, in that plants transformed with modified forms of NIM1 exhibit constitutive expression of the SAR gene, and thus a CIM phenotype. . Because of the position that the NIM1 gene retains in the SAR signal transduction pathway, it is expected that a number of gene modifications, beyond those specifically described in the invention will be expected. , appear in the constitutive expression of the genes of the SAR, and therefore a CIM phenotype.

There is a need for phosphorylation of serine residues in human IKBa for stimulus-activated IKBa degradation, which activates NF-κB. Mutagenesis of serine residues (S32 and S36) in human IKBa to alanine residues inhibits stimulus-induced phosphorylation, which blocks IKBa proteosome-mediated degradation (Traenckner et al., 1995).
Brown et al. 1996; Brockman et al. 1995; Wang et al.

1996). This modified form of IKBa can act as a dominant-negative form, retaining NF-KB in the cytoplasm, and thereby blocking downstream signaling events. On the basis of a comparison of the amino acid sequences between NIM1 and IaB shown in Figure 9, serines 55 (S55) and 59 (S59) of NIM1 (SEQ ID NO: 3) are homologous to S32 and S36 in human IKBα. To synthesize dominant-negative forms of NIM1, serines at amino acid positions 55 and 59 are mutagenized to alanine residues. Thus, in a preferred embodiment of the present invention, the NIM1 gene is modified so that the encoded product has alanines instead of serines at the amino acid positions corresponding to positions 55 and 59 of the acid sequence. amines of
NIM1 of Arabidopsis. The present invention also relates to disease-resistant transgenic plants transformed with such a modified form of the NIM1 gene, as well as methods for using this modified form of the NIM1 gene for the purpose of conferring on plants transformed with the latter. disease resistance, by activating the expression of the SAR gene.

 The deletion of amino acids 1 to 36 (Brockman et al., 1995, Sun et al., 1996) or 1-72 (Sun compared to the native amino acid sequence NIM1 of Arabidopsis. Disease-resistant transgenics, transformed with such a modified form of the NIAI gene, as well as methods for using this modified form of the NIM1 gene, to confer disease resistance and to activate the expression of the SAR gene in the transformed plants. help from the latter.

 The deletion of amino acids 261-317 from human IaBa can lead to enhanced intrinsic stability by blocking the constitutive phosphorylation of serine and threonine residues in the C-terminal site. This modified form of IKBA should function as a dominant-negative form. A serine and threonine rich region is present at amino acids 522-593 on the C-terminal site of NIM1. Thus, in a preferred embodiment of the present invention, the NIM1 gene is modified such that the coded product lacks approximately its terminal portion, which comprises amino acids 522-593, as compared to amino acid sequence NIM1. native to Arabidopsis. The present invention also relates to disease-resistant transgenic plants transformed with such a modified form of the NIM1 gene, as well as methods for using this modified form of the NIM1 gene to confer disease resistance and to activate SAR gene expression. in plants transformed with the latter.

 In another embodiment of the present invention, modified forms of the NIM1 gene product are produced as a result of deletions or chimeras of the C-terminal segment and the N-terminal segment. In yet another embodiment of the present invention, synthetic products comprising the ankyrin domains from the NIM1 gene are provided. The present invention also relates to disease-resistant transgenic plants, transformed with such synthetic products or ankyrin NIM1, as well as methods for using these variants of the NIM1 gene to confer disease resistance and to activate gene expression. of SAR in plants transformed with the latter.

 The present invention relates to DNA molecules encoding modified forms of the NIM1 gene, such as those described above, expression vectors containing these DNA molecules, and plants and plant cells transformed therewith. The invention also relates to methods for activating SAR in plants, and for imparting a CIM phenotype and resistance to a broad spectrum of diseases to plants by transforming plants using DNA molecules encoding modified forms. of the gene product NIM1. The present invention further relates to plants transformed with a modified form of the NiMi gene.

Disease resistance
Overexpression of the wild-type NIM1 gene in plants and the expression of modified forms of the NiMi gene in plants lead to immunity to a broad set of phytopathogenic microorganisms that include, but are not limited to, viruses. or viroids, tobacco mosaic virus or cucumber virus, ring spot or necrosis virus, foliar necrosis virus of geraniaceae, clover violet variegation virus, tomato blight virus, and virus analogous pathogenic fungi, for example Phytophthora parasitica and Peronospora tabacina; bacteria, Pseudomonas syringae and Pseudomonas tabaci, insects such as aphids, eg Myzus persicae, and Lepidoptera, Heliothus spp. ; and nematodes, for example Meloidogyne incognita. The vectors and methods of the invention can be used against a number of pathogenic organisms, which include but are not limited to downy mildews such as Sclerophthora macrospora, Sclerophthora rayissiae, Sclerospora graminicola,
Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora sacchari and Peronosclerospora maydis; rust such as Puccinia sorphi, Puccinia polysora and Physopella zeae; other pathogenic fungi such as Cercospora zeae-maydis, Colletotrichum graminicola, Fusarium monoliforme, Gibberella zeae, Exserohilum turcicum, Kabatiellu zeae, Erysiphe graminis,
Septoria and Bipolaris maydis; and bacteria, such as Erwinia stewartii.

 The methods of the present invention can be used to confer disease resistance to a broad range of plants, including gymnosperms, monocotyledons and dicotyledons. Although disease resistance can be conferred on any plant in these broad categories, it is particularly useful in agronomically important crops, such as the following crops: rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, aubergine, pepper, celery, carrot squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soy, tobacco, tomato , sorghum and sugar cane. Transformed cells can be regenerated in whole plants, so that the gene confers disease resistance to intact transgenic plants. The expression system may be modified so that the disease resistance gene can be expressed in a continuous or constitutive manner.

Recombinant DNA technology
The DNA molecule or the NIM1 gene fragment that confers disease resistance to plants by allowing the induction of SAR gene expression, or the modified form of the NIM1 gene conferring resistance to plant diseases. by enhancing the expression of the SAR gene, can be incorporated into plant or bacterial cells using conventional recombinant DNA technology. In general, this technique consists in inserting the DNA molecule, included in the sequence SEQ ID NO: 1, or a functional variant thereof, or a molecule encoding one of the modified forms of NIM1 described above, in an expression system whose DNA molecule is heterologous (i.e., is not normally present). The heterologous DNA molecule is inserted into the system or expression vector in a correct orientation and in a correct reading frame. The vector contains the elements necessary for transcription and translation of the inserted sequences encoding the protein. A wide variety of vector systems known in the art can be used, such as plasmids, bacteriophage viruses and other modified viruses.

Suitable vectors include, but are not limited to, viral vectors such as X, kgt11, hgt10, and Charon 4 vector systems; plasmid vectors such as pBI121, pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101, pCDNAII; and other similar systems. The coding sequence of the NIM1 and the modified coding sequences of the NIM1 described in the invention can be cloned into the vector using conventional cloning techniques, as described by
Maniatis et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Laboratory, Cold Spring Harbor, New York (1982).

 To achieve efficient expression of the gene or gene fragment of the present invention, a promoter that will lead to a sufficient level of expression or sufficient constitutive expression must be present in the expression vector. The RNA polymerase normally binds to the promoter and triggers the transcription of a gene. Promoters vary in their strength, that is, in their ability to promote transcription. Depending on the host cell system used, any one of a number of suitable promoters may be used. The components of the expression cassette can be modified to increase the expression. For example, truncated sequences, nucleotide substitutions, or other modifications may be used. Plant cells transformed with these modified expression systems thus overexpress or constitutively express the genes necessary for the activation of SAR.

A. Synthesis of plant transformation vectors
Many transformation vectors are available for plant transformation, and the genes of the present invention may be used in conjunction with any one or more of these vectors. Vector selection will depend on the preferred transformation technique and the target species of the transformation. For certain target species, different selection markers for antibiotics or herbicides may be preferred. Selection markers routinely used in transformation include the nptil gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra, Gene 19: 259-268 (1982), Bevan et al., Nature 304 184-187 (1983)), the bar gene, which confers resistance to the phosphinothricin herbicide (White et al., Nucl Acid Res 18: 1062 (1990), Spencer et al.
Theor. Appl. Genet 79: 625-631 (1990)), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931), and the dhfr gene, which confers methotrexate resistance (Bourouis et al., EMBO J. 2 (7): 1099-1104 (1983)); and the EPSPS gene, which confers resistance to glyphosate (US Pat. Nos. 4,940,935 and 5,188,642).

1. Vectors Suitable for Agrobacterium Transformation
Many vectors exist which allow transformation using Agrobacterium tumefaciens. They usually carry a T-DNA restriction sequence and include vectors such as pBIN19 (Bevan, Nucl Acid Res (1984)) and pXYZ. The synthesis of two representative vectors will be described below.
at. pCBI200 and pCIB2001
The binary vectors pCIB200 and pCIB2001 are used to synthesize recombinant vectors for use with Agrobacterium, and are synthesized as follows. PTJS75kan was created by digestion of pTJS75 by NarI (Schmidhauser & Helinski, J.

Bacteriol. 164: 446-455 (1985), which allows excision of the tetracycline resistance gene, followed by insertion of an AccI fragment from pUC4K carrying an NPTII (Messing & Vierra, Gene 19: 259- 268 (1982), Bevan et al., Nature 304: 184-187 (1983)
McBride et al., Plant Molecular Biology 14: 266-276 (1990)). XhoI linkers are ligated to the EcoRV fragment of pCIB7, which contains the left and right edges of T-DNA, a selectable plant-selectable chimeric gene nos / nptII, and the pUC multisite linker (Rothstein et al. al., Gene 53: 153-161 (1987)) and the fragment digested with
XhoI is cloned into SalI digested pTJS75kan to create pCIB200 (see also EP 0 332 104, Example 19). PCIB200 contains the following unique polysite restriction sites: EcoRi, SstI, Kpni, BglII, XbaI and
Sall. PCIB2001 is a derivative of pCIB200, created by insertion of additional restriction sites into the multisite linker. The unique restriction sites of the pCIB2001 multisite linker are EcoRi, SstI, KpnI, BglII,
XbaI, SalI, MluI, BclI, AvrII, ApaI, HpaI and StuI. PCIB2001, in addition to containing these unique restriction sites, also has left and right T-DNA edges for selection against plant and bacterial kanamycin for Agrobacterium-mediated transformation. the trfA function derived from RK2 for mobilization between E. coli and other hosts, and the OriT and OriV functions, which also come from RK2. The multisite linker pCIB2001 is suitable for cloning plant expression cassettes containing their own regulatory signals.
b. PCIB10, and its Hygromycin Selection Derivatives
The binary vector pCIB10 contains a gene encoding kanamycin resistance for selection in plants, and the left and right border sequences of DNA
T, and includes sequences from plasmid pRK252, which corresponds to a wide host range, which allows replication in both E. coli and Agrobacterium. Its synthesis is described by Rothstein et al.

(Gene 53: 153-161 (1987)). Various derivatives of pCIB10 are synthesized, which include the hygromycin-B-phosphotransferase gene, described by Gritz et al.

(Gene 25: 179-188 (1983)). These derivatives allow a selection of transgenic plant cells, on hygromycin alone (pCIB743) or on hygromycin and kanamycin (pCIB715, pCIB717).

2. Vectors suitable for processing other than
Agrobacterium
A transformation not using Agrobacterium tumefaciens avoids using T-DNA sequences in the chosen transformation vector, so that vectors lacking these sequences can be used, in addition to vectors such as those described herein. above and which contain T-DNA sequences. Transformation techniques that are not based on
Agrobacterium include particle bombardment transformation, protoplast uptake (eg PEG and electroporation), and microinjection.

The choice of the vector depends largely on the preferred choice of the species undergoing transformation.
at. pCIB3064
PCIB3064 is a pUC-derived vector that can be used in direct gene transfer techniques in combination with selection by basta herbicide (or phosphinothricin). Plasmid pCIB246 comprises the 35S promoter of CaMV, operatively fused to the E. coli GUS gene and the 35S transcription terminator of CaMV, and is described in published PCT application number WO 93/07278. The 35S promoter of this vector contains two ATG sequences, in the 5 'direction from the start site. These sites are mutated using standard PCR amplification techniques to remove ATGs and produce the SspI and PvuII restriction sites. The new restriction sites are 96 and 37 bp from the single SalI site, and 101 and 42 bp from the actual site of departure. The derivative obtained from pCIB246 is called pCIB3025. The GUS gene is then excised from pCIB3025 by digestion with SalI and SacI, the terminal sites are blunt-ended and religated to produce plasmid pCIB3060. The plasmid pJIT82 is obtained from John Innes Center,
Norwich, and the 400 bp SmaI fragment containing the bar gene from Streptomyces viridochromogenes is excised and inserted into the HpaI site of pCIB3060 (Thomson et al., EMBO J 6: 2519-2523 (1987)). This pCIB3064 thus produced comprises the bar gene under the control of the CaMV 35S promoter and the herbicide selection promoter, an ampicillin resistance gene (for selection in E. coli) and a multisite linker, with the unique sites SphI, Psti, HindIII and PainHi. This vector allows the cloning of plant expression cassettes containing their own regulatory signals.
b. pSOG19 and pSOG35
PSOG35 is a transformation vector that uses the dihydrofolate reductase (DFR) of the E. coli gene as a selectable marker conferring resistance to methotrexate. PCR amplification was used to amplify the 35S promoter (-800 bp), the intron 6 from the corn acid (-550 bp) gene, and 18 bp of the GUS untranslated leader sequence from pSOG10. A 250 bp fragment, which encodes the E. coli dihydrofolate reductase type II gene, is also amplified by PCR amplification, and these two PCR fragments are assembled to a SacI-PstI fragment from pB1221 (Clontech), which comprises the pUC19 vector backbone and the nopaline synthase terminator. The assembly of these fragments produces pSOG19, which contains the 35S promoter in fusion with the sequence of intron 6, the GUS leader sequence, the DHFR gene and the nopaline synthase terminator. The replacement of the GUS leader sequence in pSOG19 by the leader sequence from corn chlorotic mottle virus (MCMV) produces the vector pSOG35. PSOG19 and pSOG35 carry the ampicillin resistance pUC gene and possess the HindIII, SphI, PstI and EcoRI sites, which allow the cloning of foreign substances.

B. Requirements for the synthesis of plant expression cassettes
The gene sequences that are to be expressed in the transgenic plants are first assembled into expression cassettes, behind a suitable high level expression promoter, and upstream of an appropriate transcription terminator. These expression cassettes can then be easily transferred into the plant transformation vectors described above.

1. Selection of the promoter
Selection of the promoter used in the expression cassettes will determine the spatial and temporal expression sequence of the transgene in the transgenic plant. Selected promoters will express transgenes in specific plant cells (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (eg roots, leaves or flowers), and the selection will reflect the desired location of the accumulation of the NIM1 gene product or the modified NIM1 gene product. Alternatively, the selected promoter can result in the expression of the gene under a photoinduced promoter, or another promoter with provisional regulation.
at. Constitutive expression, CaMV 35S promoter
The synthesis of plasmid pCGN1761 is described in published patent application EP 0 392 225 (Example 23), which is incorporated herein by reference. PCGN1761 contains the 35S "double" promoter and the tml transcription terminator, with a unique EcoRI site between promoter and terminator, and has a pUC backbone. A derivative of pCGN1761, which has a modified multisite linker, which includes NotI and XhoI sites is synthesized in addition to the existing EcoRI site. This derivative is called pCGN1761ENX. PCGN1761ENX can be used to clone cDNA sequences or gene sequences (including microbial ORF sequences) within its polylinker, to allow their expression under the control of the 35S promoter in transgenic plants. The entire 35S promoter-gene sequence-terminator tml cassette of such a synthetic product can be excised by the HindIII sites,
SphI, SalI and XbaI, located in the 5 'direction relative to the promoter, and the XbaI, BamHI and BglI sites located in the 3' direction relative to the terminator, to allow transfer to transformation vectors such as those described above. In addition, the double 35S promoter fragment can be removed by 5 'excision using HindIII, SphI, SalI, XbalI or Psti, and by 3' excision with any of the restriction sites of the linker. multisite (EcoRi, NotI or XhoI) for replacement by another promoter.
b. Modification of pCGN1761ENX by optimizing the translation initiation site
For any of the synthetic products described in the invention, modifications can be made around the cloning sites by introducing sequences that can enhance translation. This way of doing things is particularly interesting when you want overexpression.

PCGN1761ENX is cleaved with SphI, treated with T4 DNA polymerase and religated, and the SphI site located in the 5 'direction relative to the double 35S promoter is destroyed. The vector pCGN1761ENX / Sph- is thus obtained. PCGN1761ENX / Sph- is cleaved with EcoRi, and ligated onto a ringed molecular adapter of the sequence 5'-ATTCTAAAGCATGCCGATCGG-3 '/ 5
AATTCCGATCGGCATGCTTTA-3 '(SEQ ID NO: 12 and 13). The vector pCGNSENX, which comprises the quasi-optimized plant translation initiation sequence TAAA, is thus obtained.
C, adjacent to the ATG, which itself is part of a site
SphaI, which is suitable for cloning heterologous genes at the level of their starting methionine. Downstream of the site
SphI, we keep the sites EcoRi, NotI and Chou.

We synthesize another vector, which uses a site
NcoI at the starting ATG. This vector, called pCGN1761NENX, is obtained by inserting a ringed molecular adapter of the 5 'sequence.
AATTCCGATCGCCATGGTTTA-3 '(SEQ ID NO: 14 and 15) at the EcoRI site of pCGN1761ENX. Thus, this vector comprises the entire quasi-optimized TAAACC sequence adjacent to the starting ATG which lies within the NcoI site. Downstream sites are EcoRi, NotI and XhoI.

Before this manipulation, the two NcoI sites of the vector pCGN1761ENX (at the upstream positions of the 35S promoter at the 5 'position) are, however, destroyed using techniques analogous to those described above for SphI, or else using a amplification
"Indoor-outdoor" PCR. Innes et al., PRC Protocols
A guide to methods and applications, Academic Press, New
York, (1990). This manipulation may be evaluated for possible possible annoying effects on expression by inserting any plant cDNA or reporter gene sequence into the cloning site after routine expression analysis in the cloning sites. plants.
c. Expression under a chemically regulatable promoter / pathogenic microorganism
The double 35S promoter of pCGN1761ENX can be replaced by any other promoter, which leads to sufficiently high levels of expression. By way of example, a chemically regulated PR-1 promoter, which is described in US Patent No. 5,614,395, which is incorporated herein by reference in its entirety, can replace the dual 35S promoter. The choice is preferably excised from its source by restriction enzymes, but it can also be amplified by PCR amplification using primers that carry the appropriate terminal restriction sites. the promoter should be resequenced to control for amplification errors after cloning the amplified promoter into the target vector The tobacco PR-la promoter, which can be chemically or pathogenically regulated, is cleaved off plasmid pCIB1004 (see EP 0 332 104, Example 21, for synthesis, this document is incorporated herein by reference), and transferred to plasmid pCGN1761ENX (Uknes et al., 1992). pCIB1004 is cleaved with Nicol, and the 3 'end of the linearized fragment is blunted by treatment with T4 DNA polymerase. This fragment is then cleaved using HindIII, and the fragment obtained, containing the promoter
PR-1α, was gel purified and cloned into pCGN1761ENX from which the double 35S promoter was removed. This operation is carried out by cleavage with XhoI, and making a blunt end with the T4 polymerase, then cleaving with HindIII and isolating the larger fragment containing the terminator of the vector, in which the fragment is cloned. pCIB1004 promoter. A pCGN1761ENX derivative is obtained, with the promoter PR-1a and the terminator tml, and an intermediate polysite linker having unique sites EcoRI and NotI. Selected NIM1 genes may be inserted into this vector, and the fusion products (i.e., promoter-gene-terminator) may then be transferred to any selected transformation vector, including those described herein. request.

Various chemical regulators can be used to induce the expression of the coding sequence of NIM1 in transformed plants according to the present invention. In the context of the invention, "chemical regulators" include chemical compounds known to be promoter of the PR-1 promoter in plants, or their closely related derivatives. A preferred group of regulators of the PR-1 promoter is based on the benzo-1,2,3-thiadiazole (BTH) structure, and includes but is not limited to the following types of compounds: benzo-1,2,3-thiadiazolecarboxylic acid, benzo-1,2,3-thiadiazolethiocarboxylic acid, cyanobenzo-1,2,3-thiadiazole, benzo-1,2,3-thiadiazolecarboxamide, benzo-1,2,3-thiadiazolecarboxylic acid hydrazide, 1,2-benzoic acid , 3-thiadiazole-7-carboxylic acid, benzo-1,2,3-thiadiazole-7-thiocarboxylic acid, 7-cyanobenzo-1,2,3-thiadiazole, benzo-1,2,3-thiadiazolecarboxylate, wherein the alkyl group contains 1 to 6 carbon atoms, methyl benzo-1,2,3-thiadiazole-7-carboxylate, n-propyl benzo1,2,3-thiadiazole-7-carboxylate, benzo1,2,3-thiadiazole-7-carboxylate benzyl, sec-butylhydrazide of benzo-1,2,3-thiadiazole-7-carboxylic acid, and their appropriate derivatives. Other chemical inducers may include, for example, benzoic acid, salicylic acid (SA), poly (acrylic acid) and their substituted derivatives; suitable substituents include lower alkyl, lower alkoxy, lower alkylthio and halo. Yet another group of regulators for the chemically-inducible DNA sequences of the present invention is based on the pyridinecarboxylic acid structure, for example the isonicotinic acid structure, and preferably the halogenoisonicotinic acid structure. Dichloroisonicotinic acids and their derivatives, for example lower alkyl esters, are preferred. Suitable members of this class of regulatory compounds are, for example, 2,6-dichloroisonicotinic acid (INA) and its lower alkyl esters, especially its methyl ester.
d. Constitutive expression, actin promoter
Several isoforms of actin are known to be expressed in most cell types, so that the actin promoter is an appropriate choice for a constitutive promoter. In particular, the promoter from the rice Acti gene has been cloned and characterized (McElroy et al., Plant Cell 2: 163-171 (1990)). A 1.3 kb fragment of the promoter was found to contain all the regulatory elements required for rice protoplast expression. In addition, many expression vectors based on the ActI promoter have been specifically synthesized for use in monocotyledons (McElroy et al., Mon. Gen. Genet 231: 150160 (1991)). They include intron 1 of the act, the 5'-side sequence of the AdhI and the intron 1 of the Acid (from the corn alcohol dehydrogenase gene), and the sequence from the promoter. 35S CaMV. The vectors exhibiting the greatest expression are the 35S and intron fusions of the act, or the 5'-side sequence of the ActI and the intron of the act. Optimization of the sequences around the initiation ATG (of the GUS reporter gene) also enhances the expression. The promoter expression cassettes described by McElroy et al. (Mol Gen. Genet 231: 150-160 (1991)) can be easily modified to allow expression of cellulase genes and are particularly suitable for use in monocotyledonous hosts. For example, fragments containing the promoter are removed from McElroy constructs and are used to replace the double 35S promoter of pCGN1761ENX, which is then available for insertion of specific gene sequences. The fusion genes thus synthesized can then be transferred to appropriate transformation vectors. In a separate article, the ActI promoter of rice, with its first intron, has also been shown to direct high expression in cultured barley cells (Chibbar et al., Plant Cell Rep.

12: 506-509 (1993)).
e. Constitutive expression, the ubiquitin promoter
Ubiquitin is another gene product known to accumulate in many cell types, and its promoter has been cloned from several species for use in transgenic plants (eg sunflower - Binet et al. al., Plant Science 79: 8794 (1991) and corn - 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 vectors, synthesized for monocotyledon processing, are described in EP 0 342 926 (in the name of Lubrizol), which is incorporated herein. for reference. Taylor et al. (Plant
Cell Rep. 12: 491-495 (1993)) discloses a vector (pAHC25) which comprises the maize ubiquitin promoter and a first intron, as well as its high activity in cell suspensions of many monocotyledons after introduction by bombing of microprojectiles. The ubiquitin promoter is responsible for the expression of cellulase genes in transgenic plants, especially monocotyledons. Suitable vectors are derivatives of pAHC25 or any of the transformation vectors described herein, modified by the introduction of appropriate promoter and / or intron sequences of ubiquitin.
f. Specific expression of roots
Another model of expression of the NIM1 gene of the present invention is the root expression. A suitable root promoter is described by Framond (FEBS 290: 103-106 (1991)), and also in published patent EP 0 452 269 (in the name of Ciba-Geigy), which is incorporated herein by reference. This promoter is transferred to an appropriate vector such as pCGN1761ENX for insertion of a cellulase gene, and then transfer of the entire promoter-gene-terminator cassette to a transformation vector of interest.
g. Promoters inducible by injury
Inhibitors inducible by a lesion may also be suitable for the expression of the NIM1 genes according to the invention. 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 & Lehle Plant Molec Biol 22: 783792 (1993), Firek et al., Plant Molec Biol 22: 129-142 (1993), Warner et al., Plant J. 3: 191-201 (1993)), and all are suitable for use in the context of the present invention. Logemann & al. describe the 5'-upstream sequences of the wunI gene of the potato dicotyledonous plant. Xu et al. show that a lesion inducible promoter, derived from potato dicotyledonous (pin2) is active in monocotyledonous rice. In addition,
Rohrmeier & Lehle describe the cloning of corn WipI cDNA, which is induced by injury, and which can be used to isolate the cognate promoter using conventional techniques. Similarly, Firek et al., And Warner et al., Have described a lesion-induced gene from the monocotyledon Asparagus officinalis that is expressed at local sites of injury and invasion by a microorganism. pathogenic.

Using well-known starting cloning techniques, these promoters can be transferred to appropriate vectors fused to the NIM1 gene of the invention, and used to express these genes at plant injury sites.
h. Medullo-preferential expression
Patent Application WO 93/07278 (in the name of Ciba
Geigy), which is incorporated herein by reference, describes the isolation of the maize trpA gene, which shows preferential expression in bone marrow cells.

This reference shows the gene sequence and promoter up to -1726 bp from the start of transcription. Using standard molecular biology techniques, this promoter, or portions thereof, can be transferred to a vector such as pCGN1761, where it can replace the 35S promoter and be used to drive the expression of a foreign gene in a medullo-preferential way. In fact, fragments containing the medullo-preferential promoter, or portions thereof, may be transferred to any vector and modified for use in transgenic plants.
i. Specific expression of leaf
A corn gene encoding phosphoenol carboxylate (PEPC) has been described by Hudspeth & Grula (Plant Molec
Biol 12: 579-589 (1989)). Using standard molecular biology techniques, the promoter of this gene can be used to drive the expression of any gene in a leaf specific manner in transgenic plants.
j. Targeted expression of the chloroplast
Chen & Jagendorf (J. Biol Chem 268: 2323-2367 (1993)) described the successful use of a chloroplast transit peptide for importing a heterologous transgene. This peptide as used is the transit peptide from the rbcS gene of Nicotiana plumbaginifolia (Poulsen et al., Mol. Gen. Genet 205: 193-200 (1986)). Using the DraI restriction enzymes and
SphI prorated to Tsp509I and SphI, the DNA sequence encoding this transit peptide can be excised from plasmid prbcS-8B and manipulated for use with any of the constructs described above. The DraI-SphI fragment extends from -58, relative to the ATG of the initiation rbsS, up to and including the first amino acid (and also a methionine) of the mature peptide immediately after the cleavage site. import, while the Tsp509I-SphI fragment extends from position 8 relative to the ATG of the initiation rbcS to and including the first acid of the mature peptide.

Thus, these fragments may conveniently be inserted into the multisite linker of any selected expression cassette that produces a transcriptional fusion with the untranslated leader sequence of the selected promoter (e.g., 35S, PR-1A, actin, ubiquitin, etc.), while allowing the insertion of a correctly merged NIM1 gene downstream of the transit peptide. Synthesis products of this type are customary in the art. For example, while the end of the DraI is already blunt, the 5 'Tsp509I site can be made blunt by T4 polymerase treatment, or it can be ligated to a linker or adaptation sequence to facilitate its fusion. to the chosen promoter. The 3 'site
SphI can be maintained as it is, or it can be ligated to the adapter or linker sequence to facilitate its insertion into the chosen vector so as to provide appropriate restriction sites for the subsequent insertion of a NIM1 gene. selected. Ideally, the ATG of the SphI site is maintained and comprises the first ATG of the selected NIM1 gene. Chen & Jagendorf provide consensus sequences for the ideal cleavage for importing chloroplasts, and in each case a methionine is preferred over the first position of the mature protein. On subsequent positions, there is a greater variation, and the amino acid may not be so critical. In any case, fusion constructs can be evaluated for their in vitro import efficiency using the methods described by Bartlett et al. (in: Edelmann et al (Eds), Methods in Chloroplast Molecular Biology,
Elsevier, pp. 1081-1091 (1982)), and Wasmann et al.

 (Mol Gen. Genet 205: 446-453 (1986)). Usually, the best approach may be to produce fusions using the selected NIM1 gene or a modified form of the NIM1 gene without any modification at the amino-terminal site, and only to incorporate modifications when it is clear that these fusions do not correspond to a chloroplast import with a high yield, in which case modifications can be made according to the established literature (Chen & Jagendorf, Wasmann et al., Ko & Ko, J. Biol.

Chem. 267: 13910-13916 (1992)).

 A preferred vector is synthesized by transferring the fragment encoding the DraI-SphI transit peptide from prbcS-8B into the cloning vector pCGN1761ENX / Sph-.

This plasmid is cleaved with EcoRI, and the ends are made blunt by treatment with T4 DNA polymerase. The plasmid prbcS-8B is cleaved with SphI and ligated to a ringed molecular adapter of the sequence 5'-CCAGCTGGAATTCCG-3 '/ 5'-CGGAATTCCAGCTGGCATG-3' (SEQ ID
NO: 16 and 17). The product obtained is subjected to 5'-terminal phosphorylation by treatment with the kinase of
T4. Subsequent cleavage with DraI releases the fragment encoding the transit peptide, which fragment is ligated into the ex-EcoRI-blunt sites of the modified vector described above. Clones oriented to the 5 'end of the 3S end-adjacent insert of the 35S promoter are identified by sequencing. These clones carry a DNA fusion of the 35S leader sequence and the rbcS-8A promoter peptide sequence of the -58 to the ATG of rbcS to the ATG of the mature protein, and comprising in this region a unique SphI site, and a newly created EcoRi site, as well as existing sites
NotI and XhoI of pCGN1761ENX. This new vector is called pCGN1761 / CT. DNA sequences are transferred to pCGN1761 / CT from the reading frame by PCR amplification and incorporation of a SphI, NSphI or N1AIII site at the amplified ATG which, after cleavage with the appropriate restriction enzyme, is ligated in the pCGN1761 /
CT cleaved by Spahi. To facilitate synthesis, it may be necessary to modify the second amino acid of the product of the cloned gene; however, in most cases, the use of PCR amplification, coupled with site-directed standard mutagenesis, will allow the synthesis of any desired sequence around the cleavage site and the first methionine of the protein. mature.

Another preferred vector is synthesized by replacing the double 35S promoter of pCGN1761ENX with the BamHI-SphI fragment of prbcS-8A which contains the complete photoregulated rbcS-8A promoter ranging from position 1038 to the transcription start site at. the first methionine of the mature protein. The modified pCGN1761, containing the destroyed SphI, is cleaved with PstI and EcoRi, and treated with T4 DNA polymerase to make the ends blunt. PrbcS-8A is cleaved with the SphI enzyme and ligated to the annealed molecular adapter of the sequence described above. The product obtained is subjected to 5-terminal phosphorylation by treatment with T4 kinase. Subsequent cleavage with BamHI releases the fragment containing the transit peptide promoter, which fragment is treated with T4 DNA polymer to make the ends blunt.
Bam. The promoter-transit peptide fragment thus produced is cloned into the vector pCGN1761ENX thus prepared, producing a product comprising the rbcS-8A promoter and the transient peptide, with one site.
SphI located at the cleavage site for insertion of heterologous genes. In addition, downstream of the SphI site, there are EcoRI (recreated), NotI and XhoI cloning sites. This synthetic product is called pCGN176lrbcS / CT.

 Analogous manipulations can be made to use other GS2 sequences encoding the chloroplast transit peptide and from other sources (monocotyledonous and dicotyledonous), and other genes. In addition, analogous procedures can be used to target other subcellular compartments such as mitochondria.

2. Transcription Terminators
A large number of transcription terminators are available for use in expression cassettes. They are responsible for terminating transcription beyond the transgene and its correct polyadenylation. Suitable transcription terminators are those known to function in plants, and include CaMV 35S terminator, tml terminator, nopaline synthase terminator, and pea rbcS E9 terminator. They can be used in monocotyledons and dicotyledons.

3. Sequences to amplify or regulate expression
Many sequences have been shown to enhance gene expression from the transcription unit, and these sequences can be used in conjunction with the genes of the present invention to increase their expression in transgenic plants.

Many intron sequences have been found to enhance expression, particularly in monocotyledonous cells. For example, the corn Adhi gene introns have been shown to significantly enhance the expression of the wild-type gene in the presence of its cognate promoter after introduction into these corn cells. Intron 1 was found to be particularly effective, and enhanced expression in the fusion products with the chloramphenicol acetyltransferase gene (Callis et al., Genoa
Develop. 1: 1183-1200 (1987)). In the same experimental system, the corn bronzel gene intron has a similar effect on enhancement of expression. Intron sequences are systematically incorporated into plant transformation vectors, usually with the untranslated leader sequence.

 A number of untranslated, virus-derived, expression enhancing, and non-translated leader sequences are known to be particularly effective in dicotyledonous cells. Specifically, the tobacco mosaic virus (TMV, the "W sequence"), the corneal mottle virus (MCMV), and the alfalfa mosaic virus (AMV) virus leader sequences have been shown to be effective in enhancing expression (eg Gallie et al., Nucl Acids Res 8693-8711 (1987), Skuzeski et al., Plant Molec Biol.

15: 65-79 (1990)).

4. Targeting the gene product inside the cell
It is known that there are different mechanisms for targeting gene products in plants, and the sequences that control the functioning of these mechanisms have been characterized in some detail. For example, targeting of chloroplast gene products is driven by a signal sequence found at the amino terminus of different proteins, which is cleaved during import of the chloroplast to yield the mature protein (e.g. Comai et al., J.

Biol. Chem. 263: 104-150 (1988)). These signal sequences can be fused to heterologous gene products to ensure the import of heterologous products into the chloroplast (van den Broeck et al.,
Nature 313: 358-363 (1985)). The DNA coding for the appropriate signal sequences can be isolated from the 5 'end of the cDNAs encoding the RUBISCO protein, the protein
CAB, EPSP synthase enzyme, GS2 protein and many other proteins known to be chloroplast localized.

 Other gene products are localized in other organelles, including mitochondria and peroxisome (eg Unger et al., Plant Molec Biol., 411-418 (1989)). The cDNAs encoding these products can also be manipulated to target heterologous gene products to these organelles. Exemplary of these sequences include nuclear-coded ATPases and isoforms specific for aspartate aminotransferase for mitochondria. Targeting of cellular protein bodies has been described by Rogers et al. (Proc Natl Acad Sci USA 82: 6512-6516 (1985)).

In addition, sequences have been characterized which cause the targeting of gene products to other cellular compartments. The amino-terminal sequences are responsible for ER, apoplast and extracellular secretion targeting from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)). In addition, amino-terminal sequences, together with carboxy-terminal sequences, are responsible for vacuolar targeting of gene products (Shinshi et al., Plant, Molec Biol., 14: 357-368 (1990)).
By fusing the appropriate targeting sequences described above to the transgenic sequences of interest, it is possible to direct the transgenic product to any organelle or cell compartment. For example, for chloroplast targeting, the signal sequence of the chloroplast from the
RUBISCO, the CAB gene, the EPSP synthase gene or the GS2 gene, is fused in frame to the transgene aminoterminal ATG. The signal sequence selected must include the known cleavage site, and the fusion construct must take into account all amino acids after the cleavage site necessary for cleavage. In some cases, this requirement can be satisfied by adding a small number of amino acids between the cleavage site and the transgene ATG, or by replacing certain amino acids with the transgene sequence. Mergers synthesized for the import of chloroplasts can be evaluated for the effectiveness of chloroplast uptake by in vitro translation of in vitro transcribed synthesized products, followed by in vitro absorption of chloroplast using techniques described by
Bartlett et al. in: Edelmann et al. (Eds.), Methods in
Chloroplast Molecular Biology, Elsevier, pp. 1081-1091 (1982) and Wasmann et al. Mol. Gen. Broom. 205: 446-453 (1986). These synthetic techniques are well known and apply to both mitochondria and peroxisomes.

 The above-described mechanisms of cell targeting can be used not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters, so as to achieve a specific goal of cellular targeting in the regulation of transcription of a promoter. which has an expression pattern different from that of the promoter from which the targeting signal is derived.

C. Transformation
Once the coding sequence of the NIM1 has been cloned into an expression system, it is transformed into a plant cell. Plant tissues suitable for transformation include leaf tissue, root tissue, meristems and protoplasts. The system of the present invention can be used in any plant that can be transformed and regenerated. These transformation and regeneration processes are well known in the art. Methods for synthesizing plant expression cassettes and introducing foreign DNA into plants are generally described in the art. In general, to introduce foreign DNA into plants, plasmid vectors of Ti are used to liberate foreign DNA. Direct DNA uptake, liposomes, electroporation, microinjection and microprojectiles were also used for this release. These methods are already published, and reference may be made for example to: Bilang et al. (1991) Gene 100: 247-250; Scheid et al. (1991) Mol. Gen. Broom.

228: 104-112; Guerche et al., (1987) Plant Science 52: 111-116; Neuhause et al., (1987) Theor. Appl. Broom.

75: 30-36; Klein et al., (1987) Nature 327-70-73; Howell et al., (1980) Science 208: 1265; Horsch et al. (1985)
Science 227: 1229-1231; DeBlock et al. (1989) Plant Physiology 91: 694-701; Methods for Plant Molecular
Biology (Weisbach and Weissbach, eds.) Academic Press,
Inc. (1988); and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc.

(1989). Reference can also be made to US Patent No. 5,625,136, which is incorporated herein by reference in its entirety. It is understood that the transformation process will depend on the plant cell to be transformed.

Transformation of tobacco, tomato, potato and Arabidopsis thaliana using a binary vector system Ti. Plant Physiol. 81: 301-305, 1986; Fry J., Barnason A. and Horsch RB, Transformation of Brassica napus with vectors based on Agrobacterium tumefaciens. P1. Cell Rep. 6: 321-325, 1987; Block
Md Transformation of leaf discs of the potato, independent of the genotype (Solanum tuberosum) using Agrobacterium tumefaciens. Theor. appl. broom. 76: 767-774, 1988; Deblock M., Brouwer DD and
Tenning P. Transformation of Brassica napus and Brassica oleracea by use of Agrobacterium tumefa cens, and expression of bar and neo genes in transgenic plants. Plant Physiol. 91: 694-701, 1989
Baribault TJ, Skene KGM, Cain PA and Scott NS

Transgenic grapes: regeneration of shoots expressing -glucuronidase. Pl. Cell Rep. 41: 1045-1049, 1990; Hinchee, MAW, Newell CA, ConnorWard DV,
Armstrong TA, Deaton WR, Sato SS and Rozman RJ

Transformation and regeneration of non-solanaceous crop plants. Stadler. Broom. Symp. 203201.203212, 1990
Barfield DG and Pua EC, Gene Transfer in Brassica juncea Plants Using Agrobacterium tumefaciens-mediated Transformation. Pi. Cell
Rep. 10: 308-314, 1991; Cousins YL, Lyon BR and
Llewellyn DJ, processing of a variety of Australian coto; Perspective of improvement of cotton by genetic engineering. Aust. J. Plant. Physiol. 18: 481-494. 1991
Chee PP and Slightom JL Cucumber tissue transformation by identification by microprojectile bombardment of plants containing functional and non-functional transferred genes. GENE 118: 255-260, 1992; Christou P., Ford TL and Kofron M. The development of a gene transfer method for rice, independent of the variety. Trends. Biotechnol. 10: 239246, 1992; Halluin K., Bossut M., Good E., Mazur B.,
Leemans J. and Botterman J., Transformation of sugar beet (Beta vulgaris L.) and evaluation of herbicide resistance of transgenic plants.

Bio / Technol. 10: 309-314. 1992; Dhir SK, Dhir S., Savka
MA, Belanger F., Kriz AL, Farrand SK and Whidholm
JM Regeneration of transgenic soybean plants (Glycine
Max) from electroporated protoplasts. Plant.

Physiol. 99: 81-88, 1992; Ha SB, Wu FS and Thorne
TK, transgenic plants of grass-type tall fescue (Festuca arundinacea Schreb.) Regenerated from protoplasts. Pi. Cell Rep. 11: 601-604 (1992); Blechl
AE, Genetic Transformation: The New Wheat Improvement Tool, 78th Annual Meeting, Keynote
Address, Cereal Food World 38: 846-847, 1993; Casas AM,
Kononowicz AK, Zehr UB, Tomes DT, Axtell JD,
LG Butler, Bressan RA and Hasegawa PM, transgenic sorghum plants by microprojectile bombardment. Proc. Nat. Acad. Sci. USA 90: 11212-11216, Christou
P., Philosophy and practice of gene transfer independent of the variety in recalcitrant cultures.

In Vitro Cell Dev Biol-Plant 29P: 119-124, 1993; Damiani
F., Nenz E., Paolocci F. and Arcioni S. Introduction of hygromycin resistance in Lotus spp. by transformation with Agrobacterium rhizogenes. Transgenic Res 2: 330-335, 1993; Davies DR, Hamilton J. and Mullineaux
P., Pea transformation, Pi. Cell Rep. 12: 180-183, 1993; Dong JZ and Mchughen A., Agrobacterium-mediated transgenic linseed flax, frequency of chimeric regenerants, and inheritance of transgenic plants. Plant Sci 91: 139-148, 1993; Fitch
MMM, Manshardt RM, Gonsalves D., and Slightom JL,
Transgenic papaya plants, obtained by Agrobacterium-mediated transformation of somatic embryos. Ft.

Cell Rep. 12: 245-249, 1993; Franklin CI and Trieu TN,
Transformation of the Caucasian Bluestem used as forage grass by DNA transfer mediated by biolistic bombardment. Plant Physiol 102: 167, 1993; Golovkin,
MV, Abraham, M., Morocz, S. Bottka S., Feher A., and
Dudits D., Production of transgenic maize plants by direct DNA absorption by embryogenic protoplasts. Sci Plant 90: 41-52, 1993; Guo, GQ, Xu ZH, Wei
ZM and Chen HM, transgenic plants obtained from wheat protoplasts transformed by direct gene transfer mediated by peg. Chin Sci Bull 38: 20722078, 1993; Asano Y. and Ugaki M. Agrostis alba transgenic plants obtained by direct gene transfer, electroporation-mediated, in protoplasts. Ft.

Cell Rep. 13, 1994; Ayres NM and Park WD, Genetic transformation of rice, Crit. Rev. Plant. Sci 13: 213-239, 1994; Barcelo P. Hagel C., Becker D., Martin A. and Lorz
H. Transgenic cereal plants (Tritordeum) obtained with high efficiency by microprojectile bombardment of an inflorescence tissue. PLANT J, 5: 583592, 1994; Becker D., Brettschneider R. and Lorz H; Fertile transgenic wheat obtained by microprojectile bombardment of scutellar tissue. Plant J 5: 299307, 1994; GCG Biswas, Iglesias VA Datta SK and
Potrykys I., Transgenic Rice Plants of Aquatious Rice Field Mediated by Agrobacterium tumefaciens. Pi. Cell Rep.

13: 582-586, 1994; Hartman CL, Lee L., PR Day and
Tumer NE, herbicide-resistant grass (Agrostis Palustris Huds) by biolistic transformation.

Bid-Technology (12: 919-923), 1994; Howe GT, Goldfarb
B. and Strauss SH, Agrobacterium-mediated transformation of hybrid poplar suspension cultures and regeneration of transformed plants. Cell Tissue Plant &
Orgarn Culture 36: 59-71, 1994; Konwar BK, Genetic transformation, mediated by Agrobacterium tumefaciens, sugar beet (Beta vulgaris L) J Plantbiochem
Biotechnol 3: 37-41, 1994; Ritala, A. Aspegren K., Kurten
U., Salmenkalliomerttila M., Mannomen L., Hannus R.,
Kauppinen V, Teeri TH and Enari TM Fertile transgenic barley obtained by particle bombardment of immature embryos. Plant Mol Biol 24: 317-325, 1994; Scorza R.,
JM Cordts, Ramming DW and Emershad RL, Transformation of Somatic Grape Embryos (Vitis vinifera L), and Regeneration of Transgenic Plants. J. Cell
Biochem: 102, 1994; Shimamoto K., Gene Expression in Transgenic Monocotyledons. Curr. Spin.

Biotechnol. 5: 158-162, 1994; Spangenberg G., Wang ZY,
Nagel J. and Potrykus I., Gene transfer and regeneration of transgenic plants in forage grasses. J. Cell Biochem: 102, 1994; Wan YC and Lemaux
PG, Production of a large number of fertile barley plants undergoing independent processing. seedling
Physiol 104: 3748, 1994; Weeks JT, Anderson OD and
Blechl AE, Stable transformation of wheat (Triticum aestivum L) by microprojectile bombardment. J.

Cell. Biochem: 104, 1994; Ye, XJ, Brown SK, Scorza
R., Cordts J. and Sanford JC, Genetic transformation of pea tissue by particle bombardment. J. Amer.

Soc. Hort. Sci. 119: 367-373, 1994; Spangenberg G., Wang
ZY, Nagel J. and Potrykus I., Protoplast culture and production of transgenic plants in red fescue (Festuca rubra L). Plant Science 1994, 97: 83-94, 1995.

See also U.S. Patent No. 5,639,949, incorporated herein by reference in its entirety.

 Bacteria of the genus Agrobacterium can be used to transform plant cells. Suitable species of this bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogens. Agrobacterium tumefaciens (ie, BAC404 or EHA1OS strains) is particularly useful because of its well-known ability to transform plants.

1. Dicotyledonous transformation
The dicotyledonous transformation techniques are well known and include Agrobacterium techniques, and techniques that do not require Agrobacterium. Techniques not using
Agrobacterium involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be done by PEG-mediated or electroporated uptake, particle-mediated release, or microinjection. Examples of these techniques are described in Paskowski et al., EMBO J 3: 2717-2722 (1984), Potrykus et al., Mol. Gen. Broom. 199: 169-177 (1985), Reich et al., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327: 70-73 (1987). In each case, the transformed cells are regenerated into total plants using standard methods well known in the art.

 Agrobacterium-mediated transformation is a preferred technique for dicotyledonous transformation because of its high processing efficiency and its great utility with many different species, which may be alfalfa and poplar. Further mention may be made of EP 0 317 511 (cotton), EP 0 249 432 (tomato, in the name of Calgene), WO 87/07299 (Brassica, in the name of Calgene), and US Pat. No. 4,795,855 (poplar). ). Transformation with Agrobacterium usually involves transfer of the binary vector carrying the foreign DNA of interest (e.g., pCIB200 or pCIB2001) to an appropriate Agrobacterium strain, which may be dependent on the complement of the vir genes carried by the Agrobacterium strain. in a co-resident Ti plasmid, or chromosomally (for example, strain CIB542 for pCIB200 and pCIB2001 (Uknes et al., Plant Cell 5: 159-169 (1993)) .The transfer of the recombinant binary vector to Agrobacterium is carried out by a triparental pairing procedure using an E. coli carrying the recombinant binary vector, an E. coli helper (helper) strain which carries a plasmid, for example pRK2013, and which can mobilize the vector. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Höfgen & Willmitzer, Nucl Acids.

Res. 16: 9877 (1988)).

The transformation of the target plant species by
Recombinant Agrobacterium usually involves simultaneous cultivation of Agrobacterium with explants from the plant, and follows procedures well known in the art. The transformed tissue is regenerated on a selectable medium carrying the antibiotic or herbicide resistance marker, which is present between the edges of the T DNA of the binary plasmid.

 Another approach to the transformation of plant cells using a gene involves the propulsion of inert or biologically active particles on the tissues or plant cells. This technique is described in U.S. Patent Nos. 4,945,050, 5,036,006 and 5,100,792, all in the name of Sanford et al. In general, this technique involves the propulsion of inert or biologically active particles towards the cells, under conditions permitting the penetration of the outer surface of the cell, and leading to the incorporation of the particles into the internal volume of the cells. cells.

When inert particles are used, the vector can be introduced into the cell by coating the particles with the vector containing the desired gene. Alternatively, the target cell may be surrounded by the vector, so that the vector is transported into the cell following the particle. Biologically active particles (e.g., dried yeast cells, dried bacteria or bacteriophage, each of which contains the DNA to be introduced) can also be propelled into the plant cell tissue.

2. Monocotyledon processing
Transformation of most monocotyledonous species is maintained as a routine technique. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and bombardment of particles in callus tissue. The transformations can be performed with a single DNA species or with several species of DNA (i.e., a cotransformation), and both of these techniques can be used in the present invention. The cotransformation may have the advantage of avoiding a complete synthesis of the vector and of producing transgenic plants having unbound sites for the desired gene and the selectable marker, which makes it possible to eliminate the selectable marker during subsequent generations. if it proves to be desirable. However, a disadvantage of using cotransformation is the frequency, less than 100%, with which separate DNA species integrate into the genome (Schocher et al., Biotechnology 4: 1093-1096 ( 1986)).

 Patent applications EP 0 292 435 ([1280/1281] in the name of Ciba-Geigy), EP 0 392 225 (in the name of Ciba-Geigy) and WO 93/07278 (in the name of Ciba-Geigy) describe techniques to prepare callus and protoplasts from an elongated corn line, the transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts. Gordon-Kamm et al.

(Plant Cell 2: 603-618 (1990)) and Fromm et al. (Biotechnology 8: 833-839 (1990)) have published techniques for transforming the A188-derived maize line by use of particle bombardment.

In addition, the application WO 93/07278 (in the name of Ciba-Geigy) and Koziel et al. (Biotechnology 11: 194-200 (1993)) describe techniques for transforming elite inbred maize lines by particle bombardment. This technique uses immature maize embryos 1.5 to 2.5 mm long, excised from a corn cob 14 to 15 days after pollination, and a PDS-1000He biolistic device for bombardment.

 Rice processing can also be accomplished by direct gene transfer techniques, using protoplasts or particle bombardment. Transformation using protoplasts is described for Japonica types and Indica types (Zhang et al., P1.

Cell Rep. 7: 379-384 (1988); Shimamoto et al., Nature 338: 274-277 (1989); Datta et al., Biotechnology 8, 736-740 (1990)). Both types can also be routinely transformed using particulate bombardment (Christou et al., Biotechnology 9: 957-962 (1991)).
Patent Application EP 0 332 581 (in the name of Ciba
Geigy) describes techniques for the production, transformation and regeneration of
Pooideae. These techniques allow the transformation of
Dactylis and wheat. In addition, the transformation of wheat is described by Vasil et al. (Biotechnology 10: 667-674 (1992)) using particulate bombardment in cells of a long-term regenerable callus of type C, and also by Vasil et al. (Biotechnology 11 1553-1558 (1993)) and Weeks et al. (Plant Physiol 102: 1077-1084 (1993)) by particle bombardment of immature embryos and a callus derived from an immature embryo. A preferred technique for transforming wheat, however, involves the transformation of wheat by particulate bombardment of immature embryos, and includes a high sucrose stage or a high maltose stage prior to release of the gene. Prior to bombardment, any number of embryos (0.75 to 1 mm long) are plated on MS medium containing 3% sucrose (Murashiga & Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg / l 2,4-D for induction of somatic embryos, and the operation is allowed to proceed in the dark. On the day chosen for the bombardment, the embryos are removed from the induction medium and placed on the osmotic medium (i.e., an induction medium to which sucrose and maltose have been added at the desired concentration, usually 15%). The embryos are allowed to undergo plasmolysis for 2 to 3 hours and then bombarded. Usually, 20 embryos per target plate are used, this figure being non-critical. A suitable plasmid carrying the gene (such as pCIB3064 or pCG35) is precipitated on gold particles of micrometric size using conventional techniques. Each embryo plate is shot by a DuPont Biolistics X helium device, the burst pressure being about 7 MPa (about 1000 psi), in which case a screen having a mesh size of 177 μm (80 μm) is used. mesh). After the bombardment, the embryos are placed again in the dark, for recovery for about 24 hours (always on the osmotic medium). After 24 hours, the embryos are removed from the osmotic medium and placed again on the induction medium, where they are left for one month before regeneration. About one month later, embryo explants with developing embryogenic callus are transferred to the regeneration medium (MS + 1 mg / l NAA, 5 mg / l GA), which further contains the appropriate selection (10 mg / l of basta in the case of pCIB3064 and 2 mg / l of methotrexate in the case of pSOG35). After about 1 month, the developed shoots are transferred to larger sterile containers, known as "GA7", which contain MS having a strength less than half the maximum strength, 2% sucrose, and the same concentration of the selection agent. Patent application 08 / 147,161 discloses methods for transforming wheat and is therefore incorporated herein by reference.

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

Selection
The isolated gene fragment of the present invention, or the altered forms of the NIM1 gene, can be used to confer disease resistance to a broad range of plant cells, including those of gymnosperms, monocotyledons and dicotyledons.

Although the gene can be inserted into any plant cell falling into the broad categories above, it can be used in particular in the cells of the crop plants, in particular the following plants: rice, wheat, barley, rye, corn , potato, carrot, sweet potato, sugar beet, bean, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, aubergine, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, ripe, pineapple, avocado, papaya, mango, banana, soy, tobacco, tomato, sorghum and sugar cane.

 The high level of expression of the NIM1 gene and its mutants, necessary for constitutive expression of SAR genes, in combination with other important production and quality characteristics, can be incorporated into plant lines by selection. . Thus, another embodiment of the present invention relates to a method for producing transgenic progeny of a transgenic parent plant comprising an isolated DNA molecule encoding a modified form of a NIM1 protein according to the invention, which comprises transforming said parent plant with a recombinant vector molecule according to the invention, and to transfer the characteristic to the descendants of said transgenic parent plant, using known techniques of plant breeding.

The techniques and methods of selection are known, and reference may be made, for example, to Welsh JR, Fundamentals of Plant Genetics and Breeding, John Wiley & Sons,
NY (1981); Crop Breeding, Wood DR (Ed.) American
Society of Agronomy Madison, Wisconsin (1983); Mayo O.,
The Theory of Planting Breeding, Second Edition, Clarendon
Press, Oxford (1987); Singh DP, Breeding for Resistance to Disease and Insect Pests, Springer-Verlag, NY (1986); and Wricke and Weber, Quantitative Genetics and
Plant Breeding Selection, Walter de Gruyter and Co.,
Berlin (1986).

Propagation of genetic properties, introduced into transgenic seeds and transgenic plants, and maintenance in descending plants
Genetically engineered genetic properties in transgenic seeds and plants, described above, are transmitted by sexual reproduction or vegetative growth, and can therefore be maintained and propagated in offspring. In general, this maintenance and propagation use known agricultural methods, developed to adapt to particular needs such as tillage, seeding or harvesting. Specialized processes such as hydroponics or greenhouse technologies can also be applied. As the growing plant is vulnerable to attack and injury from insects or infections, as well as competition from weeds, measures are taken to control weeds, plant diseases, insects, nematodes and other undesirable conditions, with the aim of improving yield. They include mechanical measures such as tillage or removal of weeds and infected plants, as well as the application of phytosanitary products such as herbicides, fungicides, gameticides, nematicides, growth regulators, curing agents and insecticides.

 The use of the advantageous genetic properties of the transgenic plants and seeds according to the invention may furthermore be used in plant breeding, the objective of which is the development of plants with improved properties, such as the tolerance towards pests, herbicides or constraints, better nutritional value, increased yield, or improved structure, leading to lower losses due to lodging or failure. The different stages of selection are characterized by a perfectly defined human intervention, such as the selection of lines to cross, the orientation of the pollination of parental lines, or the selection of appropriate descendants. Depending on the desired properties, different selection techniques will be used. Suitable techniques are well known and include but are not limited to hybridization, inbreeding, re-crossing, multi-line selection, variety mixing, interspecific hybridization, aneuploid techniques, etc. Hybridization techniques also include the sterilization of plants to give sterile male or female plants by mechanical, chemical or biochemical means. Cross-pollination of a male sterile plant with pollen from a different strain ensures that the genome of the plant, sterile male but sterile female, will uniformly present the properties of both parental lines. Thus, the transgenic seeds and plants according to the present invention can be used for the selection of improved plant lines, which for example will increase the yield of conventional methods such as treatment with a herbicide or a pesticide, or will allow omit these techniques because of their modified genetic properties. Or again, new crops, with improved tolerance to stress, can be produced which, because of their optimized genetic "equipment", will yield a better quality harvested product than products that have not been able to grow. support comparable adverse developmental conditions.

 In seed production, germination quality and seed uniformity are essential characteristics of the products, while seed quality and uniformity of seed harvested and marketed by the farmer is of no importance. . Since it is difficult to maintain a seed-free plant of other plants and weeds, and to control seed-borne diseases and produce seeds with good germination, very extensive and well-defined seed production practices have been established. Developed by seed growers who have gained the necessary experience in growing, packaging and marketing pure seeds. Thus, it is common practice for farmers to buy certified seed that meets specific quality standards, rather than using seed harvested from their own crops. The propagation material to be used as seed is usually treated with a protective coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures thereof. The protective coatings usually used include compounds such as captan, carboxin, thiram (TMTD @), methalaxyl (Apron @), and pirimiphos-methyl (Actellic @). If desired, these compounds are formulated with other carriers, surfactants or application enhancing adjuvants commonly used in the formulation art to provide protection against damage from bacterial, fungal or animal pests. The protective coatings may be applied by impregnating, on the propagating material, a liquid formulation, or by coating with a combined wet or dry formulation. Other application techniques can also be used, for example the direct treatment of buds or fruit.

 Another aspect of the present invention is to provide novel agricultural methods, such as the above-exemplified methods, which are characterized by the use of transgenic plants, transgenic plant material or transgenic seeds in accordance with the present invention. present invention.

The seeds may be provided in a bag, container or container of suitable packaging material, the bag or container being closable to enclose the seeds therein. The bag, container or container may be designed for short-term or long-term storage, or both, of seeds. Examples of suitable packaging materials include paper, such as kraft paper, rigid or collapsible plastics, or other polymeric materials, glass or metals. It is desirable for the bag, container or container to consist of several layers of packaging material, of the same type or of different types. In one embodiment, the bag, container or container is provided to exclude or limit water or moisture from contacting the seeds. In one example, the bag, container or container is hermetically sealed, for example heat sealed, to prevent the ingress of water or moisture. In another embodiment, water absorbing materials are disposed between or adjacent to layers of the packaging material. In still another embodiment, the bag, container or container, or the packaging material of which it is made, is treated to limit, suppress or prevent diseases, contaminations or other undesirable effects of storage. or seed transportation. An example of such treatment is sterilization, for example by chemical means or by exposure to radiation. The present invention encompasses a commercial bag comprising seeds of a transgenic plant comprising at least one modified form of a protein
NIM1, or else a NIM1 protein which is expressed in said transformed plant at higher levels than in a wild-type plant, together with a suitable carrier, with instructions on the label regarding their use, so to give plants resistance to a broad spectrum of diseases.

Disease resistance
The evaluation of disease resistance is carried out by methods known in the art. For example, refer to Uknes et al. (1993),
Molecular Plant Microbe Interaction 6: 680-685; Gorlach et al. (1996) Plant Cell 8: 629-643; Alexander et al.
Proc. Natl. Acad. Sci. USA 90: 7327-7331.

A. Phytophthora parasitaca resistance test (blackleg)
Phytophthora parasitica, the microorganism that causes the blackleg, is tested for resistance to 6-week-old plants grown as described in Alexander et al., Proc. Natl. Acad.

Sci. USA 90: 7327-7331. The plants are watered, until draining, and then seeded by applying 10 ml of a suspension of sporangia (300 sporangia per ml) on the soil. The inoculated plants are kept in a greenhouse maintained at a temperature of 23-25 ° C during the day and 20-220 ° C during the night.The tests use the following notes as a wilting scale: 0 = no symptoms; no symptoms, 1 = some signs of wilting, with reduced turgor, 2 = marked symptoms of wilting, but no putrefaction or stunting, 3 = definite symptoms of wilting with stunting, but no sign of stem rot, 4 = strong wilting , with visible rot of the stem and some damage to the radiocular system, 5 = like 4, but the plants are close to death or have died, with a sharp reduction of the root system. on plants arranged in a random pattern.

B. Pseudomonas syringae resistance test
Pseudomonas syringae pv tabaci strain No. 551 was injected into the two lower leaves of several 6- to 7-week-old plants at a concentration of 106 or 3.10 6 / ml in H 2 O. At each instant, six individual plants were evaluated. plants infected with Pseudomonas tabaci are scored on a 5-point disease severity scale, namely: 5 = 100% tissue mortality, 0 = no symptoms, t-test (LSD) is performed on the assessments performed each day, and groupings are shown after the mean score Values that follow the same letter on the day of the evaluation do not show a statistically significant difference.

C. Cercospora nicotianae resistance test
A suspension of spores of Cercospora nicotianae (ATCC No. 18366) (100,000 to 150,000 spores per ml) is sprayed to imminent runoff on leaf surfaces. The plants are kept in a 100% humidity atmosphere for 5 days. Then the plants receive a mist of water 5 to 10 times a day. At each moment, 6 individual plants are evaluated. Cercospora nicotianae is noted as a percentage of leaf area showing the basic symptoms of the disease. A t test (LSD) is performed on daily assessments, and groupings are shown after the average score. Values that follow the same letter on the day of the evaluation do not show a statistically significant difference.

D. Peronospora parasitica resistance test
Tests are being conducted on resistance to
Peronospora parasitica on plants, as described in
Uknes et al. (1993). Plants are inoculated with a compatible P. parasitica isolate by spraying a suspension of conidia (approximately 5.104 spores per ml). The inoculated plants are incubated under humid conditions at 17 ° C. in a growth chamber with a cycle of 14 hours of day and 10 hours of night.The presence of conidiophores is examined by examining the plants 3 to 14 and preferably 7 to 12 days after inoculation In addition, several plants of each treatment are randomly selected and stained with lactophenol-trypan blue (Keogh et al., Trans Br Br Mycol Soc.

74: 329-333 (1980)) to allow microscopic examination.

The invention will be better understood with reference to the examples below and the attached drawings, in which
Figure 1 shows the effect of chemical inducers on the induction of SAR gene expression in wild type and niml plants. The chemical induction of SAR genes is decreased in niml plants. Water, SA, INA or BTH are applied to wild type (WT) and niml plants. After 3 days, an RNA is prepared from these plants, and the expression of PR-1, PR-2 and PR-5 is determined.

Figure 2 shows the expression of the PR-1 gene in WS-O and nim1 plants infected with a pathogenic microorganism. Induction of the pathogenic microorganism of PR-1 decreases in niml plants. Wild type (WT) and niml plants are sputtered by the P. parasitica Emwa breed. Samples are collected in nimi mutants and wild-type plants after infection with a pathogenic microorganism or chemical treatment. Plants containing alleles niml ie niml-1, -2, -3, -4, -5 and -6, and plants Ws-O (Ws) are treated with water (C), AS, of INA or BTH 3 days before isolation of RNA. The sample
Emwa consists of an isolated plant RNA 14 days after inoculation with P. parasitica isolate Emwa.

The blots are hybridized by use as a probe of Arabidopsis PR-1 cDNA (Uknes et al., 1992).

Figure 4 shows the accumulation levels of the
SA in Ws-O and niml plants infected with P. syringae. Niml plants accumulate SNA after exposure to the pathogenic microorganism. Wild and niml-like plant leaves are infiltrated with Pst DC3000 (avrRpt2) or carrier medium (10mM MgCl2) alone. After 2 days, samples were taken from untreated plants, MgCl2 treated plants and DC3000 treated plants (avrRpt2). The samples treated with bacteria are separated into primary (infiltrated) leaves and secondary (non-infiltrated) leaves. The free AS and the total SA after hydrolysis by β-glucosidase were quantified by HPLC. Error bars indicate the standard deviation of three replicate samples.

 Figs. 5A-D show an overall map of the levels of NIMl-centered chromosomal region resolution increase, with the indicated recombinants, which include BACs, YACs, and cosmids, in the NIM1 region.

(A) Position on the NM1 map on the chromosome
1. The total number of gametes scored is 2276.

(B) Artificial chromosome of yeast (zebra), chromo
artificial bacterial (BAC), and clones P1
used to clone the NIMl.

(C) Cosmid clones that cover the NIMl site
The three cosmids that complete the niml-l are
bold lines.

(D) The four putative gene regions of frag
the smallest of the complementary genomic DNA
silent. The four open reading frames that
include the NiMi gene are indicated by
open bars. The arrows indicate the direc
transcription. The numbering is
referred to the first base of genomic DNA
of Arabidopsis present in the cosmid D7.

 Figure 6 shows the nucleic acid sequence of the NIM1 gene and the amino acid sequence of the NIM1 gene product, including changes in different alleles. This amino acid sequence, which is on the opposite strand in the form of the 9.9 kb sequence presented in the sequence SEQ ID NO: 1, and also presented in the sequence SEQ ID NO: 2, and the sequence of amino acids of the NiMi gene product is also presented in the sequence SEQ ID NO: 3.

 Figure 7 shows the accumulation of NIM1 caused by INA, BTH, SA and treatment with pathogenic microorganisms of wild type plants and niml mutant alleles. The RNA gel transfers of Figure 3 are probed to allow expression of RNA using a probe derived from positions 2081 to 3266 of the sequence shown in Figure 6.

 Figure 8 is a comparison of the amino acid sequences of the expressed sequence marker regions of the NIM1 protein products and cDNA protein of four rice gene sequences (SEQ ID NO: 4-11); the numbers correspond to the amino acid positions of the sequence SEQ ID NO: 3.

 Figure 9 is an alignment of sequences corresponding to the sequence of the NIM1 protein with mouse, rat and porcine IKBa. The vertical bars (I) above the sequences indicate the amino acid identity between NIM1 and IKBa sequences (the matrix score is 1.5); double dots (:) above the sequences indicate a similarity score greater than 0.5; the single points (.) above the sequences indicate a similarity score less than 0.5 but greater than 0.0; and a score of less than 0.0 indicates the lack of similarity, with no index above the sequences (see examples). The location of the ankyrin domains of the mammalian baba is identified according to Martin et al., Gene 152, 253-255 (1995).

The points within a sequence indicate gaps between the NIM1 and the cba proteins. The 5 ankyrin repeats of IKBa are indicated by the dashed lines below the sequences. The amino acids are numbered relative to the protein NIM1, with gaps introduced whenever necessary. The plus signs (+) are placed above the sequences every 10 friendly acid.

deposits
The following vector molecules have been deposited with the American Type Culture Collection, 12301
Parklawn Drive Rockville, MD 20852, USA, at the indicated dates: Plasmid BAC-04 was deposited with the ATCC on May 8, 1996 under ATCC No. 97543.

 Plasmid P1-18 was deposited with the ATCC on June 13, 1996 under ATCC No. 97606.

 Cosmid D7 was deposited with ATCC on September 25, 1996 under ATCC No. 97736.

Brief description of the sequences presented in the list below
SEQ ID NO: 1: 9919 bp genomic sequence of region 2 of the NIM1 gene of Figure 5D.

SEQ ID NO: 2: 5655 bp genomic sequence of Figure 6 (strand opposite to that of SEQ ID NO: 1) comprising the coding region of the wild-type Arabidopsis thaliana NIM1 gene.

SEQ ID NO: 3: AA sequence of the wild-type NIM1 protein encoded by the cd of SEQ ID NO: 2.

SEQ ID NO: 4: Amino acid sequence 33-155 of rice 1 of Figure 8.

SEQ ID NO: 5: Amino acid sequence 215-328 of rice 1 of Figure 8.

SEQ ID NO: 6: Amino acid sequence 33-155 of rice 2 of Figure 8.

SEQ ID NO: 7: Amino acid sequence 208-288 of rice 2 of Figure 8.

SEQ ID NO: 8: Amino acid sequence 33-155 of rice 3 of Figure 8.

SEQ ID NO: 9: Amino acid sequence 208-288 of rice 3 of Figure 8.

SEQ ID NO: 10: Amino acid sequence 33-155 of rice 4 of Figure 8.

SEQ ID NO: 11: Amino acid sequence 215-271 of rice 4 of Figure 8.

SEQ ID NO: 12: Oligonucleotide
SEQ ID NO: 13: Oligonucleotide
SEQ ID NO: 14: Oligonucleotide
SEQ ID NO: 15: Oligonucleotide
SEQ ID NO: 16: Oligonucleotide
SEQ ID NO: 17: Oligonucleotide
SEQ ID NO: 18: is the amino acid sequence of IIIKBa of the mouse of Figure 8.

SEQ ID NO: 19: is the amino acid sequence of the rat IKBα of Figure 8.

SEQ ID NO: 20: is the amino acid sequence of IkB α pork from Figure 8.

SEQ ID NO: 21: is the cDNA sequence of the NIM1 gene of Arabidopsis thaliana.

The sequences SEQ ID NOs: 22 and 23 are respectively the coding sequence encoding the DNA and the coded amino acid sequence, of a dominant-negative form of the NIM1 protein having alanine residues instead of serine residues. on amino acid positions 55 and 59.

SEQ ID NO: 24 and 25 respectively represent the DNA coding sequence and the encoded amino acid sequence of a dominant-negative form of the NIM1 protein having an N-terminal deletion.

The sequences SEQ ID NO: 26 and 27 respectively represent the coding sequence encoding the DNA and the coded amino acid sequence, of a dominant-negative form of the NIM1 protein having a C-terminal deletion.

The sequences SEQ ID NO: 28 and 29 respectively represent the coding sequence encoding the DNA and the coded amino acid sequence, of a modified form of the NIM1 gene comprising deletions of amino acids, both N-terminal and
C-terminal.

The sequences SEQ ID NO: 30 and 31 respectively represent the sequence coding for DNA and the coded amino acid sequence of the ankyrin domain of NIM1.

The sequences SEQ ID NO: 32 to 39 are oligonucleotide primers.

Definitions
Acd: Mutant plant with accelerated death of cells
AFLP: Amplifier fragment size polymorphism
AprRpt2: Rpt2 avirulence gene, isolated from Pseudomonas
syringae
BAC: Artificial bacterial chromosome
BTH: S-methyl ester of benzo acid (1,2,3)
thiadiazole-7-carbothioic
ICD: Phenotype of Constitutive Immunity (SAR
undergoes constitutive activation) cim: Mutant plant with constitutive immunity cM: Centimorgans cprl: Constitutive expression of a mutant plant at
PR genes
Col-O: Arabidopsis, Columbia ecotype
EC: Enzymatic combination
Emwa: Isolate of Peronospora parasitica, compatible
in the Ws-O ecotype of Arabidopsis
EMS: Ethyl methanesulfonate
INA: 2,6-dichloroisonicotinic acid
Ler: Arabidopsis ecotype Landsberg erecta lds: Mutant plant with disease simulating lesions nahG: Salicylate hydroxylase of Pseudomonas putida
which converts salicylic acid into catechol.

NahG: Arabidopsis line transformed with the gene
nahG ndr: Mutant plant resistant to non-spe
breeds nim: Mutant plant with non inducible immunity
NIM1: The wild-type gene involved in the
SAR signal transduction cascade
NIM1: Protein encoded by the gene NIM1 type sau
vage niml: Mutant allele of NIM1, which confers on the plant
susceptibility to diseases also means
Arabidopsis thaliana mutant plants
with the niml mutant allele of NIM1
Noco: Isolate of Peronospora parasitica compatible
in the Col-O ecotype of Arabidopsis
ORF: Open reading frame
PC: Primary Combination
PR: Related to a pathogenesis
SA: Salicylic acid
SAR: Acquired Systemic Resistance
SSLP: Polymorphism of size of simple sequences
UDS: Phenotype of universal sensitivity to mala
dies
Wela: Isolate of Peronospora parasitica compatible
in the Weiningen ecotype of Arabidopsis
Ws-O: Arabidopsis ecotype Issilewskija
WT: Wild type
YAC: Artificial Yeast Chromosome
The invention is further illustrated by the detailed procedures, preparations and examples hereinafter.

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

Silhavy, ML Berman and LW Enquist, Experiments with
Gene Fusions, Cold Spring Harbor Laboratory, Cold Sprind
Harbor, NY (1984) and by Ausubel FM et al., Current
Protocols in Molecular Biology, published by Greene Publishing Assoc. And Wiley-Interscience (1987).

A. Characterization of mutants niml
Example 1 Vegetable Lines and Fungal Strains
Seeds of Arabidopsis thaliana ecotype Isilewskija (Ws-O, stock number CS 2360) and fourth generation (T4) from T-DNA transformed lines are obtained from Ohio State University Arabidopsis Biological Resource Center. (Columbus,
OH). It is from Lehle Seeds (Round Rock, TX) that second generation seeds (M2) are obtained from Ws-O plants mutagenized by ethyl methanesulfonate (EMS).

The test strain 3000 of Pseudomonas syringae pv. tomato (Pst) containing the cloned avrRpt2 gene [DC3000 (avrRpt2) 1 is obtained from B. Staskawicz, University of California, Berkeley. The pathogenic varieties of P. parasitica and their sources are as follows: Emwa is from E. Holub and IR Crute, Horticultural
Research Station, East Malling, Kent; Wela comes from A.

Slusarenko and B. Mauch-Mani, Institut für Pflanzenbiologie, Zurich, Switzerland; and Noco comes from J. Parker,
Sainsbury Laboratory, Norwich, England. The fungal cultures are maintained by weekly culture on
Arabidopsis ecotype Ws-O, Weiningen, and Col-O, for the pathogenic varieties of P. parasitica respectively Emwa,
Wela and Noco.

Example 2: Selection of Mutants
M2 or T seeds were grown on soil for two weeks with 14 hours of light per day, nebulized with 0.33 mM INA (0.25 mg / ml), obtained from INA at 25 ° C. % in a wettable powder; Ciba,
Basel, Switzerland), and are inoculated 4 days later by spraying a conidia suspension of P. parasitica containing 5-10 .104 conidiospores per ml of water. This pathogen is normally virulent on the Ws ecotype
O of Arabidopsis, unless resistance has been previously induced in these plants using isonicotinic acid (INA) or a similar compound. The plants are kept in humid conditions at 180C for a week, then their fungal sporulation is noted.

Plants that support fungi proliferation after INA treatment are selected as putative mutants.

 After incubation in a high humidity atmosphere, plants with visible symptoms of disease are identified, usually 7 days after infection. These plants do not exhibit any resistance to the fungus, despite the application of the resistance-inducing chemical, and thus represent potential mutant nim (non-inducible immunity) plants.

On 360 000 plants, 75 potential nim mutants are identified.

 These potential mutant plants are isolated from the plot, placed in a low moisture atmosphere, and allowed to germinate. The plants derived from this seed are selected in an identical manner according to their susceptibility to the Emwa fungus, once again after INA treatment. Descending plants that show symptoms of infection are defined as nim mutants. Six lineages nim are thus identified. A lineage (nimI-1) is isolated from the T-DNA population, and five (nimI-2, nimI-3, nimi-4, nimI-5 and nimI-6) from the EMS population.

Example 3: Resistance to plant diseases niml
Salicylic acid (SA) and the S-methyl ester of benzo (1,2,3) thiadiazole-7-carbothioic acid (BTH) are chemicals which, like IAI, induce resistance to a broad spectrum of diseases (SAR) in wild type plants. The mutant plants are treated with SA, INA and BTH, and their resistance to Peronospora parasitica is evaluated. The isolate of P. parasitica "Emwa" is a Pp isolate that is compatible in the Ws ecotype. Compatible isolates are those that are capable of creating disease in a particular host. The isolate of P. parasitica "Noco" is incompatible on Ws but compatible on the Columbia ecotype. Incompatible pathogenic microorganisms are recognized by potential haste, triggering a hasty response that prevents the development of the disease.

Wild-type seeds, and seeds for each of the nimI (nimI-1, -2, -3, -4, -5, -6) alleles are sown on MetroMix 300 growth media, covered with a plastic dome transparent and placed at 40C in the dark for 3 days. After 3 days of treatment at 4 ° C, the plants are sent to a phytotron for 2 weeks and, about two weeks after planting, the sprouted seedlings produced four true leaves.
H2O, 5 mM SA, 300 WM BTH or INA 300 AM. The chemicals are applied as a fine mist to completely cover the seedlings with a "Chromister". The watered control plants are returned to the growth phytotron, while the chemically treated plants are kept in a separate but identical phytotron. Three days after application of the chemical, water-washed and chemically treated plants are inoculated with the compatible "Emwa" isolate. "Noco" inoculation is performed only on the plants treated with water. After inoculation, the plants are covered with a clear plastic dome to maintain the high humidity required for successful infection with P. parasitica, and placed in a culture chamber at 19 ° C during the day and 17 days. "C during the night, with cycles of 8 hours of light and 16 hours of darkness.

 To determine the relative strength of the different niml alleles, each mutant is microscopically analyzed at different times following inoculation to determine the growth of P. parasitica under normal growth conditions and after prior treatment with the parasite. SA, INA or BTH. After enlargement, sporulation of the fungus can be observed at the very early stage of disease development. The percentage of plants per pot with sporulation, 5, 6, 7, 11 and 14 days after inoculation, is determined, and the density of sporulation is also recorded.

 Table 1 shows, for each of the niml mutant plant lines, the percentage of plants that have some conidia at the surface on at least one leaf after infection with the P. parasitica Emwa race. P. parasitica is inoculated on plants 3 days after treatment with water or chemicals.

The table shows the number of days after infection at which endurance is assessed.

Table 1
Percentage of infection - Emwa / control

<tb> Mutant <SEP> Day <SEP> O <SEP> Day <SEP> 5 <SEP> Day <SEP> 6 <SEP> Day <SEP> 7 <SEP> Day <SEP> 11
<tb> Ws <SEP> WT <SEP> 0 <SEP> 10 <SEP> 25 <SEP> 100 <SEP> 90
<tb> nim1-1 <SEP> 0 <SEP> 75 <SEP> 95 <SEP> 100 <SEP> 100
<tb> nim1-2 <SEP> 0 <SEP> 30 <SEP> 85 <SEP> 100 <SEP> 100
<tb> nim1-3 <SEP> 0 <SEP> 30 <SEP> 90 <SEP> 100 <SEP> 100
<tb> nim1-4 <SEP> 4 <SEP> 80 <SEP> 100 <SEP> 100 <SEP> 100
<tb> nim1-5 <SEP> 0 <SEP> 0 <SEP> 5 <SEP> 100 <SEP> 100
<tb> nim1-6 <SEP> 0 <SEP> 5 <SEP> 70 <SEP> 80 <SEP> 100
<Tb>
Percentage of infection - Emwa / SA

<tb> Mutant <SEP> @ <SEP> Day <SEP> 0 <SEP> Day <SEP> 5 <SEP> | <SEP> Day <SEP> 6 <SEP> Day <SEP> 7 <SEP> Day <SEP> 11
<tb> Ws <SEP> WT <SEP> 0 <SEP> S <SEP> 30 <SEP> 70 <SEP> 100
<tb> nim1-1 <SEP> 0 <SEP> 5 <SEP> 95 <SEP> 100 <SEP> 100
<tb> nim1-2 <SEP> 0 <SEP> 5 <SEP> 95 <SEP> 100 <SEP> 100
<tb> nim1-3 <SEP> 0 <SEP> 10 <SEP> 90 <SEP> 100 <SEP> 100
<tb> nim1-4 <SEP> 0 <SEP> 75 <SEP> 100 <SEP> 100 <SEP> 100
<tb> nim1-5 <SEP> 0 <SEP> 0 <SEP> 20 <SEP> 75 <SEP> 100
<tb> nim1-6 <SEP> 0 <SEP> 80 <SEP> 100 <SEP> 100 <SEP> 100
<Tb>
Percentage of infection - Emwa / INA

<tb><SEP> Mutant <SEP> Day <SEP> 0 <SEP> Day <SEP> 5 <SEP> Day <SEP> 6 <SEP> Day <SEP> 7 <SEP> Day <SEP> 11
<tb> WsWT <SEP> O <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0
<tb><SEP> nim1-1 <SEP> 0 <SEP> 5 <SEP> 80 <SEP> 100 <SEP> 100
<tb><SEP> niai-2 <SEP> 0 <SEP> 15 <SEP> 95 <SEP> 100 <SEP> 100
<tb><SEP> nim1-3 <SEP> 0 <SEP> 10 <SEP> 60 <SEP> 100 <SEP> 100
<tb><SEP> nimi-4 <SEP> 0 <SEP> 80 <SEP> 100 <SEP> 100 <SEP> 100
<tb><SEP> nim1-5 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 5 <SEP> 5
<tb><SEP> nimi-6 <SEP> 0 <SEP> 1 <SEP> 50 <SEP> 90 <SEP> 100
<Tb>
Percentage of infection - Emwa / BTH

<tb><SEP> Mutant <SEP> Day <SEP> 0 <SEP> Day <SEP> 5 <SEP> Day <SEP> 6 <SEP> Day <SEP> 7 <SEP> Day <SEP> 11
<tb><SEP> Ws <SEP> WT <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0
<tb><SEP> nim1-1 <SEP> 0 <SEP> 1 <SEP> 5 <SEP> 30 <SEP> 100
<tb><SEP> nim1-2 <SEP> 0 <SEP> O <SEP> 25 <SEP> 90 <SEP> 100
<tb> nim1-3 <SEP> 0 <SEP> X <SEP><SEP> 15 <SEP> 70 <SEP> 10 <SEP> 100 <SEP> 100
<tb><SEP> niml-4 <SEP> 0 <SEP> 80 <SEP> 100 <SEP> 100 <SEP> 100
<tb> nim1-5 <SEP> O <SEP> 0 <SEP> i <SEP><SEP> 1 <SEP> 10
<tb><SEP> ni = i-6 <SEP> 0 <SEP> i <SEP><SEP> 90 <SEP> 100 <SEP> 100
<Tb>
As shown in Table 1, in normal growth, nim1-1, nim1-2, nim1-3, nim1-4, and niml-6 all have approximately the same rate of proliferation of the fungus, which is somewhat more greater than that of Witness Ws-0. The exception is the nim1-5 plants, where the proliferation of the fungus is delayed by several days compared to the other nim1 mutants and the Ws-0 control, but eventually all the niml-5 plants succumb to the fungus.

 After treatment with AS, the mutants can be grouped into three categories: nimi-4 and nim1-6 exhibit relatively rapid fungal growth; the plants nim1-l, nim1-2 and nim1-3 have a somewhat lower fungal growth rate; and proliferation of the fungus in nimi-5 plants is even slower than in untreated Ws-0 controls. After treatment with INA or BTH, the mutants once again enter three classes, where nimi-4 is the most severely compromised in its ability to limit fungal overgrowth after chemical treatment; niml-l, niml-2, nim1-3 and nim1-6 are all moderately compromised; and nim1-5 is only slightly compromised. In these experiments, Ws-0 does not promote fungal growth after treatment with INA or BTH.

Thus, in terms of inhibiting the proliferation of the fungus after chemical treatment, the mutants fall into three classes, nimi-4 being the most severely compromised, nimI-1, nimI-2, nimI-3 and nimI-4. exhibiting an intermediate inhibition of the fungus, and niml5 showing only slight degradation of fungi resistance.

Table 2 presents an assessment of disease resistance, expressed as a scoring of the infection of different niai alleles, as well as
NahG 7 and 11 days after inoculation with Peronospora parasitica. WsWT indicates the Ws wild type parental line, in which the niml alleles are found. The different alleles niml are indicated on the table, as well as the NahG plant.

 A description of the NahG plant has already been published (Delaney et al., Science 266, pp. 1247-1250 (1994)).

Arabidopsis NahG is likewise described in U.S. Patent Application Serial No. 08 / 454,876 and incorporated herein by reference. NahG is a gene from Pseudomonas putida that encodes a salicylate hydroxylase that converts salicylic acid to catechol, eliminating the accumulation of salicylic acid, a necessary signal transducing component for SAR in plants. Thus, Arabidopsis NahG plants do not exhibit normal SAR, and generally have a much higher sensitivity to pathogenic microorganisms. However, NahG plants still respond to INA and BTH chemical inducers. NahG plants therefore represent a type of universal sensitivity control.

Table 2
Severity of infection - Emwa / water

<tb> Mutant <SEP> Day <SEP> 7 <SEP> Day <SEP> it
<tb> Ws <SEP> WT <SEP> 3 <SEP> 3
<tb> nim1-1 <SEP> 4 <SEP> 4,5
<tb> nim1-2 <SEP> 3 <SEP> 4
<tb> nim1-3 <SEP> 4 <SEP> 4
<tb> nim1-4 <SEP> 5 <SEP> 5
<tb> nim1-5 <SEP> 1 <SEP> 3,5
<tb> nim1-6 <SEP> 3 <SEP> 4,5
<tb> NahG
<Tb>
Severity of infection - Emwa / SA

<tb><SEP> Mutant <SEP> Day <SEP> 7 <SEP> Day <SEP> 11 <SEP>
<tb><SEP> Ws <SEP> WT <SEP> 3 <SEP> 4
<tb><SEP> nim1-1 <SEP> 3 <SEP> 4,5
<tb><SEP> nim1-2 <SEP> 3 <SEP> 4
<tb><SEP> nim1-3 <SEP> 3 <SEP> 4
<tb><SEP> nim1-4 <SEP> 4 <SEP> 5
<tb><SEP> nim1-5 <SEP> 3 <SEP> 3
<tb><SEP> nim1-6 <SEP> 4 <SEP> 4,5
<tb> NahG <SEP> 4 <SEP> 5
<Tb>
Severity of infection - Emwa / INA

<tb><SEP> Mutant <SEP> Day <SEP> 7 <SEP> Day <SEP> 11 <SEP>
<tb><SEP> Wn <SEP> WT <SE> O <SEP> O
<tb><SEP> nim1-1 <SEP> 2.5 <SEP> 4
<tb> nim1-2 <SEP> 4 <SEP> 4
<tb> nim1-3 <SEP> 3 <SEP> 3,5
<tb><SEP> nim1-4 <SEP> 4 <SEP> 5
<tb><SEP> nim1-5 <SEP> 1 <SEP> 2
<tb><SEP> 3 <SEP> 4,5
<tb><SEP> NahG <SEP> 3 <SEP> 3
<Tb>
Severity of infection - Emwa / BTH

<tb> Mutant <SEP> Day <SEP> 7 <SEP> Day <SEP> 11
<tb> Ws <SEP> WT <SEP> O <SEP> O <SEP>
<tb> nirni-1 <SEP> 2.5 <SEP> 4
<tb> niai-2 <SEP> 3.5 <SEP> 4
<tb> ni = i-3 <SEP> 3 <SEP> 3.5
<tb> nim1-4 <SEP> 4 <SEP> 5
<tb> nim1-5 <SEP> 1.5 <SEP> 2
<tb> nîmi-6 <SEP> 3 <SEP> 4 <SEP>
<tb> NahG <SEP> o <SEP><SEP> O
<Tb>
It can be seen from Table 2 that the nimI-4 and nimI-6 alleles are either those with the most severe infection with Peronospora parasitica; it can be observed most easily in the early stages. In addition, the niml-5 allele is the one that has the greatest response to both INA and BTH, so it is said to be the weakest niml allele. NahG plants have a very good response to both INA and BTH, and look very similar to the niml allele. However, at later times, for example on day 11 of the table, the resistance to disease induced in NahG plants begins to decrease, and there is a big difference between INA and BTH, in that sense that the resistance induced by the INA disappears much more rapidly and much more strongly than the resistance induced in the NahG plants by the BTH. It is also seen in these experiments that the INA and the BTH lead to a very good resistance of Ws to Emwa, and that the alleles nim1-l, nim1-2 and the other alleles nim1-l present, so to speak, no response to SA or AIIA for disease resistance.

The lack of response of niml plants to SAR-inducing chemicals such as SA, IAA and
BTH, implies that the mutation is downstream of the entry point (s) of these chemicals in the signal transduction cascade leading to acquired systemic resistance.

Example 4 Northern Analysis of SAR Gene Expression
Like SA, INA and BTH do not cause the
SAR, or the expression of the SAR gene in any of the niml plants, it is interesting to study whether the infection by a pathogenic microorganism could induce the expression of the SAR gene in these plants. as in wild-type plants. Thus, the SAR mRNA accumulation is also used as a criterion for characterizing the different RNA alleles that is transparent to maintain the high humidity required for successful P infection. parasitica, and placed in a growth chamber at 19 ° C during the day and 17 ° C during the night, with cycles of 8 hours of light and 16 hours of darkness. The RNA is extracted from the plants 3 days after treatment with water or chemicals, or 14 days after inoculation with the compatible P. parasitica Emwa isolate. RNA is size-fractionated by agarose gel electrophoresis and transferred to GeneScreenPlus membranes (DuPont).

 Figures 1 to 3 show different RNA gel shifts that indicate that SA, INA and BTH do not cause SAR or expression of the SAR gene in nimI plants. In Figure 1, replicative blots are hybridized to the Arabidopsis PR1, PR-2 and PR-5 gene probes, as described in Uknes et al. (1992).

In contrast to the wild-type plants, the chemicals do not induce RNA accumulation from any of the three SAR genes in nimI-1 plants.

 As seen in Figure 2, infection with a pathogenic microorganism (Emwa) of wild-type Ws-O plant causes expression of the PR-1 gene within four days of infection. In nim1-1 plants, the expression of the PR-1 gene is not induced until 6 days before infection, and the level is reduced compared to that of the wild type at the same date. Thus, after infection with a pathogenic microorganism, the expression of the PR-1 gene in nimI-1 plants is delayed, and is reduced compared to the wild-type.

 The gel RNA fingerprint of Figure 3 shows that PR-1 mRNA accumulates in high amounts after treatment of wild-type plants with SA, INA or BTH. or after infection with P. parasitica. In the nimI-1, nimi-2 and nimI-3 plants, the accumulation of PR-1 mRNA undergoes a large reduction compared to the wild type after chemical treatment. The mRNA of PR-1 is also reduced after infection with P. parasitica, but there is still some accumulation in these mutants. In the niml-4 and nimi-6 plants, the PR-1 rRNA accumulation is reduced more than in the other alleles after chemical treatment (which is manifested by longer exposures) and the amount of PR-1 mRNA that accumulates after infection with P. parasitica is significantly lower, supporting the idea that these are particularly strong alleles. The accumulation of PR-1 mRNA is high in the nimI-5 mutant, but is only slightly induced after chemical treatment or infection with P. parasitica. Based on the accumulation of PR-1 mRNA and a fungal infection, mutants appear to fall into three classes: highly compromised alleles (nim1-4 and nim1-6), moderately compromised alleles ( nimI-1, nimI-2, nimI-3); and a slightly compromised allele (niml-5).

Example 5: Determination of the accumulation of SA in niml plants
The accumulation of wild type plants by pathogenic microorganisms causing a necrotic reaction leads to the accumulation of SA in the infected tissues. Endogenous SA is required for signal transduction in the SAR pathway, as a break in endogenous SA leads to a decrease in disease resistance. This defines the accumulation of AS as a marker in the SAR pathway (Gaffney et al., 1993, Science 261, 754-756). The phenotype of niml plants indicates a break in a component of the SAR pathway downstream of the AS and upstream of the induction of the
SAR.

Niml plants are tested for their ability to accumulate AS after infection with a pathogenic microorganism. The test strain of Pseudomonas syringae from tomato, carrying the avrRpt2 gene, is injected into four-week-old plant leaves. The leaves are harvested two days later to allow SA analysis as described by Delaney et al., 1995, PNAS 92,6602-6606. This analysis shows that nimi plants accumulate high levels of AS in the infected leaves, as seen on the
Figure 4. Uninfected leaves also accumulate
But not in the same amounts as the infected leaves, which is similar to what is observed with
Arabidopsis wild type. This result indicates that the nim mutation is on the map downstream of the SA marker in the signal transduction pathway. In addition, INA and BTH (inactive in niml plants) are shown to stimulate a component of the SAR pathway downstream of SA (Venooij et al., 1995), Friedrich et al (1996), and Lawton et al. al (1996)). In addition, as mentioned above, exogenously applied SA does not protect niml plants from Emwa infection.

Example 6: Genetic Analysis
To determine the dominance of the different mutants that have the niml phenotype, pollen from wild-type plants is transferred to the stigmas of nimi-i, -2, -3, -4, -5, -6. If the mutation is dominant, then the nim1 phenotype will be observed in the resulting F1 plants. If the mutation is recessive, then F1 plants obtained will have a wild-type phenotype.

 The data presented in Table 3 show that when nimI-1, -2, -3, -4 and -6 are crossed with the wild type, F1 plants obtained have the wild-type phenotype. Thus, these mutations are recessive. In contrast, F1 wild type nimI-5 X mutant progeny all have the nimI phenotype, indicating that it is a dominant mutation. After treatment with INA, no sporulation of P. parasitica was observed on wild-type plants, whereas F1 plants favor proliferation and some sporulation of P. parasitica. However, the niml phenotype of these F1 plants is less severe than that observed when niml-1 is homozygous.

To determine allelism, pollen from kanamycin-resistant nim1-1 mutant plants is transferred to the nimI-2, -3, -4, -5, f6 stigmas.

Seeds from the cross are plated on Murashige-Skoog B5 plates containing kanamycin at 25 μg / ml to verify the hybrid origin of the seed. Kanamycin resistant F1 plants are transferred to soil, and their nimI phenotype is determined. Since the F1 descendants of the crossing of the nimI-5 mutant with the wild-type Ws have a nimI phenotype, the F2 nimI-5 X nimI-1 descendant is also analyzed.

 As can be seen from Table 3, all F1 plants obtained have the nimI phenotype. Thus, the mutation in nimI-2, -3, -4, -5, -6 is not completed by nimI1; these plants all fall into the same complementation group and are therefore allelic. The F2 offspring of the nimI-5X nimI-1 cross have also the nimI phenotype, confirming that nim1-5 is a nim1 allele.

Table 3
Genetic segregation of mutants nim

<tb> Mutant <SEP> Generation <SEP> Female <SEP> Male <SEP> Phenotype
<tb><SEP> Type <SEP> saved) <SEP> Nim1b)
<tb><SEP> n
<tb> nim1-1 <SEP> F1 <SEP> Type <SEP> saved) <SEP> nim1-1 <SEP> 24 <SEP> 0
<tb><SEP> F2 <SEP> 98 <SEP> 32
<tb> nim1-2 <SEP> F1 <SEP> nim1-2 <SEP> Type <SEP> wild <SEP> 3 <SEP> 0
<tb> nim1-3 <SEP> F1 <SEP> nim1-3 <SEP> Type <SEP> wild <SEP> 3 <SEP> 0
<tb> nim1-4 <SEP> F1 <SEP> nim1-4 <SEP> Type <SEP> wild <SEP> 3 <SEP> 0
<tb> nim1-5 <SEP> F1 <SEP> nim1-5 <SEP> Type <SEP> wild <SEP> 0 <SEP> 35
<tb><SEP> F1 <SEP> Type <SEP> wild <SEP> nim1-5 <SEP> 0 <SEP> 18
<tb> nim1-6 <SEP> F1 <SEP> nim1-6 <SEP> Type <SEP> wild <SEP> 3 <SEP> 0
<tb> nim1-2 <SEP> F1 <SEP> nim1-2 <SEP> nim1-1 <SEP> 0 <SEP> 15
<tb> nim1-3 <SEP> F1 <SEP> nim1-3 <SEP> nim1-1 <SEP> 0 <SEP> 10
<tb> nim1-4 <SEP> F1 <SEP> nim1-4 <SEP> nim1-1 <SEP> 0 <SEP> 15
<tb> nim1-5 <SEP> F1 <SEP> nim1-5 <SEP> nim1-1 <SEP> 0 <SEP> 14
<tb><SEP> F2 <SEP> 9 <SEP> 85
<tb> nim1-6 <SEP> F1 <SEP> nim1-6 <SEP> nim1-1 <SEP> 0 <SEP> 12
<Tb>
a) Number of plants with a high accumulation of PR-1 mRNA, with absence of P. parasitica after INA treatment.
b) Number of plants with no accumulation of PR-1 mRNA, with presence of P. parasitica after INA treatment.
c) Wild type refers to the wild-type Ws-O strain.

B. Mapping the niml mutation
The mapping of the nim1 mutation is described in great detail in the United States patent application filed by the Applicant under number 08 / 773,559 on December 27, 1996, this application being incorporated herein by reference in its entirety.

Example 7 Identification of Markers of the NOM Site and Genetic Mapping of the NIMl Site
To determine a crude position of the NIM1 on the map, the susceptibility to P. parasitica and the absence of PR-1 mRNA accumulation after INA treatment were identified on 74 NiMi F2 plants from crossing between niml-l (Ws-O) and Landsberg erecta (Ler). By using simple sequence size polymorphism (SSLP) markers (Bell and Ecker 1994), nimI-1 is found to be about 8.2 cmorgans (cM) nga 128 and 8.2 cM nga 111 on the lower arm of chromosome 1. In addition, it turns out that niml-l is found between nga 111 and about 4 cM from the marker of
SSLP ATHGENEA. (Figure 5A).

 For fine structure mapping, 1138 nim plants from a F2 population derived from a cross between nimI-1 and LerDP23 were identified based on their ability to accumulate PR-1 mRNA and their mRNA. ability to promote fungal growth after treatment with INA. The DNA is extracted from these plants, and we note its zygosity at ATNGENEA and nga 111. As seen in Figure 5A, 93 recombinant chromosomes are identified between ATHGENEA and niml-1, which gives a distance Genetics of about 4.1 cM (93 of 2276), and 239 recombinant chromosomes are identified between nga 111 and nim1-1, giving a genetic distance of about 10.5 cM (239 of 2276). Further analysis of informative recombinants ranging from ATHGENEA to nga 111 is carried out using amplified fragment size polymorphism (AFLP) analysis (Vos et al., 1995).

AFLP markers between ATHGENEA and nga 111 are identified and are used to construct a low resolution map of the region (Figures 5A and 5B). AFLP markers W84.2 (at 1 cM nimI-1) and W85.1 (at 0.6 cM nimI-1) were used to isolate clones of the artificial yeast chromosome (YAC) from CIC (Center for the Study of Human Polymorphism,
INRA and CNRS) (Creusot et al., 1995). Two YAC clones, CIC 12H07 and CIC12F04, are identified with the
W84.2, and two YAC clones, CIC7E03 and CIC1OG07, are identified with the W85.1 tag (Figure 5B). To cross the gap between the two sets of YAC lateral clones, Bacterial Artificial Chromosome (BAC) clones and P1 clones overlapping CIC12H07 and CIC12F04 are isolated and mapped, and successive step steps are performed. extending the area contiguous with BAC / P1 to NIM1 (Figure 5C, Liu et al., 1995, Chio et al., 1995). New polymorphisms
AFLP are developed during walking, which are specific for the BAC or P1 clones, and are used to determine if the nim1 gene has been crossed. The nimi was crossed when the BAC and P1 clones were isolated, giving rise to the two markers of AFLP L84.6a and
L84.8. The marker of AFLP L84.6a found on clone P1, namely P1-18, P1-17 and P1-26, identifies three recombinants, and the L84.8 marker, which is found on the P1 clones, namely P1-20, P1-22, P1-23 and P1-24, and the BAC clones, namely BAC-04, BAC-05 and BAC-06, identify a recombinant. Since these clones overlap to form a large contiguous area (greater than 100 kb) and include AFLP markers in the lateral position relative to nml, it turns out that the gene is located on the contiguous zone. The BAC and P1 clones that comprise the contiguous zone are used to produce additional markers of AFLP, showing that nim1 is located between L84.Y1 and L84.8, representing a range of about 0.09 cM.

C. Isolation of the NIM1 gene
Example 8 Synthesis of a Contiguous Cosmic Zone
A cosmid library of the NIM1 region is synthesized in the cosmid vector of T-DNA compatible with
Agrobacterium pCLD04541 using CsCl purified DNA from BAC-06, BAC-04 and P1-18. The DNAs of the three clones are mixed in equimolar amounts and are subjected to partial digestion with the Sau3A restriction enzyme. The 20-25 kb fragments are isolated using a sucrose gradient, combined and filled with dATP and dGTP. Plasmid pCLD04541 is used as a cosmid T-DNA vector. This plasmid contains a pRK290-based replicon, corresponding to a wide host range, a tetracycline resistance gene for bacterial selection, and the nptII gene for plant breeding. The vector is cleaved with XhoI and filled with dCTP and dTTP.

The prepared fragments are then ligated into the vector. The ligation mixture is packaged and transduced into E. coli strain XL1-blue MR (Statagene). The transformants obtained are screened by hybridization with clones BAC04, BAC06 and P1-18, and the positive clones are isolated. The cosmid DNA is isolated from these clones, and the template DNA is prepared using EcoRI / MseI and HindIII / enzyme combinations.
MseI. The fingerprints obtained from the AFLP polymorphism are analyzed to determine the order of the cosmid clones.

A set of 15 semi-overlapping cosmids are selected which cover the nim region (Figure 5D).

Cosmid DNAs are also restricted by EcoRI,
PstI, BssHII and SgrAI. It is thus possible to estimate the size of the cosmid inserts, and to verify the overlaps between the different cosmids, as determined by the AFLP borrows.

 Physical mapping shows that the physical distance between L84.Y1 and L84.8 is greater than 90 kb, which gives a relationship between genetic distance and physical distance of about 1 megabase per cM. To facilitate identification of the NIM1 gene, the DNA sequence of BAC04 is determined.

Example 9 Identification of a Clone Containing the NIMi Gene
Cosmids produced from clones that cover the NDM1 region are displaced in Agrobacterium tumefaciens AGL-1 by conjugative transfer, as part of a triparental pairing with the HB101 assist strain (pRK2013). These cosmids are then used to transform a kanamycin-sensitive kanamycin-sensitive Arabidopsis line by use of vacuum infiltration (Bechtold et al., 1993, Mindrinos et al., 1994).

Seeds from the infiltrated plants are harvested and allowed to germinate on agar plates
GM containing 50 mg / ml kanamycin as a selection agent. Only seedlings transformed with cosmid DNA can detoxify the selection agent and survive. Seedlings that survive selection are transferred to soil approximately two weeks after plating, and are tested for the nimI phenotype as described below.

Transformed plants that no longer have the niml phenotype identify the cosmid (s) containing a functional NIMl gene.

Example 10: Complementation of the niml phenotype
Plants transferred to the soil are grown in a phytotron for about a week after transfer. AIN 300 WM was applied as a fine mist to completely cover the plants with a chromister. After two days, the leaves are harvested to allow extraction of RNA and analysis of PR-1 expression. The plants are then sprayed with Peronospora parasitica (Emwa isolate), and cultured in the presence of high humidity in a culture chamber, at 19 ° C during the day and 17 ° C during the night, with cycles of 8 hours of light and 16 hours of darkness. 8 to 10 days after the fungal infection, the plants are evaluated and given a positive or negative rating for fungal growth. The plants Ws and niml are treated in the same way, to serve as a control for each experiment.

Total RNA is extracted from the recovered tissue using a LiCl / phenol extraction buffer (Verwoerd et al., 1989). The RNA samples are placed on a formaldehyde-agarose gel, and transferred to GeneScreen Plus membranes (DuPont). The fingerprints of the blots are hybridized using a cDNA probe of
PR-1 labeled at 32P. The transfers obtained are exposed to a film to determine the transformants which are capable of inducing the expression of PR-1 after treatment with INA. The results are summarized in Table 4, which shows the complementation of the nim1 phenotype by cosmid clones D5, E1 and D7.

Table 4

<tb> Name <SEP> of <SEP> clone <SEP> Number <SEP> of <SEP> Number <SEP> of <SEP> Plants <SEP> to <SEP> PR-1 <SEP> induced <SEP> by
<tb><SEP> transformants <SEP> INA / nb <SEP> total <SEP> of <SEP> plants <SEP> tested <SEP> (%)
<tb> A8 <SEP> 3 <SEP> 0/3 <SEP> (O <SEP> 4) <SEP>
<tb> All <SEP> 8 <SEP> 4/18 <SEP> (22 <SEP>%) <SEP>
<tb> C2 <SEP> 10 <SEP> 1/10 <SEP> (10 <SEP>%)
<tb> C7 <SEP> 33 <SEP> 1/32 <SEP> (3 <SEP> 8) <SEP>
<tb> D2 <SEP> 81 <SEP> 4/49 <SEP> (8 <SEP>%) <SEP>
<tb> DS <SEP> 6 <SEP> 5/6 <SEP> (83 <SEP>%) <SEP>
<tb> El <SEP> 10 <SEP> 10/10 <SEP> (100 <SEP>%)
<tb> D7 <SEP> 129 <SEP> 36/36 <SEP> (100 <SEP>%)
<tb> E8 <SEP> 9 <SEP> 0/9 <SEP> (0 <SEP>%)
<tb> F12 <SEP> 6 <SEP> 0/6 <SEP> (O <SEP> 4) <SEP>
<tb> E6 <SEP> 1 <SEP> 0/1 <SEP> (O <SEP>%) <SEP>
<tb> E7 <SEP> 34 <SEP> 0/4 <SEP> (O <SEP> 4) <SEP>
<tb> Control <SEP> Ws <SEP> NA <SEP> 28/28 <SEP> (100 <SEP>%) <SEP>
<tb> (type <SEP> wild)
<tb> Control <SEP> pheno- <SEP> NA <SEP> 0/34 <SEP> (O <SEP>%)
<tb> type <SEP> nim1-1
<Tb>
NA: Not applicable
Example 11: Sequencing of the Region of the NIM1 Gene
BACO4 DNA (25 μg, obtained from KeyGene) is used as a source of DNA used for sequence analysis because this BAC is the clone that completely overlaps the region that complements the genes. mutants niml. The BAC04 DNA is randomly sheared in a fogger to produce fragments having an average size of about 2 kb. The ends of the sheared fragments are repaired, and the fragments are purified. The prepared DNA is ligated with EcoRV digested pRBKanF4 (a derivative of pBRKanF1) (Bhat 1993)). The colonies obtained, resistant to kanamycin, are selected according to the isolation of the plasmids by using the Wizard Plus 9600 Miniprep system.
System (Promega). Plasmids are sequenced using the dye terminator technique (Applied Biosystems, Foster City, CA) with primers designed to sequence both plasmid strands (M1321 forward and T7 rearward, Applied BioSystems). The data is collected on ABI377 DNA sequencers. The sequences are edited and assembled in contiguous areas using Sequencher 3.0 (GeneCodes Corp., Ann Arbor, MI), Staden, Phed, Phrap and Crossmatch genome assembly programs (Phil Green, Washington University, St. Louis,
MO) and consed (David Gordon, Washington University, Saint
Louis, MO). Cosmid-derived DNA, which is found to complement the nimi-i mutation, is sequenced using primers developed by Oligo 5.0 priming analysis software (National Biosciences Inc., Plymouth,
MN). Sequencing of DNA from the Ws-0 and nXml alleles, as well as that of the cDNAs, is performed essentially as described above.

A region of about 9.9 kb, defined by the overlap of E1 and D7 cosmids, is identified by complementation analysis as containing the nimI region. Primers which are in a lateral position with respect to the vector insertion site and which are specific to the cosmic skeleton are developed using the Oligo 5.0 primers analysis software (National Biosciences Inc.). The DNA is isolated from cosmids D7 and E1 using a variant of the ammonium acetate method (Traynor P.L., 1990, BioTechniques 9 (6): 676).

This DNA is sequenced directly using the above dye terminator chemistry. The sequence obtained makes it possible to determine the end points of the complementation region. The region defined by the overlap of cosmids E1 and D7 is presented under the name of sequence SEQ ID NO: 1.

 A truncated version of the BamHI-EcoRV fragment is also synthesized, and gives a synthetic product which does not contain the region 3 of the gene (FIG. 5D).

The following approach is necessary, because of the presence of HindIII sites in the Bam-Spe region of DNA. The BamHI-EcoRV synthesis product is fully digested with SpeI, and then subdivided into two separate reactions by double digestion. One aliquot is digested with BamHI and the other with HindIII. Agarose gels (QIAquick Gel Extraction Kit) are isolated
2816 bp BamHI-SpeI and a 1588 bp HindIII-SpeI fragment, and ligated with BamHI-HindIII digested pSGCG01. DHSa is transformed with the ligation mixture. The colonies obtained are screened to determine the correct insertion by digestion with HindIII after preparation of the DNA using Wizard Magic.
MiniPreps (Promega). A clone containing the correct synthetic product is electroporated into Agrobacterium strain GV3101 for transformation of Arabidopsis plants.

Example 12: Sequence Analysis and Subcloning of the NiMi Region
The 9.9 kb region containing the NiMi gene is analyzed to determine the presence of open reading frames in the six frames, using Sequencher 3.0 and GCG software. Four regions containing large open reading frames are identified as possible genes (regions 1-4 of the genes in Figure 5D). These four regions are subjected to PCR amplification from the wild-type parent DNA, and different nimI, nimI-1, -2, -3, -4, -5 and -6 allelic variant sites. The primers used for these amplifications are selected using Oligo 5.0 (National Biosciences
Inc.) and are synthesized by Integrated DNA Technologies Inc. The PCR amplification products are separated on 1.0% agarose gels which are purified by the QIAquick Gel Extraction Gel Extraction Kit. The purified genomic products of the PCR amplification are directly sequenced by using the primers used for initial amplification, and with additional primers for sequencing across all regions not covered by the initial primers. The average coverage of these gene regions is about 3.5 readings / base.

 The sequences are edited and are assembled using the Sequencher 3.0. The specific base changes of the different niml alleles are identified only in the region called Region 2 of the gene, as seen below in Table 5, which shows the sequence variations among the six niml alleles.

Table 5

<tb><SEP> Region <SEP> of the <SEP> annoyance
<tb><SEP> Allele / Ecotype <SEP> 1 <SEP> (bases <SEP> 590-1090) <SEP> 2 <SEP> (NIM1) <SEP> (bases <SEP> 13804100) <SEP> 3 <SEP> (bases <SEP> 4 <SEP> (bases <SEP> 8140
<tb><SEP> 5870-6840) <SEP> 9210)
<tb><SEP> nim1-1 <SEP> No <SEP> of <SEP> change <SEP> t <SEP> inserted <SEP> in <SEP> 2981: <SEP> change <SEP> No <SEP> of <SEP> No <SEP> of
<tb><SEP> of <SEP> 7AA <SEP> and <SEP> Termination <SEP> Change <SEP> Change
<tb><SEP> premature <SEP> of <SEP> the <SEP> protein
<tb><SEP> nim1-2 <SEP> No <SEP> of <SEP> change <SEP> Replace <SEP> from <SEP> g <SEP> by <SEP> a <SEP> by <SEP> Step <SEP> from <SEP> No <SEP> from
<tb><SEP> 2799 <SEP>: <SEP> His <SEP> in <SEP> Tyr <SEP> change <SEP> change
<tb><SEP> nim1-3 <SEP> No <SEP> of <SEP> change <SEP><SEP> Deletion of <SEP> t <SEP> in <SEP> 3261 <SEP>;<SEP> No <SEP> of <SEP> No <SEP> of
<tb><SEP> change <SEP> of <SEP> 10AA <SEP> and <SEP> change <SEP> change
<tb><SEP><SEP> premature <SEP> termination of <SEP> la
<tb><SEP> protein
<tb><SEP> nim1-4 <SEP> No <SEP> of <SEP> change <SEP> c <SEP> in <SEP> t <SEP> to <SEP> 2402 <SEP>: <SEP> Arg <SEP> in <SEP> lys <SEP> No <SEP> of <SEP> No <SEP> of
<tb><SEP> change <SEP> change
<tb><SEP> nim1-5 <SEP> No <SEP> of <SEP> change <SEP> c <SEP> in <SEP> t <SEP> to <SEP> 2402 <SEP>: <SEP> Arg <SEP> in <SEP> lys <SEP> No <SEP> of <SEP> No <SEP> of
<tb><SEP> change <SEP> change
<tb><SEP> nim1-6 <SEP> g <SEP> in <SEP> a <SEP> to <SEP> 734 <SEP>: <SEP> Asp <SEP> g <SEP> in <SEP> a <MS> to <SEP> 2670: <SEP> Gln <SEP> in <SEP> Stop <SEP> Not <SEP> from <SEP> No <SEP> from
<tb><SEP> in <SEP> lys <SEP> ~ <SEP> change <SEP> change
<tb> Ws <SEP> Not <SEP> of <SEP> change <SEP> a <SEP> in <SEP> g <SEP> to <SEP> 1607 <SEP>: <SEP><SEP> in <SEP > Leu <SEP> t <SEP> in <SEP> a <SEP> to <SEP> 5746 <SEP> t <SEP> in <SEP> g <SEP> to <SEP> 8705
<tb><SEP> (comparison
<tb><SEP> of <SEP> d <SEP> to
<tb><SEP> Columbia)
<tb><SEP> a <SEP> in <SEP> c <SEP> to <SEP> niml-6 belong to the strain Ws. Columbia-0 represents the wild type.

 It is apparent that the NIM1 gene is within Region 2 of the gene because there are amino acid changes or sequence changes within the Region 2 open reading frame of the gene. in the six alleles niml. Simultaneously, at least one of the niml alleles shows no change in open reading frames in regions 1, 3 and 4 of the gene. Accordingly, the only region of the gene within the 9.9 kb region likely to contain the NiMi gene is region 2 of the gene.

The Ws section of Table 5 shows the changes in the Arabidopsis Ws ecotype relative to the ecotype
Colombia of Arabidopsis. The sequences presented therein relate to the Columbia ecotype of Arabidopsis, which contains the wild-type gene in the experiments described in the invention. The changes are referred to as amino acid changes in region 2 of the gene (the NIAI region) and are indicated as a change in base pairs in the other regions.

The cosmid region containing the nimI gene is represented by a BamHI-EcoRV restriction fragment having a size of about 5.3 kb. D7 cosmid DNA and pBlueScriptII plasmid DNA (pBSII) are digested with BamHI and with EcoRV (NEB). The 5.3 kb fragment from D7 is isolated from agarose gels and is purified using the gel extraction kit.
QIAquick (NO 28796, Qiagen). The fragment is ligated overnight to Bam-EcoRV digested pBSII, and the ligation mixture is transformed into the EcoAquick strain (NO 28796, Qiagen). The fragment is ligated overnight on Bam-EcoRV digested pBSII, and the ligation mixture is transformed into the
E. coli DH5α. The colonies containing the insert are selected, the DNA is isolated, and confirmation is achieved by HindIII digestion. The Bam-EcoRV fragment is then engineered into a binary vector (pSGCG10) for transformation into Arabidopsis.

Example 13: Northern blot analysis of the four regions of the gene
Identical Northern blots were made from Rs samples isolated from Ws treated with water, SA, BTH and INA, and nimI lines, as previously described in Delaney et al. (1995).

These transfers are hybridized with PCR amplification products obtained from the four regions of the gene identified in the region of the 9.9 kb NIM1 gene (SEQ
ID NO: 1). Only the region of the gene containing the NiMi gene (region 2 of the gene) has detectable hybridization with the RNA samples, indicating that only the NIM1 region contains a detectable transcription gene (Figure 5D and Table 5).

Example 14: Complementation with Region 2 of the Gene
Additional complementation experiments also showed that region 2 of the gene (Figure 5D) contains the functional NIMi gene. A BamHI / HindIII genomic DNA fragment containing region 2 of the gene is isolated from cosmid D7 and cloned into the binary vector pSGCG01 containing the kanamycin resistance gene.

The resulting plasmid is transformed into the Agrobacterium strain GV3101, and the positive colonies are selected on kanamycin. PCR amplification is used to verify that the selected colony contains the plasmid. Kanamycin-sensitive plants are infiltrated by this bacterium, as already described. The resulting seed is harvested on a GM agar containing 50 μg / ml kanamycin. The plants surviving the selection are transferred to a soil and subjected to a complementation test. Transformed plants and control Ws and niml plants were sprayed with INA 300 WM. Two days later, the leaves are harvested for RNA extraction and analysis of PR-1 expression. The plants are then sprayed with Peronospora parasitica (Emwa isolate) and cultured as described above. Ten days after the fungal infection, the plants are evaluated and given a positive or negative rating for fungal growth. The transformed plants, as well as the Ws controls, are negative for fungal overgrowth after treatment with INA, whereas the niml controls are positive for fungal overgrowth. The RNA is extracted and analyzed as described above for these transformants and controls. Ws controls and transformants exhibit PR-1 gene induction after INA treatment, whereas nim1 controls do not induce PR-1 induction by INA.

Example 15 Isolation of a cDNA of NIMl
A library of Arabidopsis cDNA, made in an IYES expression vector (Elledge et al., 1991, PNAS 88, 1731-1735), is plated onto, and plaque transfers are performed. The filters are hybridized with a 32p-labeled PCR amplification product obtained from region 2 of the gene (Figure 5D). 14 positives are identified from a screening of approximately 150,000 plaques. Each range is purified, and the plasmid DNA is recovered. The cDNA inserts are extracted from the vector by EcoRI digestion, purified on agarose gel and sequenced. The sequence obtained from the longest cDNA is indicated on the sequence SEQ ID NO: 2 and on the
Figure 6. To confirm that the 5 'end of the cDNA was obtained, a Gibco BRL 5' RACE kit was used following the manufacturer's instructions. The RACE products obtained (by rapid amplification of the ends of the cDNA) are sequenced and appear to contain the additional bases indicated in FIG. 6. The transcribed region present in the two cDNA clones and detected by the RACE amplification is indicated. in uppercase in Figure 6. The changes in the alleles are shown above the DNA strand. The uppercase indicates the presence of the sequence in a cDNA clone, or its detection after RACE PCR amplification.

 The same RNA samples as those produced in the induction studies (Figure 3) are probed with the NIM1 gene by using, as a probe, a complete cDNA clone. In Figure 7, it can be seen that the INA induces the NIM1 gene in the wild-type Ws allele. However, the nimI-1 mutant allele has a baseline level of expression lower than that of the NIM1 gene, and can not be induced by INA. This result is analogous to what is observed in the niml-3 allele and the nimi-6 allele. The nimI-2 allele has approximately normal levels in the untreated sample and is similar in induction to the wild-type sample, as is the nimi-4 allele.

The nimI-5 allele appears to have a higher level of basic expression than the NIMi gene, and a much stronger expression after induction by chemical inducers.

D. Counterparts of the NIMl
Example 16: BLAST search with the NIM1 sequence
A multiple sequence alignment is synthesized using Clustal V (Higgins, Desmond G, and Paul
M. Sharp (1989), Fast and Sensitive Multiple Sequence Alignments, Microcomputer, CABIOS 5: 151-153) as part of the software package for Macintosh (1994) Lasergene
Biocomputing Software by DNA * (1228 South Park Street,
Madison Wisconsin, 53715). Certain regions of the NIM1 protein are homologous, in terms of their amino acid sequence, of 4 cDNA proteins different from rice. Homologies are identified by using NSMl sequences in a Gen BLAST search
Bank. Comparisons of homology regions of NIMI and rice cDNA products are shown in Figure 8 (see also SEQ ID NO: 3 and SEQ ID NO: 4-11). The NIM1 protein fragments have amino acid sequences having an identification level of 36 to 48% with the 4 rice products.

Example 17 Isolation of homologous genes from other plants
By using NIAI cDNA as a probe, Arabidopsis NiMi homologues are identified by screening libraries or cDNA libraries from different cultures, such as, but not limited to, those mentioned above. Examples of conventional techniques to achieve this are hybridization screening of plaque-based DNA libraries (plaques or colonies, see, for example, Sambrook et al., Molecular Cloning, eds. Cold
Spring Harbor Laboratory Press (1989)), and amplification by PCR using oligonucleotide primers (see, for example, Innis et al., PCR Protocols, a Guide to
Methods and Applications, eds., Academic Press (1990)).

The homologs identified are genetically engineered in the expression vectors according to the invention, and transformed in the cultures above. Transformants are evaluated for improved disease resistance by using appropriate pathogenic microorganisms of the crop plant being tested.

 NIM1 homologs in the genomes of cucumber, tomato, tobacco, maize and barley are detected by DNA blot analysis. The genomic DNA is isolated from cucumber, tomato, tobacco, maize, wheat and barley, digested with restriction enzymes BamHI, HindIII, XbaI or SalI, electrophoretically separated on 0.8% agarose gels and transferred to a nylon membrane by capillary transfer. After crosslinking with UV to fix the DNA, the membrane is hybridized under low stringency conditions [1% BSA; 520 mM NaPO4, pH 7.2; sodium lauryl sulphate 7%; EDTA 1 mM; 250 mM sodium chloride) at 55 ° C for 18 to 24 hours using the 32 P-radiolabelled Arabidopsis thaliana NIM1 cDNA. After hybridization, the blots are washed under low stringency conditions [6XSSC for 15 min. minutes (X3), 3XSSC for 15 minutes (X1) at 550C, 1XSSC is 0.15M NaCl, 15mM Na citrate (pH 7.0), and exposed to X-ray film to visualize the corresponding bands. at NIMl.

In addition, expressed sequence markers (ESTs), identified by analogy with the NIM1 gene, including the
EST rice described in Example 16, can also be used to isolate homologues. Rice ESTs can be used in particular to isolate NiMi homologs from other monocots.

 Homologs can be obtained by PCR amplification. In this process, comparisons are made between known homologs (eg rice and Arabi dopsis). PCR regions are then used to make regions having a high similarity or identity with the amino acids and the DNA. The M and W rich regions are best followed by F, Y, C, H, Q, K, and E rich regions, since these amino acids are coded by a limited number of codons. Once an appropriate region has been identified, primers are prepared for this region, with a variety of substitutions on the position of the third codon. This substitution diversity on the third position may be limited depending on the target species. For example, because corn is rich in GC, primers are developed that use G or C, if possible, in the third position.

 The PCR amplification reaction is carried out from cDNA or genomic DNA under many standard conditions. When a band is apparent, it is cloned and / or sequenced to determine if it is a NIAI homolog.

E. A high level of expression of NIMi confers on plants resistance to diseases
A high level of expression of the NIM1 gene in transgenic plants, intended to confer a CIX phenotype, is likewise described in the US patent application filed by the applicant under serial number 08 / 773,554 on December 27, 1996, which document is incorporated herein by reference in its entirety.

Example 18: High level of expression of NIM1 due to the effect of the insertion site
To determine whether any of the transformants described above in Example 10 and Table 4 have a level of NiMi expression due to the effect of the insertion site, primary transformants are self-fertilized. containing the cosmids D7, D5 or
El (containing the NOM gene), and the seed is collected
T2. Seeds of an E1 line, four D5 lines and 95 D7 lines are sown on the soil, and grown as described above. When the T2 plants have arrived at at least four true leaves, a single leaf is collected separately for each plant. RNA is extracted from this tissue and assayed for expression of PR-1 and NIM1. The plants are then seeded with P. parasitica (Emwa), fungal growth is determined after 3 to 14 days, preferably 7 to 12 days after infection. Plants with higher than normal expression of both NIM1 and PR-1, with fungal resistance, show that a high level of NIMi expression confers a CIM phenotype.

 Table 6 presents the results of tests of different transformants, on resistance to fungal infection. As can be seen from the table, a number of transformants show below normal fungal growth, and many of them show no visible fungal growth. RNA is prepared from collected samples and analyzed as described above (Delaney et al., 1995). The blots are hybridized to the PR-1 of the Arabidopsis gene probe (Uknes et al., 1992).

Lines D7-74, D5-6 and El-1 exhibit early induction of PR-1 gene expression, whereas PR-1 mRNA is evident 24 or 48 hours after fungus treatment. These three lines also demonstrate resistance to fungal infection.

Table 6

<tb><SEP>line><SEP>Proliferation><SEP>line><SEP>Proliferation><SEP> Proliferation line
<tb><SEP> of <SEP> P. <SEP> of <SEP> P. <SEP> of <SEP> P
<tb> parasitica <SEP> parasitica <SEP> parasitica
<tb><SEP> D7-2 <SEP> Negative <SEP> 52 <SEP> + <SEP> 90 <SEP> +
<tb><SEP> 3 <SEP> + <SEP> 53 <SEP> + <SEP> 91 <SEP> +
<tb><SEP> 9 <SEP> + <SEP> 54 <SEP> +/- <SEP> 92 <SEP> +
<tb><SEP> 11 <SEP> + <SEP> 56 <SEP> + <SEP> 93 <SEP> +
<tb><SEP> 12 <SEP> + <SEP> 57 <SEP> + <SEP> 94 <SEP> +
<tb><SEP> 13 <SEP> + <SEP> 58 <SEP> + <SEP> 95 <SEP> +
<tb><SEP> 14 <SEP> 59 <SEP> 96 <SEP> +
<tb><SEP> 17 <SEP> + <SEP> 60 <SEP> + <SEP> 97 <SEP> +
<tb><SEP> 18 <SEP> + <SEP> 61 <SEP> + <SEP> 98 <SEP> + /
<tb><SEP> 19 <SEP> + <SEP> 62 <SEP> + <SEP> 100 <SEP> + /
<tb><SEP> 20 <SEP> + <SEP> 63 <SEP> + <SEP> 101
<tb><SEP> 21 <SEP> + <SEP> 64 <SEP> + <SEP> 102 <SEP> | <SEP> + /
<tb><SEP> 22 <SEP> + <SEP> 66 <SEP> + <SEP> 103 <SEP> + <SEP>
<tb><SEP> 23 <SEP> + <SEP> 67 <SEP> + <SEP> 104 <SEP> +
<tb><SEP> 24 <SEP> + <SEP> 68 <SEP> + <SEP> 106 <SEP> +
<tb><SEP> 25 <SEP> + <SEP> 69 <SEP> + <SEP> 107 <SEP> +
<tb><SEP> 28 <SEP> + <SEP> 70 <SEP> + <SEP> 108 <SEP> +
<tb><SEP> 29 <SEP> ~ <SEP> + <SEP> 71 <SEP> + <SEP> 114 <SEP> +
<tb><SEP> 31 <SEP> + <SEP> 72 <SEP> + <SEP> 115 <SEP> +
<tb><SEP> 32 <SEP> | <SEP> + <SEP> 73 <SEP> + <SEP> 118 <SEP> +
<tb><SEP> 33 <SEP> + <SEP> 74 <SEP> Negative <SEP> 119 <SEP> +
<tb><SEP> 34 <SEP> ~ <SEP> + <SEP> 75 <SEP> + <SEP> 122 <SEP> +
<tb><SEP> 35 <SEP> ~ <SEP> + <SEP> 77 <SEP> + <SEP> 123 <SEP> +
<tb><SEP> 36 <SEP> + <SEP> 78 <SEP> + <SEP> 124 <SEP> +
<tb><SEP> 38 <SEP> + <SEP> 79 <SEP> + <SEP> 125 <SEP> +
<tb><SEP> 39 <SEP> + <SEP> 80 <SEP> +/- <SEP> 126 <SEP> +
<tb><SEP> 42 <SEP> + <SEP> 81 <SEP> + <SEP> 128 <SEP> +
<tb><SEP> 43 <SEP> + <SEP> 82 <SEP> + <SEP> 129 <SEP> +
<tb><SEP> 46 <SEP> + <SEP> 83 <SEP> + <SEP> 130 <SEP> +
<tb><SEP> 47 <SEP> + <SEP> 84 <SEP> + <SEP> | <SEP> D5-1 <SEP> +
<tb><SEP> 48 <SEP> + <SEP> 85 <SEP> + <SEP> 2 <SEP> +
<tb><SEP> 49 <SEP> + <SEP> 86 <SEP> + <SEP> 4 <SEP> +
<tb><SEP> 50 <SEP> + <SEP> 87 <SEP> + <SEP> 6
<tb><SEP> 51 <SEP> + <SEP> 89 <SEP> + <SEP> E1-1 <SEP> Negative
<Tb>
The plants are treated with P. parasitica isolate Emwa and noted 10 days later.

+: Normal fungal growth
+/-: Below normal fungal growth
-: No visible fungal growth.

Example 19: Expression of NIMi caused by 35S
Plants that constitutively express the NIM1 gene are likewise produced by transformation of wild-type Ws or Col plants, using the Arabidopsis BamHI-HindIII genomic fragment (Figure 6) cloned into pSGCG01 transformed into the strain of Agrobacterium
GV 3101 (pMP90, Koncz and Schell 1986, Mol Gen Genet 204: 383-396). In another synthetic product, complete NIM1 cDNA is cloned into the EcoRI site of pCGN1761 ENX (Comai et al., 1990, Plant Mol Biol 15, 373-381). From the plasmid thus obtained, a fragment
XbaI containing a strengthened CaMV 35S promoter, the NIM1 cDNA in the correct orientation for transcription, and a 3'-terminator of tml. This fragment is cloned into the binary vector pCIB200 and transformed into
GV3101. Ws or Col plants are subjected as described above to infiltration by each of these transformed strains. The resulting seed is harvested and placed on a GM agar plate containing 50 mg / ml kanamycin. Surviving seedlings are transferred to the soil and tested.

Example 20: Expression of the NIMl caused by the 35S other embodiment
A genomic fragment of Arabidopsis BamHI / HindIII (FIG. 6, bases 1249-5655) containing the NIM1 promoter, the gene and the downstream sequence cloned into pSGCG01, is subjected to restriction endonuclease digestion PstI, and the ends are made blunt with T4 polymerase. The fragment is further digested with SpeI restriction endonuclease to give a 2262 bp fragment. The NIM1 cDNA cloned into the EcoRI site of pCGN1761ENX (see above) is digested with the NotI restriction endonuclease, and the ends are blunted by T4 polymerase.

The fragment is further subjected to restriction endonuclease digestion SpeI. The 2262 bp genomic NIMI fragment obtained above is ligated with the pCGN1761ENX vector containing 91 bp of the NIM1 cDNA.

The transformation cassette comprises the 35S promoter, and the terminator of the tml is released from pCGN1761ENX by partial digestion with the restriction enzyme XbaI, and ligated into the XbaI site of the dephosphorylated pCIB200.

The resulting plasmid is transformed into GV3101, and the Ws or Col plants are infiltrated as described above. The seeds are placed on a plate, and the surviving seedlings are selected as described above.

Example 21 Evaluation of the CIM Phenotype in the High Level of Expression of NIM1 Transformants
One leaf of each primary transformant is harvested, RNA is isolated (Verwoerd et al., 1989, Nuc Acid
Res, 2362), and the constitutive expression of PR-1 and the expression of NIM1 by RNA transfer analysis (Uknes et al., 1992) are determined. Each transformant is assessed for enhanced disease resistance response, which is indicative of an analysis of constitutive expression of SAR (Uknes et al., 1992).

Conidial suspensions of 5-10.104 spores per ml from two compatible isolates of P. parasitica, Emwa and Noco (i.e., these fungal strains create disease on the seedlings respectively) are prepared.
Wild-type Ws-O and Col-O), and the transformants are sprapped with the appropriate isolate, depending on the ecotype of the transformant. Inoculated plants are incubated in the presence of high humidity for 7 days. The plant disease rate is evaluated on day 7, and a single leaf is collected for RNA analysis using a probe which is a means of measuring fungal infection.

Transformants that have a CIM phenotype are conducted to F1 generation, and homozygous plants are identified. Transformants are subjected to a bacterium for disease resistance tests. Noco and Emwa are fungal infections, and the leaves are stained with lactophenol blue to identify the presence of fungal hyphae, as described in Dietrich et al. (1994). Transformants are infected with the pathogenic bacterium Pseudomonas syringae
DC3000 to evaluate the resistance spectrum, which is manifest as described in Uknes et al. (1993). Uninfected plants are evaluated for free AS and glucose-conjugated SA, and the leaves are stained with lactophenol blue to assess the presence of microscopic lesions. Resistant plants are sexually cross-linked with SAR mutants such as NahG and ndrl, to establish the epistatic relationship of the resistance phenotype to that of other mutants, and to evaluate how these dominant-negative NIM1 mutants can influence the loop. of feedback depending on
HER.

Example 22: High Level of Expression of NIM1 in Culture Plants
Synthetic products imparting a CIM phenotype to Col-O and Ws-0 plants, and other plants, are transformed into culture plants for evaluation.

Although the NIM1 gene can be inserted into any plant cell within these broad categories, it is particularly useful in plant cells of crop plants, such as the following rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini , cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, ripe, pineapple, avocado, papaya, mango, banana, soy, tobacco, tomato, sorghum and sugarcane . Transformants are assessed for improved resistance to disease. In a preferred embodiment of the invention, the expression of the NIM1 gene corresponds to a level which is at least two times greater than the level of expression of the NLM1 gene of the wild-type plants, and which is preferably 10 times higher. at the expression level of the wild type.

F. Other general uses of plants with phenotype nim
Example 23: Use of nim mutants in the evaluation of diseases
Nim mutants are induced with different pathogenic microorganisms, and these mutants appear to develop larger lesions faster than wild-type plants. This phenotype is called UDS (ie universal susceptibility to disease) and is the result of mutants that can not express the genes of SAR, to ensure the defense of plants against pathogenic microorganisms. The UDS phenotype of the nim mutants makes them useful as control plants for the evaluation of disease symptoms in experimental lines as part of the field pathogenesis assays, where the natural resistance phenotype of so-called Wild type lines can vary (ie, different pathogenic microorganisms, with different pathotypes of the same pathogenic microorganism). Thus, in the field, where the natural infection by pathogenic micro-organisms is based on an evaluation of the resistance of experimental lines, the incorporation, in the experiment, of nim mutant lines of the appropriate culture plant should allow a evaluation of the true level and pressure spectrum of pathogenic micro-organisms, without the inherent variations in the use of non-experimental lines.

Example 24: Evaluation of the utility of transgenes for disease resistance
Nim mutants are used as host plants to transform transgenes and facilitate their evaluation in the context of use in disease resistance. For example, an Arabidopsis nim mutant line, characterized by its UDS phenotype, is used for subsequent transformations with disease resistance candidate genes, which allows for evaluation of the contribution of an individual gene to resistance against the basic level of mutant plants nim UDS.

EXAMPLE 25 Mutants NIM, a Tool for Understanding Plant-Pathogenic Microorganism Interactions
The nim mutants can be used to understand pathogenic plant-microorganism interactions, and in particular to understand the processes used by the pathogenic microorganism to invade plant cells. This is because nim mutants do not mount an attack response by pathogenic microorganisms, and the unrestrained development of the pathogenic microorganism is an ideal scenario for studying its biological interaction with the host.

 Of even greater importance is the observation that a mutant host may be susceptible to pathogenic microorganisms that normally are not associated with that particular host, but rather are associated with a different one. For example, a nim mutant of Arabidopsis, such as nimI-1, -2, -3, -4, -5 or -6, is caused by a number of pathogenic microorganisms that normally only affect tobacco. , and turns out to be sensitive. Thus, the nim mutation causing the UDS phenotype results in a change in sensitivity to a certain range of pathogenic microorganisms, which is of great utility in the molecular, genetic and biochemical analysis of the host-pathogen interaction.

Example 26: Mutants nim for use in the selection of fungicides
Nim mutants are particularly useful in the selection of new micro-organism compounds of late blight), Rhizoctonia solani (the mite-footer microorganism), Pseudocercosporella herpotrichoides (the microorganism of pseudocercosporellosis), Puccinia spp. (the microorganism of rust), and Septoria nodorum. Similarly, maize nim mutants are expected to be highly sensitive to maize pathogenic microorganisms, and should therefore be useful in the selection of fungicides with activity against maize diseases.

 The nim mutants are further useful for selecting a wide range of pathogenic microorganisms and pathotypes in a heterologous host, i.e., in a host that can not normally enter the range of the species of the host of a particular pathogenic microorganism, and which can be particularly easy to handle (such as Arabidopsis). Due to its UDS phenotype, the heterologous host is susceptible to pathogenic microorganisms of other plant species, including economically important crop plant species. Thus, by way of example, the same nim mutant of Arabidopsis could be infected by a wheat pathogenic microorganism such as Erisyphe graminis (the microorganism responsible for late blight) or a pathogenic microorganism of corn, such as Helminthosporium mardis, and could be used to test the efficacy of candidate fungicides. Such an approach leads to considerable improvements in yield over currently used techniques for selection of individual plant species and different varieties of species with different pathogenic micro-organisms and pathotypes likely to exhibit different virulence on different varieties of plant species. culture. In addition, the use of Arabidopsis has advantages because of its small size and the possibility, therefore, to perform a greater number of trials with limited space resources.

Example 27: NIM1 is a homologue of IaB
A multiple sequence alignment is performed between the NIM1 and IKB protein gene products, which makes it possible to determine that the NIMi gene product is a homologue of the IKBα (FIG. 9). Sequence homology searches are performed using BLAST (Altschul et al., J. Mol Biol 215, 403410 (1990)). Multiple sequence alignment is achieved using Clustal V (Higgins et al., CABIOS 5, 151-153 (1989)), which is part of Lasergene software.
Biocomputing Software from DNASTAR (Madison, WI). The sequences used in the alignment are NIM1 (SEQ ID
NO: 3), mouse I <Ba (SEQ ID NO: 18; GenBank registration number 1,022,734), rat IKBa (SEQ ID NO: 19, GenBank registration number 57674 and X63594; Tewari et al., Nucleic Acids Res 20, 607 (1992)) and porcine IKBa (SEQ ID NO: 20, GenBank registration number
Z21968; Martin et al., EMBO J. 12, 2773-2779 (1993), registration number GenBank 517193, by Martin et al.,
Gene 152, 253-255 (1995)). The parameters used in the Clustal analysis are a gap penalty of 10, and a gap length penalty of 10. The evolution divergence distances are calculated using the PAM250 weight table (Dayhoff et al. Matrices for detecting distant relationships in Atlas of Protein Sequence and
Structure, Volume 5, Suppl. 3, MO Dayhoff, ed (National
Biomedical Research Foundation, Washington, DC, pp. 345-358 (1978)). The similarity of the residues is calculated using a modified Dayhoff table (Schwartz and Dayhoff "A model of evolutionary change in proteins", in Atlas of Protein Sequence and Structure, MO

Dayhoff, ed (National Biomedical Research Foundation,
Washington, DC), pp. 353-358 (1979); Gribskov and
Burgess, Nucleic Acids Res. 14, 6745-6763 (1986)).

Homology research indicates that similarity of NIM1 to the ankyrin domains of different proteins, including ankyrin, NF-KB and IKB. The best overall homology is homology to IKB and related molecules (Figure 9). NIM1 contains 2 serines at amino acid positions 55 and 59, serine at position 59 is in a context (D / ExxxxxS) and at an (N-terminal) position consistent with a role in inducible, dependent degradation of phosphorylation and mediated by ubiquitin. All IKBs possess these N-terminal serines, and they are necessary for inactivation of IKB and subsequent release of
NF-KB. NIM1 has ankyrin domains (amino acids 262-290 and 323-371) It is believed that the ankyrin domains are involved in protein protein interactions and represent a ubiquitous feature of the IKB and NF-KB molecules. The endpoints of IKBs may be dissimilar and NIM1 has some homology with the QL-rich region (amino acids 491-499) found on the C-terminal sites of some IKBs.

Example 28: Production of modified forms of NIMl
Changes in serine residues 55 and 59 in alanine residues
Phosphorylation of serine residues in IkB &alpha; This is necessary for stimulatory activation of Ia3a, which activates NF-κB. Mutagenesis of serine residues (S32-S36) in human IIKBα to alanine residues inhibits stimulus-induced phosphorylation, which blocks proteome-mediated degradation of IaBa (E. Britta-Mareen Traenckner et al. , EMBO J. 14: 2876-2883 (1995), Brown et al.
Science 267: 1485-1488 (1996); Brockman et al., Molecular and Cellular Biology 15: 2809-2818 (1995); Wang et al., Science 274: 784-787 (1996)).

This modified form of IKBa acts as a dominant-negative form by retaining NF-KB in the cytoplasm, which blocks downstream signaling events. On the basis of sequence comparisons between the
NIM1 and IKB, NIMi serine 55 (S55) and 59 (S59) are homologous to S32 and S36 in human isba. To synthesize dominant-negative forms of NIM1, serines at amino acid positions 55 and 59 are mutagenized to alanine residues. This can be done by any method known to those skilled in the art, for example by using the QuikChange Site Directed Site Directive mutagenesis kit.
Mutagenesis Kit (# 200,518: Stratagene).

Using a complete NIM1 cDNA (SEQ ID NO: 21) comprising 42 bp of the 5'-untranslated (UTR) sequence and 187 bp of the 3'-UTR sequence, the mutagenized construct can be made according to the instructions. from the manufacturer, using the following primers (SEQ ID NO: 21, positions 192-226): 5'-CAA CAG CTT CGA AGC CGT TGA CGC
GCC GGA TG-3 '(SEQ ID NO: 32) and 5'-CAT CCG GCG CGT CAA
AGA CGC CTT CGA AGC TCT TG-3 '(SEQ ID NO: 33), where the underlined bases denote mutations. The strategy is as follows: the NiMi cDNA cloned in the vector pSE936 (Elledge et al., Proc Nat Sci USA 88: 1731-1735 (1991)) is denatured, and the primers containing modified bases are ringed. The DNA polymerase (Pfu) lengthens the primers by a non-strand displacement, which leads to cut circular strands. DNA is digested with restriction endonuclease
DpnI, which cuts only the methylated sites (non-mutagenized matrix DNA). The remaining circular dsDNA is transformed into E. coli strain XL1-Blue. The plasmids from the colonies obtained are extracted and sequenced to check the presence of the mutated bases and to confirm that no other mutation has taken place.

 The mutagenized NIM1 cDNA was digested with the restriction endonuclease EcoRI and cloned into pCGN1761 as transcriptional control of the CaSM mosaic virus 35S promoter. The transformation cassette comprising the 35S promoter, the NIM1 cDNA and the tml terminator is released from pCGN1761 by partial digestion with the restriction enzyme XbaI and ligated into the XbaI site of the dephosphorylated pCIB200. The sequences SEQ ID NO: 22 and 23 respectively show the coding sequence of the DNA and the coded amino acid sequence of this modified form of the Niai gene.

 The present invention also relates to modified forms of NIAI alleles, wherein the coding sequence of such an allele hybridizes under the following conditions to the coding sequence SEQ ID NO: 22: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; sodium lauryl sulphate 7%; EDTA 1 mM; 250mM sodium chloride at 550C for 18-24 hours, and wash in 6XSSC for 15 minutes (X3), 3XSSC for 15 minutes (X1) at 55 ° C. In these embodiments, the NIMi alleles that hybridize at sequence No. 22 under these conditions are modified so that the coded product has alanines instead of serines at the amino acid positions which correspond to positions 55 and 59 of the sequence SEQ ID NO: 22.

Example 29: Production of Modified Forms of NIMl
N-terminal deletion
The deletion of amino acids 1-36 (Brockman et al.,
Sun et al.) Or 1-72 (Sun et al.) Human IKBa, which comprises K21, K22, S32 and S36, leads to a phenotype.
IKBa dominant-negative in transfected human cell cultures. An N-terminal deletion, approximately 125 first amino acids of the coded product of the NIM1 cDNA, removes 8 lysine residues, which can serve as a potential site for ubiquitinization, and also removes the putative phosphorylation sites in
S55 and S59 (see Example 2). This modified gene product can be obtained by any means known to those skilled in the art. For example, using the method of Ho et al. Gene 77: 51-59 (1989), a form of NIM1 can be produced in which the DNA encoding approximately the first 125 acids is deleted. The following primers produce a 1612 bp PCR amplification product (SEQ ID NO: 21: 418 to 2011): 5'-gg aat TCA-ATG GAT TCG
GTT GTG ACT GTT TTG-3 '(SEQ ID NO: 34) and 5'-gga att cTA
CAA ATC TGT ATA CCA TTG G-3 '(SEQ ID NO: 35), wherein the synthesis initiation codon is underlined (ATG), the binding sequence corresponding to EcoRI being lowercase.

Fragment amplification utilizes a reaction mixture comprising from 0.1 to 100 ng of the template DNA, 10 mM Tris pH 8.5 / KCl / 50 mM / 2 mM MgCl 2 / 0.001% gelatin / 0, 25 mM of each dNTP / 0.2 mM of each primer and 1 unit of rTth DNA polymerase, with a final volume of 50 ml, as well as a Perkin PCR amplification machine
Elmer Cetus 9600. PCR amplification conditions are as follows: 94 "C 3 minutes: 35X (940C 30 seconds 52" C 1 minute: 720C 2 minutes): 720C 10 minutes. The product of the PCR amplification is directly cloned into the vector pCR2.1 (Invitrogen). The insert produced by PCR amplification in the PCR vector is released by restriction endonuclease digestion EcoRI and ligated into the EcoRI site of the dephosphorylated pCGN1761, as transcriptional regulation of the double 35S promoter. The construct is sequenced to verify the presence of the starting synthetic ATG and to confirm that no further mutation occurs during the PCR.

The transformation cassette comprising the 35S promoter, the modified NIM1 cDNA and the tml terminator is released from the pCGN1761ENX by partial digestion with the restriction enzyme XbaI and ligated into the site.
XbaI of pCIB200. The sequences SEQ ID NOs: 24 and 25 respectively show the DNA coding sequence and the encoded amino acid sequence of a modified form of the NIM1 gene having an N-terminal amino acid deletion.

The present invention also relates to modified forms of NIM1 alleles, wherein the coding sequence of such an allele hybridizes under the following conditions to the coding sequence SEQ ID NO: 24: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; sodium lauryl sulphate 7%; EDTA 1 mM; 250mM sodium chloride at 550C for 18-24 hours, and wash in 6XSSC for 15 minutes (X3), 3XSSC for 15 minutes (X1) at 55 ° C. In these embodiments, the NiMi alleles that hybridize to the sequence SEQ ID NO: 24 under these conditions are modified so that the coded product has a deletion
N-terminus that removes lysine residues that may serve as potential ubiquitinization sites in addition to serines at the amino acid positions corresponding to positions 55 and 59 of the wild-type gene product.

Example 30: Production of Modified Forms of NIMl
C-terminal deletion
It is believed that the deletion of amino acids 261-317 of human IKBα leads to enhancement of intrinsic stability by blocking the constitutive phosphorylation of serine and threonine residues at the C-terminal site. A region rich in serine and threonine is present on the amino acids 522-593 of the site
C-terminal of NIM1. The region coding for the C-terminal site of the NIM1 gene can be modified by deletion of the nucleotide sequences which encode amino acids 522-593.

Using the method of Ho et al. (1989), the region encoding the C-terminal site and the 3'UTR site of the NIMi cDNA (SEQ ID NO: 21: 1606-2011) is deleted by PCR amplification, giving a 1623 bp fragment per use. the following primers: 5'cggaattcGATCTCTTTAATTTGTGAATTT C-3 '(SEQ ID NO: 36) and 5'ggaattcTCAACAGTT CATAATCTGGTCG-3' (SEQ ID NO: 37), where a synthetic stop codon is underlined (TGA) on the complementary strand ), and the EcoRI binding sequences are in lower case. The constituents of the PCR amplification reaction are as described above, and the cycling parameters are as follows: 94 ° C 3 minutes: 30X (94 ° C 30 seconds: 52 ° C 1 minute: 720C 2 minutes) 720C 10 minutes] The product of the PCR amplification is cloned directly into the PCR vector: 1 (Invitrogen) The PCR produced insert of the PCR vector is released by restriction endonuclease digestion
EcoRI and ligated into the EcoRI site of the dephosphorylated pCGN1761, which contains the double 35S promoter. The construct is sequenced to verify the presence of the synthetic stop codon in the frame, and to confirm that no other mutation has occurred during the PCR.

The transformation cassette, which comprises the promoter, the modified NIMl cDNA and the tml terminator, is released from pCGN1761 by digestion with the partial restriction enzyme XbaI and ligated into the XbaI site of the dephosphorylated pCIB200. The sequences SEQ ID NO: 26 and 27 respectively show the coding sequence of the DNA and the coded amino acid sequence of a modified form of the NIM1 gene having a Cterminal amino acid deletion.
the present invention also relates to modified forms of NIAI alleles, wherein the coding sequence of such an allele hybridizes under the following conditions to the coding sequence SEQ ID NO: 26: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; sodium lauryl sulphate 7%; EDTA 1 mM; 250mM sodium chloride at 55 ° C for 18-24 hours, and wash in 6XSSC for 15 minutes (X3), 3XSSC for 15 minutes (X1) at 550 ° C. In these embodiments, the NIM1 alleles that hybridize SEQ ID NO: 26 under the above conditions are modified so that the coded product has a C-terminal deletion which removes serine and threonine residues.

Example 31: Production of modified forms of NIM1
N-terminal / C-terminal deletion chimera
A form of N-terminally and C-terminally deleted NIM1 is produced using a unique KpnI restriction site at position 819 (SEQ ID NO: 21). The N-terminal deletion form (Example 3) is digested with EcoRI / KpnI restriction endonucleases, and the 415 bp fragment corresponding to the modified N-terminal site is recovered by gel electrophoresis. Similarly, the C-terminal deletion form (Example 4) is subjected to restriction endonuclease digestion EcoRI /
KpnI, and the 790 bp fragment corresponding to the modified Cterminal site is recovered by gel electrophoresis.

The fragments are ligated to 150C, digested with EcoRI to remove the EcoRi concatamers, and cloned into the EcoRI site of the dephosphorylated pCGN1761. The N / C-terminal deletion form of NIM1 falls within the transcriptional regulation of the double 35S promoter. Similarly, a chimeric form of MIMI is produced, which consists of putative mutagenized phosphorylation sites.
S55 / S59 (Example 2) fused to the C-terminal deletion (Example 4). The synthetic product is produced as described above. The constructs are sequenced to verify the fidelity of the initiation codon and the stop codon and to confirm that no mutations occurred during cloning. The different transformation cassettes, comprising the 35S promoter, the NIMl chimera and the tml terminator, are released from pCGN1761 by partial digestion with the restriction enzyme XbaI and ligated into the XbaI site of the dephosphorylated pCIB200.

The sequences SEQ ID NO: 28 and 29 respectively show the coding sequence of the DNA and the coded amino acid sequence of a modified form of the NIMi gene comprising amino acid deletions that are both N-terminal and
C-terminal.

 The present invention also relates to modified forms of NIM1 alleles, wherein the coding sequence of such an allele hybridizes under the following conditions to the coding sequence SEQ ID NO: 28: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; sodium lauryl sulphate 7%; EDTA 1 mM; 250mM sodium chloride at 550C for 18-24 hours, and wash in 6XSSC for 15 minutes (X3), 3XSSC for 15 minutes (X1) at 55 ° C. In these embodiments, the NIMI alleles that hybridize to the sequence SEQ ID NO: 28 under the above conditions are modified so that the coded product has both an N-terminal deletion, which eliminates lysine residues that can serve as potential sites of ubiquitination in addition to serines on the amino acid positions corresponding to positions 55 and 59 of the wild-type gene product, which a C-terminal deletion, which removes the serine and threonine residues.

Example 32: Production of modified forms of NIM1
Ankyrine domains
NiMi has homology with the ankyrin units at approximately amino acids 103262. Using the method of Ho et al. (1989), the DNA sequence encoding the putative ankyrin domains (SEQ ID NO: 2: 3093-3951) is amplified by PCR (conditions: 94 ° C, 3 minutes: 35X (940C 30 seconds: 62 "C 30 seconds 72 "C 2 min): 720 C 10 min) from the NiMi cDNA (SEQ ID NO: 21: 349-1128) using the following primers: 5 'ggaCatcaA GACTCCAACAACACCGCCGC-3' (SEQ ID NO: 38 ) and 5 'ggaattcTCAACCTTCCAAAGTTGCTTCTGATG-3' (SEQ ID NO: 39) The product obtained is digested with the restriction endonuclease EcoRI and then spliced into the EcoRI site of the dephosphorylated pCGN1761 under the conditions of the transcriptional regulation of the double 35S promoter. The construct is sequenced to verify the presence of the synthetic initiation codon (ATG), an in-frame stop codon (TGA), and to confirm that no other mutations occurred during amplification. PCR The transformation cassette, which includes the 35S promoter, the ankyrin domains and the tml terminator is released from the pC GN1761 by partial digestion with the restriction enzyme XbaI and ligated into the XbaI site of the dephosphorylated pCIB200. The sequences SEQ ID NOs: 30 and 31 respectively show the coding sequence of the DNA and the coded amino acid sequence of the ankyrin domain of the NIAI.

 The present invention also relates to modified forms of NIM1 alleles, wherein the coding sequence of such an allele hybridizes under the following conditions to the coding sequence SEQ ID NO: 30: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; sodium lauryl sulphate 7%; EDTA 1 mM; 250mM sodium chloride at 550C for 18-24 hours, and wash in 6XSSC for 15 minutes (X3), 3XSSC for 15 minutes (X1) at 55 ° C. In these embodiments, the NiMi alleles that hybridize SEQ ID NO: 30 under the above conditions are modified so that the coded product consists essentially of the ankyrin domains of the wild-type gene product.

Example 33 Synthesis of Chimeric Genes
To increase the probability of appropriate spatial and temporal expression of modified forms of NIM1, the 4407 bp HindIII / BamHI fragment (SEQ ID NO: 2, bases 1249-5655), and / or an EcoRV / fragment is used.
5655 bp BamHI (SEQ ID NO: 2: bases 1-5655) containing the NIM1 promoter and gene, to create modified forms of NIM1 in Examples 28-32 above. Although the synthesis steps may differ, the concepts are comparable to the examples described above in the invention. A strong overexpression of the modified forms can be potentially lethal. Thus, the modified forms of the NIM1 gene described in Examples 28 to 32 may be placed within the framework of the regulation of promoters other than the endogenous NIM1 promoter, including but not limited to the promoter nos or the small subunit of the Rubisco promoter . Similarly, the modified NIM1 forms can be expressed for the regulation of the PR-1 pathogenic microorganism response promoter (US Patent No. 5,614,395) .This expression allows strong expression of the modified forms NIM1 only in presence of an attack by pathogenic microorganisms, or other conditions of activation of SAR.In addition, disease resistance may be manifest in transformants that express modified forms of NIMl in the context of regulation of the PR-1 promoter after treatment with concentrations of SAR activators (ie, BTH or INA) that do not normally activate SAR, thus activating a feedback loop (Weymann et al.

(1995) Plant Cell 7: 2013-2022).

Example 34 Transformation of Modified Forms of NIM1 in Arabidopsis thaliana
The products of synthesis obtained in Examples 28 to 33 are sent to Arabidopsis thaliana by electroporation in strain GV3101. These constructs are used to transform the Arabidopsis Col-O and Ws-O ecotypes by vacuum infiltration (Mindrinos et al., Cell 78, 1089-1099 (1994)) or by conventional root transformation techniques. Seeds from these plants are harvested, and allowed to germinate on agar plates with kanamycin (or other suitable antibiotic) as the selection agent. Other seedlings, which are transformed with cosmid DNA, can detoxify the selection agent and survive. The seedling plants that survive the selection are transferred to the soil and tested for the CIM (Constitutive Immunity) phenotype. Plants are evaluated for observable phenotypic differences from wild-type plants.

Example 35: Evaluation of the CIM Phenotype in Plants Transformed by Modified Forms of NIM1
One leaf of each primary transformant is harvested, RNA is isolated (Verwoerd et al., 1989, Nuc Acid
Res, 2362), and tested to determine constitutive PR-1 expression by RNA blot analysis (Uknes et al., 1992). Each transformant is evaluated for its improved resistance response to disease, indicative of an analysis of constitutive expression of SAR (Uknes et al., 1992).

Conidial suspensions at 5-10.104 spores / ml from 2 compatible isolates of P. parasitica are prepared.
Emwa and Noco (i.e., these fungal strains cause disease on wild-type plants, Ws-0 and Col-O, respectively), and the transformants are sprapped with the appropriate isolate, depending on the ecotype of the transformant. The inoculated plants are incubated in the presence of high humidity for 7 days.

The plants are grafted on the 7th day, and a single leaf is harvested for RNA analysis, which uses a probe that provides a means to measure fungal infection.

Transformants that exhibit a CIM phenotype are conducted at T1 generation, and homozygous plants are identified. The transformants are subjected to a disease resistance test bacterium, as described below. The fungal infection with Noco and Emwa is repeated, and the leaves are stained with lactophenol blue to identify the presence of fungal hyphae, as described in Dietrich et al. (1994). Transformants are infected with pathogenic bacteria
Pseudomonas syringae DC 3000 to evaluate the resistance spectrum, which is highlighted as described in
Uknes et al. (1993). Uninfected plants are evaluated for free AS and glucose-conjugated SA, and the leaves are stained with lactophenol blue to assess the presence of microscopic lesions.

Resistant plants are sexually cross-linked with SAR mutants such as NahG (US Patent No. 5,614,395) and indri, to establish the epistatic relationship of the resistance phenotype with other mutants, and to evaluate how these dominant-negative mutants NIMl can influence the SA dependent feedback loop.

Example 36: Isolation of NIMl homologs
Using as a probe the cDNA of NIMl (SEQ
ID NO: 21), Arabidopsis NIM1 homologues are identified by screening genothèques or cDNA libraries from different cultures, for example, but not limited to, those mentioned elsewhere in Example 11. conventional techniques to achieve this include hybridization screening of DNA libraries on plates (plaques or colonies, see for example Sambrook et al., Molecular
Cloning, eds, Cold Spring Harbor Laboratory Press.

(1989)), and PCR amplification using oligonucleotide primers (see, for example, Innis et al., PCR Protocols, Guide to Methods and Application, eds, Academic Press (1990)). The identified homologs are genetically engineered into the expression vectors of the present invention and transformed into the above cultures. Transformants are assessed for their improved resistance to disease by using pathogenic microorganisms of the crop plant being tested.

 NIM1 homologs in the genomes of cucumber, tomato, tobacco, maize and barley are detected by DNA blot analysis. The genomic DNA is isolated from cucumber, tomato, tobacco, corn, wheat and barley, digested with restriction enzymes BamHI, HindIII, XbaI or SalI, electrophoretically separated on 0.8% agarose gels and transferred to a nylon membrane by capillary transfer. After crosslinking with UV to fix the DNA, the membrane is hybridized under low stringency conditions [1% BSA; 520 mM NaPO4, pH 7.2; sodium lauryl sulphate 7%; EDTA 1 mM; 250 mM sodium chloride) at 55 ° C for 18 to 24 hours using 32 P-radiolabeled Arabidopsis thaliana NIM1 cDNA After hybridization, the blots are washed under low stringency conditions [6XSSC for 15 min. minutes (X3), 3XSSC for 15 minutes (X1) at 55 ° C; 1XSSC is 0.15 M NaCl, 15 mM Na citrate (pH 7.0)], and exposed to X-ray film to visualize the bands corresponding to NIM1.

In addition, expressed sequence markers (ESTs), identified by analogy with the NIM1 gene, can be used to isolate homologs. For example, several rice expressed sequence markers (ESTs) have been identified, which are similar to the NIM1 gene. A multiple sequence alignment is synthesized using Clustal 5 (Higgins, Desmond G. and Paul M. Sharp (1989), Fast and sensitive multiple sequence alignments on microcomputer, CABIOS 5: 151153) as part of the Lasergene package. Biocompu ting Software by DNA * (1228 South Park Street, Madison
Wisconsin, 53715) for Macintosh (1994). Certain regions of the NIM1 protein are homologous, for the amino acid sequence, to 4 different cDNA protein products, in rice. Homologies are identified by using NIMl sequences as part of a GenBank BLAST search. Comparisons of regions of homology in NNM1 and in rice ANDc products are shown in Figure 8 (see also sequences
SEQ ID NO: 3 and SEQ ID NO: 4 to 11). The NIM1 protein fragments have from 36 to 48% identical amino acid sequences with the four rice products. These
EAST rice can be especially useful for isolating homologues of NIMl from other monocots.

 Homologs can be obtained by PCR amplification. In this process, comparisons are made between known homologs (eg rice and Arabidopsis). PCR regions are then used to make regions having a high similarity or identity with amino acids and DNA. Regions rich in amino acid residues M and W are best followed by regions rich in amino acid residues F, Y, C, H, Q, K and E, since these amino acids are coded by a limited number of codons. Once an appropriate region has been identified, primers are prepared for this region, with a variety of substitutions on the position of the third codon. This diversity of substitutions on the third position may be limited depending on the target species. For example, because corn is rich in GC, primers are developed that use G or C, if possible, in the third position.

 The PCR amplification reaction is carried out from cDNA or genomic DNA under many standard conditions. When a band is apparent, it is cloned and / or sequenced to determine if it is a homologue of the NIM1.

Example 37 Expression of Modified Forms of NIM1 in Culture Plants
Synthetic products that confer a phenotype
CIM at Col-O and Ws-0 plants are transformed in culture plants for evaluation. Alternatively, modified native NIM1 genes, isolated from culture plants in the above Example, are returned to their original plants. Although the NIM1 gene can be inserted into all plant cells falling into these broad categories, it is particularly useful in plant cells of crop plants, including the following: rice, wheat, barley, rye, corn, potato, carrot , sweet potato, sugar beet, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, aubergine, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soy, tobacco, tomato, sorghum and sugar cane. Transformants are assessed for improved disease resistance. In a preferred embodiment of the invention, the expression of the modified form of the NIMi gene is at a level which is at least two times higher than the level of expression of the native NIM1 gene in wild plants, and is preferably 10 times higher than the level of expression of the wild type.

 Of course, the invention is not limited to the embodiments described above and shown, from which we can provide other modes and other embodiments, without departing from the scope of the invention. .

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LIST OF SEQUENCES (1) GENERAL INFORMATION:
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(D) SOFTWARE: Patentîn Release N 1.0, version N 1.30
(ii) TITLE OF THE INVENTION: METHODS OF USING THE NIM1 GENE
TO CONFER THE VEGETABLES WITH DISEASE RESISTANCE
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(iii) Hypothesis: No
(iv) ANTI-SENS: NO
(xi) DESCRIPTION OF THE SEQUENCE SEQ ID NO: 1 TGATCATGAA TTGCGTGTAG GGTTGTGTTT TAAAGATAGG GATGAGCTGA AGAAGGCGGT 60 GGACTGGTGT TCCATTAGAG GGCAGCAAAA GTGTGTAGTA CAAGAGATTG AGAAGGACGA 120
GTATACGTTT AAATGCATCA GATGGAAATG CAATTGGTCG CGTCGGGCAG ATTGAATAGA 180 AGAACATGGA CTTGTTAAGA TAACTAAGTG TAGTTGGTCC ACATACTTGT TGTTCTATTA 240
AGCCGGAAAA CTTCAACTTG TAATTTGCAG CAGAAGAGAT TGAGTGTCTG ATCAGGGTAC 300
AACCCACTCT AACAGCAGAG TTGAAAAGTT TGGTGACATG CTTAAAACTT CAAAGCTGCG 360
GGCAGCAGAA CAGGAAGTAA TCAAAGATCA GAGTTTCAGA GTATTGCCTA AACTAATTGG 420
CTGCATTTCA CTCATCATAT GGGCTACTTG TGGACTGCAA TATGAGCTTT TCCCTAATCC 480
TGAATTTGCA TCCTTCGGTG GCGCGTTTTG GGCGTTTCCA CAGTCCATTG AAGGGTTTCA 540 ACACTGTAGA CCTCTGATCA TAGTGGATTC AAAAGACTTG AACGGCAAGT ACCCTATGAA 600
ATTGATGATT TCCTCAGGAC TCGACGCTGA TGATTGCTTT TTCCCGCTTG CCTTTCCGCT 660
TACCAAAGAA GTGTCCACTG ATAGTTGGCG TTGGTTTTCTCT ACTAATATCA GAGAGAAGGT 720
AACACAAAGG AAAGACGTTT GCCTCGTCTC CAGTCCTCAC CCGGACATAG TTGCTGTTAT 780
TAACGAACCC GGATCACTGT GGCAAGAACC TTGGGTCTAT CACAGGTTCT GTCTGGATTG 840 TTTTGCTTA CAATTCCATG ATATTTTTGG AGACTACAAC CTGGTGAGCC TTGTGAAGCA 900
GGCTGGATCC ACAAGTCAGA AGGAAGAATT TGATTCCTAC ATAAAGGACA TCAAAAAGAA 960
GGACTCAGAA GCTCGGAAAT GGTTAGCCCA ATTCCCTCAA AATCAGTGGG CTCTGGCTCA 1020 TGACCAGTGG TCGGAGATAT GGAGTCATGA CGATAGAAAC AGAAGATTTG AGGGCAATTT 1080 GTGAAAGCTT TCAGTCTCTT GGTCTATCAG TGACAGCGAA CGCACCTGCA CATGTGGGAA 1140 GTTTCAATCG AAGAAGTTTC CATGTATGCA CCCAGAAATG GTGCAAAGGA TTGTTAACTT 1200 GTGTCATTCA CAAATGTTGG ATGCAATGGA GCTGACTAGG AGAATGCACC TTACACGCCC 1260 ACTCAGTCTT CTCTTATCTC TAGACCTGAA ACTAACTTCC TGTGTAATTC GAGTTACAAA 1320
AGGTTAAAGG AAGAATTAGG AAGATACATA TAACATGAAT GTTGCCAGAA GTTCAGGGAA 1380 CTTGAATATT CTTTTGGTTC TTGGTGGAAA ATATCCAACA GATGAACAAT TTGACATTAT 1440
TTCACACTTT GATTCTAGCA ACTCTGTAAC ACCATCATGG GTTATTGTTG ATGTACATAA 1500
ATATATATTA CAAATCTGTA TACCATTGGT TCAAATTGTT ACAACATTTG TTTGAAGCAC 1560
ACCTGCAGCA ATAATACACA GGATGCAAAA CGAAGAGCGA AACTATATGA CGCCAACGAT 1620
AGACATAAAC AGTTACAGTC ATCATGAAAA CAGAATTATA TGGTACAGCA AAAATTACAC 1680
TAAGAGGCAA GAGTCTCACC GACGACGATG AGAGAGTTTA CGGTTAGACC TCTTTCCACC 1740
GGTTGATTTC GATGTGGAAG AAGTCGAATC TGTCAGGGAC GAATTTCCTA ATTCCAAATT 1800
GTCCTCACTA AAGGCCTTCT TTAGTGTCTC TTGTATTTCC ATGTACCTTT GCTTCTTTTG 1860
TAGTCGTTTC TCAGCAGTGT CGTCTTCTCC GCAAGCCAGT TGAGTCAAGT CCTCACAGTT 1920
CATAATCTGG TCGAGCACTG CCGAACAGCG CGGGAAGAAT CGTTTCCCGA GTTCCACTGA 1980 TGATAAAAAA AACAAGGTCA GACAGCAAGT AACAAAACCA TGTTTAAAGA TCATTTAGTT 2040
TTGTTTTTTG TGATAAGGAG TCCGATGAAG TGGGTGAGAA TCCATACCGG TTTTAGAAAG 2100
CGCTTTTAGT CTACTTTGAT GCTCTTCTAG GATTCTGAAA GGTGCTATCT TTACACCCGG 2160
TGATGTTCTC TTCGTACCAG TGAGACGGTC AGGCTCCAGG CTAGTCACTA TGAACTCACA 2220
TGTTCCCTTC ATTTCGGCGA TCTCCATTGC AGCTTGTGCT TCCGTTGGAA AAAGACGTTG 2280 AGCAAGTGCA ACTAAACAGT GGACGACACA AAGAATAGTT ATCATTAGTT CACTCAGTTT 2340 CCTAATAGAG AGGACATAAA TTTAATTCAA ACATATAAGA AATAAGACTT GATAGATACC 2400
TCTATTTTCA AGATCGAGCA GCGTCATCTT CAATTCATCG GCCGCCACTG CAAAAGAGGG 2460
AGGAACATCT CTAGGAATTT GTTCTCGTTT GTCTTCTTGC TCTAGTATTT CTACACATAG 2520
TCGGCCTTTG AGAGAATGCT TGCATTGCTC CGGGATATTA TTACATTCAA CCGCCATAGT 2580
GGCTTGTTTT GCGATCATGA GTGCGGTTCT ACCTTCCAAA GTTGCTTCTG ATGCACTTGC 2640
ACCTTTTTCC AATAGAGATA GTATCAATTG TGGCTCCTTC CGCATCGCAC CAACATGAAG 2700
CACCGTATAT CCCCTCGGAT TCCTATGGTT GACATCGGCA AGATCAAGTT TTAAAAGATC 2760
TGTTGCGGTC TTCACATTGC AATATGCAAC AGCGAAATGA AGAGCACACG CATCATCTAG 2820
ATTGGTGTGA TCCTCTTTCA AAAGCAACTT GACTAACTCA ATATCATCCG AGTCAAGTGC 2880
CTTATGTACA TTCGAGACAT GTTTCTTTAC TTTAGGTACC TCCAAACCAA GCTCTTTACG 2940
TCTATCAATT ATCTCTTTAA CAAGCTCTTC CGGCAATGAC TTTTCAAGAC TAACCATATC 3000
TACATTAGAC TTGACAATAA TCTCTTTACA TCTATCCAAT AGCTTCATAC AAGCTTTACC 3060
ACATATATTA GCAAGCTTGA GTATAACCAA TGTGTCCTCT ATAACAACTT TGTCTACAAC 3120
GTCCAATAAG TGCCTCTGAA ATACAAATAC AAGTACTCAA GTAAGAACAT ATTCATGAAT 3180 GTGTAACCAT AGCTTAATGC AGATGGTGTT TTACCTGATA GAGAGTAATT AATTCAGGGA 3240 TCTTGAAGAT GAAAGCCAAA TAGAGAACCT CCAACATGAA ATCCACCGCC GGCCGGCAAG 3300
CCACGTGGCA GCAATTCTCG TCTGCGCATT CAGAAACTCC TTTAGGCGGC GGTCTCACTC 3360
TGCTGCTGTA AACATAAGCC AAAACAGTCA CAACCGAATC GAAACCGACT TCGTAATCCT 3420
TGGCAATCTC CTTAAGCTCG AGCTTCACGG CGGCGGTGTT GTTGGAGTCT TTCTCCTTCT 3480
TAGCGGCGGC TAAAGCGCTC TTGAAGAAAG AGCTTCTCGC TGACAAAACG CACCGGTGGA 3540
AAGAAACTTC CCGGCCGTCG GAGAGAACAA GCTTAGCGTC GCTGTAGAAA TCATCCGGCG 3600
AGTCAAAGAC GGATTCGAAG CTGTTGGAGA GCAATTGCAG AGCAGATACA TCAGGTCCGG 3660
TGAGTACTTG TTCGGCGGCC AGATAAACAA TAGAGGAGTC GGTGTTATCG GTAGCGACGA 3720
AACTAGTGCT GCTGATTTCA TAAGAATCGG CGAATCCATC AATGGTGGTG TCCATCAACA 3780
GGTTCCGATG AATTGAAATT CACAAATTAA AGAGATCTCT GCTAATCAAC GAAGAGACCT 3840
TATCAACTGG ATTTGGTTAA AGATCGAAGA TAACCATTGA CGAGCAGAGC CAAGTCAAGT 3900
CAACGAGAGT GGTGGTGAGA TATGAAGAAG CATCCTCGTC CCACGGTTTA CATTTCACCA 3960
AAACCGGTAA ATTTCCAGGA AAGGAATCTT TGTCAGAGAT CTTTTTTAAA AAGATATAAC 4020
AGGAAGCTAA ACCGGTTCGG GTTATAAATG TTAGTATTTA TACCGGAGAC ATTTTGTGTT 4080 GCTAATTTTT GTATATGAGA AGTTCAATCC GGTTCGGTAA GCCCCTGAAC CAAACTAGAT 4140 TTGGAGATGA TATAAATATA TAAAATTTAT TTTTCATCCG GTTCGTTATT TTCATATAAA 4200
TATATAAATA TTATTTTTTA AATTTAAGAA TTAGATTTAC ATGTGAAAGT TACATTTCTG 4260
TTTATTTTCT TTGAAGTAAA ATGATAAAGG GAACGATATAT TAAGTTTCAT GCTTTATTCA 4320
CATAAGTTTT GTAATGTATA TTATATTTTT CGTTTATTGA AAAAGTAATT TTCAGTGTTC 4380
AGCATGTTTA CACTATAATT AAATCAAGTC GAATATTTCC TGGAACTATT CTCCTTGTTC 4440
TATAGCAAAT GAAAACGCTC TTCACAACAA AATCATTATA GATATAGGAA TAAATTACAT 4500
TAAAAACATG AAAGTCATAA TGAATATATT TTTTTAATTA GGATTTGATT TAAAAACAAT 4560
TATTGTATAC ATATAAAAGA CTTCTTTAGT TATTTGCCTT CAACTTCTCG TTCTGAATCA 4620
TGCGATAAAT CAGCTTTTTC AATAACTACG ACGTAAAAGC AAATTCATAA CACGTCTAAA 4680
CAAATTTGGC TCATCCTTCA CTTGATTGGT GTTTTCCGGA CTCGATGTTG CTGGAAACTG 4740
AGAAGAAGAA GGAATCTGCA TAATCACCTC TTGGTTCCTC ACCGGTAGAC TCATTTTGTT 4800
GGATCGAAAA CGATCGAGAT CAGAAAATGA AAAGATAGGT TAAAGATGCC TATGAATACA 4860
ACAACGTAAG ATTATGTTGA ATAAACAGAG TACTTTATAT AGGAGTTATA ATAAGGTAAA 4920
TAAATTATTG CTTTCCGCGT TTTTACTTT TGTATTTCTT AAATGATAAG TTAAATTAGG 4980
ATAAGATTTG TATGATTTTA AGTAAATTTA CAATAACTCT CTATAACTCA ATAGCATCAC 5040
ATATTTAATT AATTTTACTA ATTATCTTTT GAACAATTTT ATGAAATAGT TTTCTTTTAA 5100
TTAATTTTTT AAAATGATAT ATTATAAAAT TTAATTGAAT CAATCTGATA TAATTTTTTT 5160
ATCTTCTACC ATCTATTATA GTTGATAAAT ATTGTGATAA ACTTTAGATA AACACCCAAT 5220 TGCCAAATAT TTAATAAATT TTGTGTACCA TGCGTTTTTT TTGGAGAATA TATATACGTG 5280 GACAGCATAC CGTACATATA TTGTATAAAA GCTTATAAAA CATAGATACG GGTTATATTG 5340
GTAAGCTATA AATATATTA AACAATAGTA AGATATTACG TGTTGTGTCT AAATATGTGT 5400
TGCTTTAGAT ATTATGTATA TCTAATATAT TAAAATATCT TTTATTAACT AATATATTAT 5460
TTAAGAGAGA AAATTGGGAC ACTATTTTCT ATACAGTAAC TGTTTTCAAC TATAAACAGG 5520
AACCCTTGAT ATAATAAAAT AACTAGCCAA AAAATCAGAT TAAATATTCA TAAAACAATG 5580
TTTGGTATTA TTACATAAAC CTAAGAAACA AAATTCAATA TTCCTTTTTA CCTTATAAAA 5640
AACAATTAAA CATCACTAGA TATATTTATG CCCCACAATG AGCGAGCCAA TTGAGACTTG 5700
AGACTTGAGA TCCTTGTCAA CTACGTTTGC ATTTGTCGGC CCATTTTTTT TATTTTTTTT 5760
TTAAAGTGTC GGCCCGTTGT TTCTTCCGTT CAGATCAACC CTCTCGTAAT CAGAACAAAA 5820
CGGAAAACAA ACGAAAGAAC AATCAGATCC CTCTTTTTTT GCATAAACTA AATTCAACTT 5880
CTCTGCGTTT ATGTTGTAGA GGCAACCACG ATCACTACTA CGAAACAATA CAACGTCGTT 5940
GCTTGGAGTC CACGTAATCA AATCTACTCC AATGTTTTTA ATATCTTTCA CTTTAACCCA 6000
CGACTTTTCA AAACTGCTCT TTAAAACCCA TAACTCGTGA ACATCTTCTT GATCTTTGTT 6060
TGTCCACTGA CGAATAGCAC CTAGCTTCCC TTCGTATCTG ACTAATCCTG AGAAAACATC 6120
AGAGTTCGGA GTATGGAAGA AGGACCAAGT TTCGGTTTTG AGACAAAACC GGATCACATT 6180
GTTGTTCCGT GATATCCAAT GCAAGAACCC CGAAACTTGT ATCGGGTTGG AAAAAATTAA 6240 TCTGTCTGTT TTTGGTAGAC GCAAATTTTC TAATCTCTTC CAGGTAAACG AATCAGAATC 6300 GAAAACTTCG CACATAAAAG TTCTGTGATT CAAATGGTAG ATACCCCGAG ACATACACAT 6360 ACGCCGAGAC TGCGAAAGCC TTTGTATTTT ATACCGGAAA GGGTTCAATC CGATTACCGC 6420
TAAACCCAAT GACATATCCC AACCCTTCAC TTCTGGCTTT GGTATGACCT GATACTGTTT 6480
AGTGGTTGGT TTGAAGACTA TGTATCCACG TGATGGTTTT GTATACTTAA CACAAAGCAA 6540
TATCCCATGA CTTGCATCAC AAGCTTCGAT CTTTATCATT CCGGGTGGCA GAAAGTCGAT 6600
GGAGACTCCA TTGTTTTGTA AATCACTCCT CTCATGGACA AAACTGGTTC GAAGTTCGTG 6660
TCCTTTTACT ATGTAGTGTT GTATGAAGTA TCCCGAATATA CGATTGGTTC TAAGGAGATT 6720
AAGATTGACA AACCATGACT CGTAGCTTCT CTTGTTGCAC TCTTTATTCA GGAGCCTGAA 6780
TTTTCCGATT TTTGACGCCG GAAGATAAGA AAGAAATTCT TGGATCATGT CTTGATTTAT 6840
CACCGGAGAA CTCATGATCC TGTCGGGAAT AAAGAGATGA GCACGATCAC TGAATGAGAA 6900
ATGAAAAAAT CAGGATCGGT AGAGAACAAC TTATGATGAA TAAAGTGTTT ATATATCCTT 6960
TCTTTGTTTA AGGAAAGTAT CAAAATTTGC CTTTTTCTTC GCTAGTCCTA AAACAAACAA 7020
ATTAACCAAA AGATAAAATC TTTCATGATT AATGTTACTT GTGATACCTT AAGCCAAAAC 7080
TTTATCTTTA GACTTTTAAC CAAATCTACA GTAATTTAAT TGCTAGACTT AGGAAACAAC 7140
TTTTTTTTTT ACCCAACAAT CTTTGGATTT TAATTGTTTT TTTTTCTACT AATAGATTAA 7200
CAACTCATTA TATAATAATO TTTCTATCAT AATTGACAAT TCTTTCTTTT TAATAAACAT 7260
CCAGCTTGTA TAATAATCCA CAAGTCAATT TCACCATTTT GGCCAATTTA TTTTCTTATA 7320 AAAATTAGCA CAAAAAAGAT TATCATTGTT TAGCAGATTT AATTTCTAAT TAACTTACGT 7380
AATTTCCATT TTCCATAGAT TTATCTTTCT TTTTATTTCC TTAGTTATCT TAGTACTTTC 7440
TTAGTTTCCT TAGTAATTTT AAATTTTAAG ATAATATATT GAAATTAAAA GAAGAAAAAA 7500 AACTCTAGTT ATACTTTTGT TAAATGTTTC ATCACACTAA CTAATAATTT TTTTTAGTTA 7560
AATTACAATA TATAAACACT GAAGAAAGTT TTTGGCCCAC ACTTTTTTCO GATCAATTAG 7620
TACTATAGTT AGGGGAAGAT TCTGATTTAA AGGATACCAA AAATGACTAG TTAGGACATG 7680
AATGAAAACT TATAATCTCA ATAACATACA TACGTGTTAC TGAACAATAG TAACATCTTA 7740
CGTGTTTTGT CCATATATTT GTTGCTTATA AATATATTCA TATAACAATG TTTGCATTAA 7800
GCTTTTAAGA AGCACAAAAC CATATAACAA AATTAAATAT TCCTATCCCT ACCAAAAAAA 7860
AAAATTAAAT ATTCCTACAG CCT ACCGTCTGTT CTCGGTAGAC GCAAATTTTT TAATCTCTTC CACATAAACG AATCGGAATC 8460 AAAAACTTCG CACGCAAAAG TTCTGAGATT CCGAGTCATA CCAGGCGATT TCGAAAGCCT 8520
AAATATTTTA TACCGGAAAG GCTGCAATCC GGTTACCGTT AGACCTAATG ACTTATCACA 8580
ACTCCTCACT TTTGGGTTTG GTATGATCTG ATACTGTTTT GTTGTTGGTT TGCAGACTAT 8640
GTATTCCGGT ATTGGTCTTG TATCATTATA ACAAAGCAAT ATCCCATGAC GTGCATCACA 8700
AGCTTTGATC TTTACCTCTC CTTGTGGCAG AAAATCGATG GAGACTCCTT TGTTATCCAA 8760
ATCTCTCCTC TCATGGAAAA AACTGGTATC AAGTTTGTAT CCTCTTTCGT AGCGTTCTAG 8820
GAAGTATCCA GAGATATTGT TGGTTCGATG GAGATTTAGG TTGACAAACC AAGACTCGTA 8880
GCTTCTCTTG TTGCACTCTT TATTGATGAG CCTCAATTTT CCGATTTCGG ACCCCCGAAG 8940
ATAAGAAAGA ACCTCTTGGA TCGTGTCCTG ATTTATCACC GGAGAACTCA TGATCTTATT 9000
GGAAAAAAGA AAGAAAGAGA TGAGCACGAT CAGTGAATGA GATATATAGA AATCAGGATT 9060
GGTAGAGAAC CGACGATGAT GAATATACAA GTGTTTATAA GTATCACAAA TTGCCTTTTT 9120
CTTCGCTAGT CCCAAAACAA GCAAATTAAC CAAAGATAAA ATCTTCATTA ATGTTTTCCT 9180
TTTTCTTCGC CAGTCCCAGA TAAAAATATA TATAAAATAT TTCATTAGGT TACTTGTAGT 9240
ACCTTGAGCC CAAAGTTTCT CTTTTGACTT TTAACCAAAT TAACAGTAAT TTAATAGCTA 9300
GACTTAGAAA ACAACATTTT GTATATATAT TCTTTGACAT CAAAATTCAA CAATCTTTGG 9360
GTTTCTATAG TGTTTTTTTT CTTATTCTAA TAGATTACCA CTCATTATAT CATATACAAA 9420 GTGTTTCCTT TTCAATCAAC ATCCATTTTC TTTAAAAATT AGCAAGTTTG TTCTTATATC 9480
ATCATTCAGC AGATTTCTTA ATTAAACTTA GTGATTTCCA TTTTGCACCT ATATGTTTCT 9540 CTTTCTTAGT TTAGTACTTT AAATTTTCAT ATATATAATT TATTAAAATT AAAAGTAAAA 9600 ACTCCAGTTT AACTTATGTT AAATGTTTCA TCACACTAAA AGAGCATTAA GTAATAAATA 9660 TTTTAGCTTT ATGAAAAAAA ATATCAAATC ACTGAAGACA TTTGTTGGCC TATACTCTAT 9720 TTTTTATTTG GCCAATTAGT AATAGACTAA TAGTAACTCA TATGATATCT CTCTAATTCT 9780
GGCGAAACGA ATTATCTGAT TCTAAAGATA GTAAAAATGA ATTTTGATGA AGGGAATACT 9640
ATTTCACACA CCTAGAAAGA GTAAGGTAGA AACCTTTTTT TTTTGGTCA GATTCTTGTA 9900
TCAAGAAGTT CTCATCGAT 9919 (2) INFORMATION FOR SEQUENCE SEQ ID NO: 2:
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 5655 base pairs
(B) TYPE: nucleic acid
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) Hypothesis: No
(iv) ANTI-SENS: NO
(ix) PARTICULARITY
(A) NAME / KEY: exon
(B) LOCATION: 2787..3347
(D) OTHER INFORMATION: / product 3 "1st exon of the NIM1"
(ix) PARTICULARITY
(A) NAME / KEY: exon
(B) LOCATION: 3427..4162
(D) OTHER INFORMATION: / product = "2 @@ e exon of NIM1"
(ix) PARTICULARITY
(A) NOMICLE: exon
(B) LOCATION: 4271 .. 4474
(D) OTHER INFORMATION: / product = "3 @ me exon of NIM1"
(ix) PARTICULARITY
(A) NAME / KEY: exon
(@) LOCATION: 4566 .. 4666
(D) OTHER INFORMATION: / product = "4 exon of NIM1"
(ix) PARTICULARITY
(A) NAME / KEY: CDS
(B) LOCATION: join (2787..3347, 3427 .. 4162, 4271 .. 4474, 4566 .. 4866)
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 2
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 GAACGGAGA 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 TAGTATAATAA AAGATATTTT AATATATTAG 1140
ATATACATAA TATCTAAAGC AACACATATT TAGACACAAC ACGTAATATC TTACTATTGT 1200
TTACATATAT TTATAGCTTA CCAATATAAC CCGTATCTAT GTTTTATAAG CTTTTATACA 1260
ATATATGTAC GGTATGCTGT CCACGTATAT ATATTCTCCA AAAAAAACGC ATGGTACACA 1320
AAATTTATTA AATATTTGGC AATTGGGTGT TTATCTAAAG TTTATCACAA TATTTATCAA 1380
CTATAATAGA TGGTAGAAGA TAAAAAAATT ATATCAGATT GATTCAATTA AATTTTATAA 1440
TATATCATTT TAAAAAATTA ATTAAAAGAA AACTATTTCA TAAAATTGTT CAAAAOATAA 1500 TTAGTAAAAT TAATTAAATA TGTGATGCTA TTGAGTTATA GAGAGTTATT GTAAATTTAC 1560
TTAAAATCAT ACAAATCTTA TCCTAATTTA ACTTATCATT TAAGAAATAC AAAAGTAAAA 1620 AACGCGGAAA GCAATAATTT ATTTACCTTA TTATAACTCC TATATAAAOT ACTCTGTTTA 1680
TTCAACATAA TCTTACGTTG TTGTATTCAT AGGCATCTTT AACCTATCTT TTCATTTTCT 1740 GATCTCGATC GTTTTCGATC CAACAAAATG AGTCTACCGO TGAGGAACCA AGAGGTGATT 1800 ATGCAGATTC CTTCTTCTTC TCAGTTTCCA GCAACATCGA GTCCGGAAAA CACCAATCAA 1860
GTGAAGGATG AGCCAAATTT GTTTAGACGT GTTATGAATT TGCTTTTAGG TCGTAGTTAT 1920
TGAAAAAGCT GATTTATCGC ATGATTCAGA ACGAGAAGTT GAAGGCAAAT AACTAAAGAA 1980
GTCTTTTATA TGTATACAAT AATTGTTTTT AAATCAAATC CTAATTAAAA AAATATATTC 2040
ATTATGACTT TCATGTTTTT AATGTAATTT ATTCCATATAT CTATAATGAT TTTGTTGTGA 2100
AGAGCGTTTT CATTTGCTAT AGAACAAGGA GAATAGTTCC AGGAATATATT 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 CACTTGACTT GGCTCTGCTC GTCAATGGTT ATCTTCGATC 2700
TTTAACCAAA TCCAGTTGAT AAGGTCTCTT CGTTGATTAG CAGAGATCTC TTTAATTTGT 2760
CAATTTCAAT TCATCGGAAC CTGTTG ATG ACC GAC ACC ATT GAT GGA TTC GCC 2813
Thr Asp Thr Thr Asp Asp Gly Phe Ala
1 5
GAT TCT TAT GAA ATC AGC ACT AGT TTC GTC GCT ACC GAT AAC ACC 2861
Asp Ser Tire Glu Ser Ser Ser Ser Ser Ser Val Ala Thr Asp Asn Thr
10 15 20 25
GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA GTA CTC ACC GGA CCT 2909
Asp Ser Ser Val Tyr Leu Ala Ala Glu Gln Val Leu Thr Gly Pro
30 35 40
GAT GTA TCT GCT CTG CAA TTC CTC TCC AAC AGC TTC GAA TCC GTC TT 2957
Asp Val Ser Ala Leu Gln Ser Leu Leu Ser Asn Phe Glu Ser Val Phe
45 50 55
GAC TCG CCG GAT GAT TTC TAC AGC GAC GCT AAG CTT CTT CTC TCC GAC 3005
Asp Asp Asp Ser Asp Asp Asp Asp Asp Phe Asp Ser Leu Al Leu
60 65 70
GGC CGG GAA GTT TCT INCL. CAC CGG TGC GTT TTG TCA GCG AGA AGC TCT 3053
Gly Arg Glue Val Ser Phe His Arg Cys Val Leu Ser Ser Ala Arg Ser Ser
75 80 85
TTC AAC AGC GCT TTA GCC GCC GCT GAC AAC GAG AAA GAC TCC AAC 3101
Phe Phe Lys Ser Ala Ala Ala Ala Lys Lys Glu Lys Asp Ser Asn
90 95 100 105
AAC ACC GCC GCC GTG AAG CTC GAG CTT AAC GAG ATT GCC AAC GAT TAC 3149
Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu Ile Ala Lys Asp Tyr
110 115 120
GAA GTC GGT TTC GAT TCC GT GTG ACT GTT GT GCT TAT GTT TAC AGC 3197
Glu Val Gly Phe Asp Val Ser Val Val Leu Val Leu Val Tyr Ser Val Tyr
125 130 135
AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT GAA TGC GCA GAC GAG 3245
Ser Arg Pro Arg Pro Pro Pro Lys Gly Val Ser Pro Glu Cys Ala Asp Glu
140 145 150
AAT TGC TGC CAC GTG GCT CGC CGC CCG GCG GTG GAT TTC ATG TTG GAG 3293
Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu
155 160 165
GTT CTC TAT TTG GCT TTC ATC TTC ATC ATC CCT GAA TTA ATT ACT CTC 3341
Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu 170 175 180 185
TAT CAG GTAAAACACC ATCTGCATTA AGCTATGGTT ACACATTCAT GAATATGTTC 3397
Tyr Gln TTACTTGAGT ACTTGTATTT GTATTTCAG AGG CAC TTA TTC GAC GTT GTA GAC 3450
Arg His Leu Val Val Asp
190,195
AAA GTT GTT ATA GAG GAC ACTA TTG GTT ATA CTC AAC CTT GCT AAT ATA 3498
Lys Val Val Glu Island Asp Thr Leu Leu Leu Leu Leu Ala Asn Ile
200 205 210
TGT GGT AAA ATG GCT TCT ATAC CTA TTG GAT AGT TGT AAA GAG ATT ATT 3546
Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile
215 220 225
GTC AAC TCT AAT GTA GAT ATG GTT AGT CTT GAA AAC TCA TTG CCG GAA 3594
Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu
230-235 240
GAG CTT GTT AAA GAG ATA ATT GAT AGM CGT AAA GAG CTT GGT TTG GAG 3642
Glu Leu Val Lys Glu Island Asp 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 3690
Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp 260 265 270 275
TCG GAT GAT ATT GAG TTA GTC TTG TTG TTG CTT AAA GAG GAT CAC ACC 3738
Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr
280,285,290
AAT CTA GAT GAT GCC TCT GCT CTT CAT TTC GCT GTT GCA TAT TGC AAT 3786
Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn
295 300 305
GTG AAFC ACC GCA ACA GAT CTT TAA AAA CTT GAT TTC CCC GAT GTC AAC 3834
Val Lys Thr Ala Thr Asp Leu Leu Leu Leu Leu Leu Asp Val Asn
310,315,320
CAT AGG AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT CCC ATG CGG 3882
His Arg Asn Pro Arg Gly Tire Thr Val Leu His Val Ala Ala Met Arg
325 330 335
AAG GAG CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT AGT AGT GCA 3930
Lys Glu Pro Gln Leu Leu Leu Leu Leu Glu Lys Gly Ala Ser Ala 340 345 350 355
TCA GAA GAA ACT TTG GAA GGT AGA ACC GCA CTC ATG ATC GCA AAA CAA 3978
Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile Ala Lys Gln
360 365 370
GCC ACT ATG CCC GTT GAA TGT AAT ATC ATC CCG GAG CAA TGC AAG CAT 4026
Ala Thr Met Ala Val Glu Cys Asn Asn Island Pro Glu Gln Cys Lys His
375,380,385
TCT CTC AAA GGC CCA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA 4074
Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys
390 395 400
CCA GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TT GCA GTG GCG GCC 4122
Arg Glu Gln Pro Island Arg Asp Pro Pro Val Ser Phe Ala Val Ala Ala
405 410 415
GAAT GAA TTG AAC ATG ACG CTC CTC GAT CTT GAA AAT AGA G 4162
Asp Glu Leu Lys Met Leu 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 Ala Leu
435
GCT CAA CGT CTT TT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC 4326
Ala Gln Arg Leu Phe Gla Ala Gla Ala Ala Gla Ala
440 445 450
GAA ATG AAC GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC 4374
Glu Met Lys Gly Thr Cys Glu Phe Island Thr Val Ser Ser Glu Pro Asp
455 460 465
CGT CTC ACT GGT ACG AAC AGA ACA TCA CCG GGT GTA AAG ATA GCA CCT 4422
Arg Leu Thr Gly Thr Lys Arg Thr Pro Ser Gly Val Lys Ile Ala 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 Gln Ser Arg Leu Lys Ala Leu Ser Lys
485 490 495
ACC G GTATGGATTC TCACCCACTT CATCGGACTC CTTATCACAA AAAACAAAAC 4524
Thr 500 TAAATGATCT TTAAACATGG TTTTGTTACT TGCTGTCTGA CCTTGTTTTT TTTATCATCA 4584
G GA GA CTC GGG AAA CGA TTC TTC CCC TGT TCG GCA GTG CTC 4629
Val Glu Leu Gly Lys Arg Phe Phe Arg Pro Cys Ser Ala Val Leu
505 510 515
GAC CAG ATT ATG AAC TCT GAG GAC TTC ACT CAA CTC GCT TGC GGA GAA 4677
Asp Gln Met Asn Cys Glu Asp Leu Thr Gln Leu Ala Cys Gly Glu
520,525,530
GAC GAC ACT GCT GAG AAA CGA CTA CAA AAC AAC CAA AGG TAC ATG GAA 4725
Asp Asp Thr Ala Glu Lys Arg Leu Gln Lily Lys Gln Arg Tyr Met Glu
535,540,545
ATA CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GAA TTA 4773
Gln Glu Thr Leu Leu Lys Ala Phe Ser Glu Asp Asn Leu Glu Leu
550 555 560
GGA AAT TCC TCC CTG ACA GAT TCG ACT TCT TCC ACA TCG AAA TCA ACC 4821
Ser Ser Ser Thr Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser
565 570 575
GGT CGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT CGT CGC TCA 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 AGACTTGCTA 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
CTTCTGGAT 5655
(2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 3
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 594 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: lineal
(xi) DESCRIPTION OF THE SEQUENCE SEQ ID NO: 3
Thr Asp Asp Thr Asp Asp Ply Asp Ala Ser Ser Ser Glu Gl Ser Ser Ser
1 5 10 15
Thr Ser Phe Val Ala Asp Asp Asn Thr Asp Ser Ser Val Val Leu
20 25 30
Ala Ala Glu Gln Val Leu Gly Pro Asp Ser Val Leu Ala Leu Cln Leu
35 40 45
Ser Ser Asn Ser Phe Glu Ser Phe Asp Asp Asp
50 55 60
Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His
65 70 75 80
Arg Cys Val Leu Ser Ala Ser Ser Ser Phe Phe Ser Lys Ser Ala Leu Ala
85 90 95
Ala Ala Lys Lys Glu Lys Asn Asn Thr Asp Ala Ala Val Lys Leu
100 105 110
Glu Leu Lys Glu Ala Lys Asp Asp Tyr Glu Val Gly Phe Asp Ser Val
115 120 125
Val Thr Val Leu Ala Tyr Val Tyr Ser Arg Ser Arg Arg Pro Pro Pro
130 135 140
Lys Gly Val Ser Glu Gys Asp Ala Asp Glu Asn Cys Cys His Val Ala Cys 145 150 155 160
Arg Pro Ala Val Asp Phe Met Leu Glu Leu Val Leu Leu Ala Phe Ile
165 170 175
Phe Lys Ile Pro Glu Leu Thr Leu Tyr Gln 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
Ala Asn Island Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Asp Arg Cys Lys
210 215 220
Glu Island Val Lys Island Ser Asn Val Asp Val Val Ser Leu Glu Lily Ser 225 230 235 240
Leu Pro Glu Glu Leu Val Lys Glu Ile Asp Asp Arg Arg Lys Glu Leu
245 250 255
Gly Leu Glu Val Pro Lys Val Lys Lily His Val Ser Asn Val His Lys
260,265,270
Ala Leu Asp Asp Asp Asp Glu Ile Leu Val Leu Lys Leu Leu Leu Lys Glu
275 280 285
Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala
290,295,300
Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Leu Leu Leu 305 310 315 320
Asp Val Asn His Arg Asn Pro Arg Gly Tire Thr Val Leu His Val Ala
325 330 335
Ala Met Arg Lys Glu Gln Leu Leu Leu Leu Leu Leu Glu Lys Gly
340,345,350
Ala Ser Ala Ser Ala Thr Glu Glu Gly Arg Thr Ala Leu Met Ile
355 360 365
Ala Lys Gln Ala Thr Ala Val Glu Cys Asn Asn Glu Gln Glu
370,375,380
Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Cln 385 390 395 400
Glu Asp Lys Arg Glu Gln Pro Island Arg Asp Pro Pro Val Ser Phe Ala
405 410 415
Val Ala Ala Asp Glu Leu Lys Met Leu Leu Leu Asp Leu Glu Asn Arg
420,425,430
Val Ala Leu Ala Gln Arg Leu Phe Pro Glu Ala Gla Gla Ala Ala Met
435 440 445
Glu Ile Ala Glu Met Lys Gly Thr cys Glu Phe Ile Val Thr Ser Leu
450 455 460
Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Pro Ser Gly Val Lys 465 470 475 480
Ala Island Pro Phe Arg Island Leu Glu Glu His Gln Ser Arg Leu Lys Ala
485 490 495
Leu Ser Lys Thr Glu Val Leu Gly Lys Arg Phe Pro Phe Arg Cys Ser
500 505 510
Ala Val Leu Asp Gln Met Asn Cys Glu Asp Leu Thr Gln Leu Ala
515 520 525
Cys Gly Asp Glu Asp Thr Ala Glu Arg Lys Gln Gln Lilies Lys Gln Arg
530,535,540
Tyr Met Glu Island Gln Glu Thr Leu Lily Lily Ala Phe Ser Glu Asp Asn 545 550 555 560
Leuk Leu Leuk Asn Ser Ser Leu Ser Asp Ser Ser Ser Ser Ser Ser 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 FOR SEQUENCE SEQ ID NO: 4
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 41 amino acids
(B) TYPE: amino acid
(C) NATURE OF THE STRANDS: does not apply
(D) TOPOLOGY: does not apply
(ii) TYPE OF MOLECULE: peptide
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 4
Arg Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Glu Leu Val 1 5 10 15
Lys Leu Met Val Met Gly Glu Asp Asp Asp Ala Leu Ala
20 25 30
Val His Tyr Val Ala Val Cln His Cys Asn
40 (2) INFORMATION FOR SEQUENCE SEQ ID NO: 5:
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 38 amino acids
(B) TYPE: amino acid
(C) NATURE OF THE STRANDS: does not apply
(D) TOPOLOGY: does not apply
(ii) TYPE OF MOLECULE: peptide
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 5
Pro Thr Gly Lys Thr Ala Leu His Ala Ala Ala Glu Met Val Ser Pro
1 5 10 15
Asp Met Val Ser Val Leu Leu Asp His His Asp Asp Xaa Asn Phe Arg
20 25 30
Thr Xaa Gly Val Thr Thr
35 (2) INFORMATION FOR SEQUENCE SEQ ID NO: 6
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 41 amino acids
(B) TYPE: amino acid
(C) NATURE OF THE STRANDS: does not apply
(D) TOPOLOGY: does not apply
(ii) TYPE OF MOLECULE: peptide
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 6
Arg Arg Arg Arg Arg Ala Leu Asp Ala Ala Asp Ile Clu Leu Val
1 5 10 15
Lys Leu Met Val Met Gly Glu Asp Asp Asp Ala Leu Ala
20 25 30
Val His Tyr Val Ala Val Gln His Cys Asn
35 40 (2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 7
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) NATURE OF THE STRANDS: does not apply
(D) TOPOLOGY: does not apply
(ii) TYPE OF MOLECULE: peptide
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 7
Arg Arg Pro Asp Ser Lys Thr Ala Leu His Leu Ala Ala Glu Met Val 1 5 10 15
Ser Pro Asp Met Val Ser Leu Val Leu Leu Asp Gln
(2) INFORMATION FOR SEQUENCE SEQ ID NO: 8
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 41 amino acids
(B) TYPE: amino acid
(C) NATURE OF THE STRANDS: does not apply
(D) TOPOLOGY: does not apply
(ii) TYPE OF MOLECULE: peptide
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 8
Arg Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Glu Leu Val
1 5 10 15
Lys Leu Met Val Met Gly Glu Asp Asp Asp Ala Leu Ala
20 25 30
Val His Tyr Val Ala Val Gln His Cys Asn
35 40 (2) INFORMATION FOR SEQUENCE SEQ ID NO: 9
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) NATURE OF THE STRANDS: does not apply
(D) TOPOLOGY: does not apply
(ii) TYPE OF MOLECULE: peptide
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 9
Arg Arg Pro Asp Ser Lys Thr Ala Leu His Leu Ala Ala Glu Met Val 15 10 15
Ser Pro Asp Met Val Ser Leu Val Leu Leu Asp Gln
(2) INFORMATION FOR SEQUENCE SEQ ID NO: 10
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 41 amino acids
(B) TYPE: amino acid
(C) NATURE OF THE STRANDS: does not apply
(D) TOPOLOGY: does not apply
(ii) TYPE OF MOLECULE: peptide
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 10
Arg Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Glu Leu Val
1 5 10 15
Lys Leu Met Val Met C and Glu Gly Leu Asp Asp Leu Asp Asp Ala Leu Ala
20 25 30
Val His Tyr Val Ala Val Gln His Cys Asn
35 40 (2) INFORMATION FOR THE SEQUENCE SEQ ID NO: l1
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) NATURE OF THE STRANDS: does not apply
(D) TOPOLOGY: does not apply
(ii) TYPE OF MOLECULE: peptide
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: ll:
Pro Thr Gly Lys Thr Ala Leu His Ala Ala Ala Glu Met Val Ser Pro
1 5 10 15
Asp Met Val
(2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 12
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 22 base pairs
(B) TYPE: NUCLEIC ACID
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: lineal
(ii) TYPE OF MOLECULE: other nucleic acids
(A) DESCRIPTION: / desc = "oligonucleotide"
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 12 AATTCTAAAG CATGCCGATC GG 22 (2) INFORMATION FOR SEQUENCE SEQ ID NO: 13
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 21 base pairs
(B) TYPE: NUCLEIC ACID
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: lineal
(ii) TYPE OF MOLECULE: other nucleic acids
(A) DESCRIPTION: / desc = "oligonucleotide"
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 13
AATTCCGATC GGCATGCTTT A 21 (2) INFORMATION FOR SEQUENCE SEQ ID NO: 14
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 22 base pairs
(B) TYPE: NUCLEIC ACID
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: lineal
(ii) TYPE OF MOLECULE: other nucleic acids
(A) DESCRIPTION: / desc = "oligonucl otidew"
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 14
AATTCTAAAC CATGGCGATC GC 22 (2) INFORMATION FOR SEQUENCE SEQ ID NO: 15
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 21 base pairs
(B) TYPE: NUCLEIC ACID
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: lineal
(ii) TYPE OF MOLECULE: other nucleic acids
(A) DESCRIPTION: / desc '1o igonucl otide "
(xi) DESCRIPTION OF THE SEQUENCE SEQ ID NO: 15 AATTCCGATC GCCATGGTTT A 21 (2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 16
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 15 base pairs
(B) TYPE: NUCLEIC ACID
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: lineal
(ii) TYPE OF MOLECULE: other nucleic acids
(A) DESCRIPTION: / desc - "oligonucleotide"
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 16:
CCAGCTGGAA TTCCG
(2) INFORMATION FOR SEQUENCE SEQ ID NO: 17:
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 19 base pairs
(B) TYPE: NUCLEIC ACID
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: lineal
(ii) TYPE OF MOLECULE: other nucleic acids
(A) DESCRIPTION: / desc = "oligonucleotide"
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 17 CGGAATTCCA GCTGGCATG 19 (2) INFORMATION FOR SEQUENCE SEQ ID NO: 18:
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 314 amino acids
(B) TYPE: amino acid
(C) NATURE OF THE STRANDS: does not apply
(D) TOPOLOGY: does not apply
(ii) TYPE OF MOLECULE: prot ine
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 18
Met Phe Gln Pro Ala Gly His Gly Asp Gln Gln Ala Met Glu Gly Pro 1 5 10 15
Arg Asp Gly Leu Lys Glu Arg Lys Leu Asp Val Asp Asp Arg Asp Asp Ser
20 25 30
Gly Leu Asp Ser Met Asp Lys Glu Glu Glu Tyr Glu Met Val Lys Glu
35 40 45
Leu Arg Glu Arg Island Gln Gln Gln Pro Gln Ala Pro Ala Ala Glu Gla
50 55 60
Pro Trp Lys Gln Gln Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu 65 70 75 80
Ala Ile Ile Glu Glu Lys Pro Leu Glu Glu Val Ile Gly Gln
85 90 95
Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln
100 105 110
Thr Pro Leu His Leu Ala Val Island Thr Asn Gln Pro Gly Island Ala Glu
115 120 125
Ala Leu Leu Lys Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly
130 135 140
Asn Thr Pro Leu His Leu Ala Cys Glu Gly Gly Cys Leu Ala Ser Val 145 150 155 160
Ala Val Leu Thr Gln Thr Cys Thr Gln His Leu His Ser Val Leu
165 170 175
Gln Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser Thr
180 185 190
His Gly Tyr Leu Ala Val Glu Island His Leu Val Leu Leu Gly Ala Asp
195 200 205
Val Asn Ala Gln Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala
210 215 220
Val Asp Leu Gln Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly 225 230 235 240
Ala Asp Val Asn Arg Val Thr Tyr Gln Gly Tire Ser Pro Tyr Gln Leu
245 250 255
Thr Trp Gly Arg Pro Ser Thr Arg Gln Gln Gln Leu Leu Cly Gln Leu
260,265,270
Thr Leu Glu Asn Leu Gin Met Leu Glu Pro Glu Ser Asp Glu Glu Ser
275 280 285
Asp Asp Glu Glu Ser Glu Phe Glu Glu Asp Glu Leu Pro Asp Asp Asp
290,295,300
Cys Val Phe Gly Gly Gln Arg Leu Thr Leu
305 310 (2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 19
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 314 amino acids
(B) TYPE: amino acid
(C) NATURE OF THE STRANDS: does not apply
(D) TOPOLOGY: does not apply
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 19
Met Phe <RTI ID
Leu Arg Glu Arg Island Gln Gln Gln Pro Gln Ala Pro Ala Ala Glu Gla
50 55 60
Pro Trp Lys Gln Gln Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu 65 70 75 80
Ala Ile Ile His Glu Glu Lys Thr Leu Thr Met Glu Val Ile Gly Gln
85 90 95
Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln
100 105 110
Thr Pro Leu His Leu Ala Val Island Thr Asn Gln Pro Gly Island Ala Glu
115 120 125
Ala Leu Leu Lys Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly
130 135 140
Asn Thr Pro Leu His Leu Ala cys Glu Gly Gly Cys Leu Ala Ser Val 145 150 155 160
Ala Val Leu Thr Gln Thr Cys Thr Gln His Leu His Ser Val Leu
165 170 175
Gln Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser Ile
180 185 190
His Gly Tyr Leu Gly Val Glu Island His Leu Val Leu Thr Leu Gly Ala Asp
195 200 205
Val Asn Ala Gln Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala
210 215 220
Val Asp Leu Gln Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly 225 230 235 240
Ala Asp Val Asn Arg Val Thr Tyr Gln Gly Tire Ser Pro Tyr Gln Leu
245 250 255
Thr Trp Gly Arg Ser Ser Arg Arg Gln Gln Gln Gln Leu Gly Gln Leu
260,265,270
Thr Leu Glu Asn Leu Gln Leu Leu Pro Glu Ser Asp Glu Glu Glu Ser
275 280 285
Asp Asp Glu Glu Ser Glu Phe Glu Glu Asp Glu Leu Pro Asp Asp Asp
290,295,300
Cys Val Phe Gly Gly Gln Arg Leu Thr Leu 305 310 (2) INFORMATION FOR SEQUENCE SEQ ID NO: 20
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 314 amino acids
(B) TYPE: amino acid
(C) NATURE OF THE STRANDS: does not apply
(D) TOPOLOGY: does not apply
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 20
Met Phe Gln Pro Ala Glu Pro Gly Gln Glu Trp Ala Met Glu Gly Pro 1 5 10 15
Arg Asp Ala Leu Lys Glu Arg Leu Leu Asp Asp Arg Arg Asp Asp Ser
20 25 30
Gly Leu Asp Ser Met Asp Lys Glu Glu Glu Tyr Glu Met Val Lys Glu
35 40 45
Leu Arg Glu Arg Island Glu Glu Pro Gln Glu Ala Arg Pro Gly Ala Glu
50 55 60
Pro Trp Lys Gln Gln Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu 65 70 75 80
Ala Island Island His Glu Glu Lys Ala Leu Thr Glu Met Val Val Arg Gln
85 90 95
Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln
100 105 110
Thr Pro Leu His Leu Ala Val Island Thr Asn Gln Pro Glu Island Ala Glu
115 120 125
Ala Leu Leu Glu Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly
130 135 140
Asn Thr Pro Leu His Leu Ala Cys Glu Gly Gly Cys Leu Ala Ser Val 145 150 155 160
Gly Val Leu Thr Gln Pro Arg Gly Thr Gln His Leu His Ser Ile Leu
165 170 175
Gln Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser Ile
180 185 190
Gly Tyr Leu Gly Ile Val Glu Leu Leu Ser Ser Leu Gly Ala Asp
195 200 205
Val Asn Ala Gln Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala
210 215 220
Val Asp Leu Gln Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly
225 230 235 240
Ala Asp Val Asn Arg Val Thr Tyr Gln Gly Tire Ser Pro Tyr Gln Leu
245 250 255
Thr Trp Gly Arg Ser Ser Arg Arg Gln Gln Gln Gln Leu Gly Gln Leu
260,265,270
Thr Leu Glu Asn Leu Gln Met Leu Glu Pro Glu Ser Asp Glu Glu Ser
275 280 285
Asp Asp Glu Glu Glu Phe Thr Asp Asp Aspu Asp Asp Asp
290,295,300
Cys Val Leu Gly Gly Arg Gln Leu Leu Leu
305 310 (2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 21
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 2011 base pairs
(B) TYPE: nucleic acid
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: cDNA
(vi) ORIGINAL SOURCE
(A) ORGANISM: Arabidopsis thaliana
(ix) PARTICULARITY
(A) NAME / KEY: misc ~ feature
(B) LOCATION: 1..2011
(D) OTHER INFORMATION: / note = "cDNA sequence of NIM1"
(ix) PARTICULARITY
(A) NAME / KEY: CDS
(B) LOCATION: 43..1824
(OTHER INFORMATION :

* / product = "NIM1 protein"
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 21
GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC 54
Met Asp Thr Thr
ATT GAT GGA TTC GCC GAT TCT GAAT TAT ATC AGC ACT AGT TTC GTC 102
Asp Gly Phe Island Ala Asp Ser Tire Glu Ser Ser Ser Thr Ser Phe Val
5 10 15 20
GCT ACC GAT ACC AAC GAC TCC ATT ATT GTT TAT CTC GCC GCC GAA CAA 150
Ala Thr Asp Asn Thr Asp Ser Ser Val Val Leu Ala Ala Glu Gln
25 30 35
GTA CTC ACC GGA CCT GAT GTA GCT CTG TGA CTG TTG CTC TCC AAC AGC 198
Val Leu Thr Gly Pro Asp Ser Ser Leu Leu Leu Leu Leu Ser Asn Ser
40 45 50
TTC GAA TCC GTC TTT GAC TCG CCG GAT GAT TTC TAC AGC GAC GCT AAC 246
Phe Glu Ser Val Phe Asp Asp Asp Asp Asp Phe Asp 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 Val Leu Ser Asp Gly Arg Glue 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 AAC AAC 342
Ser Ala Ser Ser Phe Phe Ser Lys Ser Ala Leu Ser Ala Ala Lys Lys
85 90 95 100
GAG AAA GAC TCC AAC AAC ACC GCC GCC GTG AAC CTC GAG CTT AAC GAG 390
Glu Lys Asp Asn Ser Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu
105 110 115
ATT GCC AAG GAT GAAT TAC GTC GGT TTC GAT GTT TTT GTG ACT GTT TTC 438
Ile Ala Lys Asp Tire Glu Val Gly Phe Asp Ser Val Val Thr Val Leu
120 125 130
GCT TAT GTT AGC TAC AGC AGG AGG AGG CCG CCG TAC AAA GCA GTT TCT 486
Ala Tyr Val Tyr Ser Ser Arg Pro Arg Pro Pro Pro Lys Gly Val Ser Pro
135 140 145
GAA TGC GCA GAC GAG AAT TGC TGC CAC GTG GCT TGC CGG CCG CCC GTG 534
Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val
150 155 160
GAT TTC ATG TTG GAG GTT CTC TAT TTC GCT TTC ATC TTC AAG ATC CCT 582
Asp Phe Met Leu Glu Val Leu Tire 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 Thr Leu Tyr Gln Arg His Leu Leu Asp Asp Val Val Asp Lys
185 190 195
GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAC CTT GCT AAT ATA TGT 678
Val Val Ile Glu Asp Thr Ile Leu Val Leu Leu Leu Ala Asn Ile Cys
200 205 210
GGT AAA GCT TGT ATG CTA AAC TTG GAT AGT TGT AAA GAG ATT ATT GTC 726
Gly Lys Ala Cys Met Leu Leu Lys Asp Arg Cys Lys Glu Ile Ile Val
215 220 225
AAG TCT AAT GTA GAT ATC GTT AGT CTT GAA AAG TCA TTG CCG GAA GAG 774
Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu
230-235 240
CTT GTT AAA GAG ATA ATT GAT AGC CGT AAA GAG CTT CGT TTG GAG GTA 822
Leu Val Lys Glu Island Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val 245 250 255 260
CCT AAA GTA AAA AAA CAT GTC TCG AAT GTA CAT AAG GCA CTT GAC TCC 870
Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser
265 270 275
GAT GAT ATT GAG TTA GTC AAG TTG TTC TTC AAA GAG GAT ACC AAT 918
Asp Asp Ile Glu Leu Val Leu Leu Leu Leu Lys Glu Asp His Thr Asn
280,285,290
CTA GAT GAT CCC TGT GCT TTC CT TTC GCT GTT GMT TAT TGC AAT GTG 966
Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val
295 300 305
AAC ACC GCA ACA GAT CTT TTA AAA CTT GAT CTT GCC GAT GTC AAC CAT 1014
Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val 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 Tire Thr Val Leu His Val Ala Ala Met Arg Lys 325 330 335 340
GAG CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA 1110
Glu Pro Gln Leu Leu Leu Leu Leu Leu Glu Lys Gly Ser Ala Ser
345 350 355
GAA GCA ACT TTG GAA GGT AGA ACC GCA CTC ATG ATC GCA AAA CAA GCC 1158
Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ala Lys Gln Ala
360 365 370
ACT ATG CCC GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT TCT 1206
Thr Met Ala Val Glu Cys Asn Asn Island Pro Glu Gln Cys Lys His Ser
375,380,385
CTC AAA GGC CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA CGA 1254
Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys Arg
390 395 400
GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TT GCA GTC GCG GCC GAT 1302
Glu Gln Pro Pro Asp Asp Pro Pro Pro Ala Val Ala Ala Ala Asp 405 410 415 420
GAA TTG AAC ATG ACG CTC CTC GAT CTT GAA AAT AGA GTT GCA CTT GCT 1350
Glu Leu Lys Met Leu Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala
425 430 435
CAA CGT CTT TT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC GAA 1398 Gln Arg Leuk Pro Thr Glu Ala Gln Ala Ala Met Glu Island Ala Glu
440 445 450
ATG AAC GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC CGT 1446
Met Lys Gly Thr Cys Glu Phe Island Thr Val Ser Ser Glu Pro Asp Asp Arg
455 460 465
CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAC ATA GCA CCT TTC 1494
Leu Thr Gly Thr Lys Arg Thr Pro Ser Gly Val Lys Ile Ala Pro Phe
470 475 480
AGA ATC CTA GAA GAG CAT CAA ACT AGA CTA AAA GCC CTT TCT AAA ACC 1542
Arg Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys Thr 485 490 495 500
GTC GAA CTC GGG AAA CGA TTC TTC CCG CGC TGT TCG GCA GTG CTC GAC 1590
Val Glu Leu Gly Lys Arg Phe Phe Arg Pro Cys Ser Ala Val Leu Asp
505 510 515
CAG ATT ATG AAC TGT GAG GAC TTG ACT CAA CTG GCT TGC GGA GAA GAC 1638 Gln Ile Met Asn Cys Glu Asp Leu Thr Gln Leu Ala Cys Gly Glu Asp
520,525,530
GAC ACT GCT GAG AAA CGA CTA CAA AAC AAG CAA AGG TAC ATG GAA ATA 1686
Asp Thr Ala Glu Lys Arg Leu Gln Lily Lys Gln Arg Tyr Met Glu Ile
535,540,545
CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GTA TTA GGA 1734 Gln Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn Leu Glu Leu Gly
550 555 560
AAT TTG TCC CTG ACA GAT TCC ACT TCT TCC ACA TCG AAA TCA ACC GGT 1782
Asn Leu Ser Leu Ser Asp Ser Ser Ser Ser Ser Ser Ser Lys Ser Thr Gly 565 570 575 580
GGA AAC 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 TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT 1944
ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG 2004
ATTTGTA 2011 (2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 22
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 2011 base pairs
(B) TYPE: nucleic acid
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: cDNA
(ix) PARTICULARITY
(A) NAME / KEY: CDS
(B) LOCATION: 43..1824
(D) OTHER INFORMATION: product = "modified form of NIM1"
/ note = "Serine residues at amino acid positions 55 and 59 of the wild type NIM1 gene product have been modified to alanine residues".

(ix) PARTICULARITY
(A) NAME / KEY: misc feature
(B) LOCATION: 205..217
(D) OTHER INFORMATION: / note = "Nucleotides 205 and 217 change from T to G compared to the wild-type sequence"
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 22
CATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TO ATG ACC ACC 54
Met Asp Thr Thr
ATT GAT GGA TTC GCC GAT TCT GAAT TAT ATC AGC ACT AGT TTC GTC 102
Asp Gly Phe Island Ala Asp Ser Tire Glu Ser Ser Ser Thr Ser Phe Val
5 10 15 20
GCT ACC GAT ACC ACC GAC TCC ATT ATT GTT TAT CT GCC GCC GAA CAA 150
Ala Thr Asp Asn Thr Asp Ser Ser Val Val Leu Ala Ala Glu Gln
25 30 35
GTA CTC ACC GGA CCT GAT GTA GCT CTG TGA CTG TTG CTC TCC AAC AGC 198
Val Leu Thr Gly Pro Asp Ser Ser Leu Leu Leu Leu Leu Ser Asn Ser
40 45 50
TTC GAA GCC GTC TT GAC CCC CCG GAT GAT TTC TAC AGC GAC GCT AAG 246
Phe Glu Ala Val Phe Asp Ala Pro Asp Asp Phe Tyr Ser Asp Asp Ala 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 Glue Val Ser Phe His Arg Cys Val Leu
70 75 80
TCA CCC AGA AGC TCT TTC TTC AAC AGC GCT TTA GCC GCC GCT AAC AAC 342
Ser Ala Ser Ser Phe Phe Ser Lys Ser Ala Leu Ser Ala Ala Lys Lys
85 90 95 100
GAG AAA GAC TCC AAC AAC ACC GCC GCC GTG AAC CTC GAG CTT AAG GAG 390
Glu Lys Asp Asn Ser Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu
105 110 115
ATT CCC AAC GAT GAAT TAC GTC GGT TTC GAT GTT GTG ACT GTT TTG 438
Ile Ala Lys Asp Tire Glu Val Gly Phe Asp Ser Val Val Thr Val Leu
120 125 130
GCT TAT GTT AGC TAC AGC AGM GTG AGM CCG CCG TAC AAA GGA GTT TCT 486
Ala Tyr Val Tyr Ser Ser Arg Pro Arg Pro Pro Pro Lys Gly Val Ser Pro
135 140 145
GAA TGC GCA GAC GAG AAT TGC TGC CAC GTG GCT TGC CGG CCC CCC GTG 534 Clu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val
150 155 160
GAT TTC ATG TTG GTT GTT CTC TAT TTG TT GCT ATC TTC ATC ATC CCT 582
Asp Phe Met Leu Glu Val Leu Tire Leu Ala Phe Ile Phe Lys Ile Pro 165 170 175 180
GAA TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC CTT GTA GAC AAA 630
Glu Leu Thr Leu Tyr Gln Arg His Leu Leu Asp Asp Val Val Asp Lys
185 190 195
GTT GTT ATA GAG GAC ACTA TTG GTT ATA CTC AAG CTT GCT AAT ATA TCT 678
Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile cys
200 205 210
GGT AAA GCT TCT ATG CTA AAC TTG GAT AGT TGT AAA GAG AT ATT GTC 726
Gly Lys Ala cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val
215 220 225
AAG TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTC CCC GAA GAG 774
Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu
230-235 240
CTT GTT AAA GAG ATA ATT GAT AGC CGT AAA GAG CTT GGT TTG GAG GTA 822
Leu Val Lys Glu Island Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val 245 250 255 260
CCT AAA GTA AAG AAA CAT GTC TCC AAT GTA CAT AAG GCA CTT GAC TCG 870
Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser
265 270 275
GAT GAT ATT GAG TTA CTC AAC TTG TTC TTG AAA GAG GAT CAC ACC AAT 918
Asp Asp Ile Glu Leu Val Leu Leu Leu Leu Lys Glu Asp His Thr Asn
280,285,290
CTA GAT GAT CCC TGT GCT TTC CT TTC GCT GTT GMT TAT TGC AAT GTG 966
Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val
295 300 305
AAG ACC GCA ACA GAT CTT TTA AAA CTT GAT CTT GCC GAT GTC AAC CAT 1014
Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His
310,315,320
AGG AAT CCG AGG GGA TAT ACC GTG CTT CAT GTT GCT GCG ATG CGC AAG 1062
Arg Asn Pro Arg Gly Tire Thr Val Leu His Val Ala Ala Met Arg Lys 325 330 335 340
GAG CCA CAA TTC ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA 1110
Glu Pro Gln Leu Leu Leu Leu Leu Leu Glu Lys Gly Ser Ala Ser
345 350 355
GAA GCA ACT TTG GAA GGT AGA ACC GCA CTC ATG ATC GCA AAA CAA GCC 1158
Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ala Lys Gln Ala
360 365 370
ACT ATG CCC GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT TCT 1206
Thr Met Ala Val Glu Cys Asn Asn Island Pro Glu Gln Cys Lys His Ser
375,380,385
CTC AAA GGC CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA CGA 1254
Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys Arg
390 395 400
GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TT GCA GTC GCG GCC GAT 1302
Glu Gln Pro Pro Asp Asp Pro Pro Pro Ala Val Ala Ala Ala Asp 405 410 415 420
GAA TTG AAG ATG ACG CTG CTC GAT CTT GAA AAT AGA GTT GCA CTT GCT 1350
Glu Leu Lys Met Leu Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala
425 430 435
CAA CGT CTT TT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC GAA 1398 Gln Arg Leuk Pro Thr Glu Ala Gln Ala Ala Met Glu Island Ala Glu
440 445 450
ATG AAC GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC CGT 1446
Met Lys Gly Thr Cys Glu Phe Island Thr Val Ser Ser Glu Pro Asp Asp Arg
455 460 465
CTC ACT GGT ACG AAG AGA ACA TCA CCC GGT GTA AAC ATA GCA CCT TTC 1494
Leu Thr Gly Thr Lys Arg Thr Pro Ser Gly Val Lys Ile Ala 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 Gln Ser Arg Leu Lys Ala Leu Ser Lys Thr 485 490 495 500
GTG GAA CTC GGG AAA CGA TTC TTC CCG CCC TGT TCG GCA GTG CTC GAC 1590
Val Glu Leu Gly Lys Arg Phe Phe Arg Pro Cys Ser Ala Val Leu Asp
505 510 515
CAG ATT ATG AAC TGT GAG GAC TTG ACT CAA CTC GCT TGC GGA GAA GAC 1638 Gln Ile Met Asn Cys Glu Asp Leu Thr Gln Leu Ala Cys Gly Glu Asp
520,525,530
GAC ACT GCT GAG AAA CGA CTA CAA AGA AGG CAA AGG TAC ATG GAA ATA 1686
Asp Thr Ala Glu Lys Arg Leu Gln Lily Lys Gln Arg Tyr Met Glu Ile
535,540,545
CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GAA TTA GCA 1734 Gln Glu Thr Leu Lys Lys Ala Phe Ser Asp Glu Asn Leu Glu Leu Gly
550 555 560
AAT TTG TCC CTG ACA GAT TCC ACT TCT TCC ACA TCG AAA TCA ACC GGT 1782
Asn Leu Ser Leu Ser Asp Ser Ser Ser Ser Ser Ser Ser Lys Ser Thr Gly 565 570 575 580
GGA AAC 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 TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT 1944
ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG 2004
ATTTGTA 2011 (2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 23
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 594 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 23
Met Asp Asp Thr Thr Asp Asp Phe Ala Asp Ser Tyr Ser Glu Ser Ser
1 5 10 15
Thr Ser Phe Val Ala Asp Asp Asn Thr Asp Ser Ser Val Val Leu
20 25 30
Ala Ala Glu Gln Val Leu Gly Pro Asp Ser Val Leu Ala Leu Cln Leu
35 40 45
Ser Ser Asn Ser Phe Glu Asp Ala Val Phe Asp Ala Asp Asp Asp Phe Tyr
50 55 60
Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His
65 70 75 80
Arg Cys Val Leu Ser Ala Ser Ser Ser Phe Phe Ser Lys Ser Ala Leu Ala
85 90 95
Ala Ala Lys Lys Glu Lys Asn Asn Thr Asp Ala Ala Val Lys Leu
100 105 110
Glu Leu Lys Glu Ala Lys Asp Asp Tyr Glu Val Gly Phe Asp Ser Val
115 120 125
Val Thr Val Leu Ala Tyr Val Tyr Ser Arg Ser Arg Arg Pro Pro Pro
130 135 140
Lys Gly Val Ser Glu Cys Asp Ala Asp Glu Asn Cys cys His Val Ala Cys 145 150 155 160
Arg Pro Ala Val Asp Phe Met Leu Clu Val Leu Tyr Leu Ala Phe Ile
165 170 175
Phe Lys Island Pro Glu Leu Island Thr Leu Tyr Gln 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
Ala Asn Island Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Asp Cys Lys
210 215 220
Glu Island Val Lys Island Ser Asn Val Asp Val Val Ser Leu Glu Lily Ser 225 230 235 240
Leu Pro Glu Glu Leu Val Lys Glu Ile Asp Asp Arg Arg Lys Glu Leu
245 250 255
Gly Leu Glu Val Pro Lys Val Lys Lily His Val Ser Asn Val His Lys
260,265,270
Ala Leu Asp Asp Asp Asp Glu Ile Leu Val Leu Lys Leu Leu Leu Lys Glu
275 280 285
Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala
290,295,300
Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Leu Leu Leu 305 310 315 320
Asp Val Asn His Arg Asn Pro Arg Gly Tire Thr Val Leu His Val Ala
325 330 335
Ala Met Arg Lys Glu Gln Leu Leu Leu Leu Leu Leu Glu Lys Gly
340,345 350%
Ala Ser Ala Ser Ala Thr Glu Glu Gly Arg Thr Ala Leu Met Ile
355 360 365
Ala Lys Gln Ala Thr Ala Val Glu cys Asn Asn Pro Glu Gln Gln
370 375 380 cys Lys His Ser Leu Lys Gly Arg Leucys Val Glu Ile Leu Glu Gln 385 390 395 400
Glu Asp Lys Arg Glu Gln Pro Island Arg Asp Pro Pro Val Ser Phe Ala
405 410 415
Val Ala Ala Asp Glu Leu Lys Met Leu Leu Leu Asp Leu Glu Asn Arg
420,425,430
Val Ala Leu Ala Gln Arg Leu Phe Pro Glu Ala Gla Gla Ala Ala Met
435 440 445
Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu
450 455 460
Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Pro Ser Gly Val Lys 465 470 475 480
Ala Island Pro Phe Arg Island Leu Glu Glu His Gln Ser Arg Leu Lys Ala
485 490 495
Leu Ser Lys Thr Glu Val Leu Gly Lys Arg Phe Pro Phe Arg Cys Ser
500 505 510
Ala Val Leu Asp Gln Met Asn Cys Glu Asp Leu Thr Gln Leu Ala
515 520 525 cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gln Lys Lys Gln Arg
530,535,540
Tyr Met Glu Island Gln Glu Thr Leu Lily Lily Ala Phe Ser Glu Asp Asn 545 550 555 560
Leu Leu Leuk Leu Leu Leu Ser Ser Asp 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 FOR SEQUENCE SEQ ID NO: 24
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 1597 base pairs
(B) TYPE: nucleic acid
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: cDNA
(ix) PARTICULARITY
(A) NAME / KEY: CDS
(B) LOCATION: 1..1410
(D) OTHER INFORMATION: / product = "Modified form of NIM1"
/ note = "N-terminal deletion compared to the sequence of the
NIM1 wild type "
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 24
ATG GAT TCC GTT GTG GTT ACT TTG GCT TAT GTT AGC TAC AGC AGA GTG 48
Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val
1 5 10 15
AGA CCG CCG CCT AAA GGA GTT TCT GAA TGC GCA GAC GAG AAT TGC TGC 96
Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Asp Ala Asp Glu Asn Cys Cys
20 25 30
CAC GTG GCT TGC CGG CCG CCC GTG GAT TTC ATG TTG GAG GTT CTC TAT 144
His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr
35 40 45
TTG GCT TTC ATC TTC AAG ATC CCT GAA TTA ACT ATT CTC TAT CAG AGG 192
Leu Ala Phe Ile Phe Lys Island Pro Glu Leu Island Thr Leu Tyr Gln Arg
50 55 60
CAC TTA TTG GAC GTT GTA GAC AAA GTT GTT GAC ATA GAC ACA TTG GTT 240
His Leu Leu Asp Asp Val Val Asp Lys Val Val Glu Asp Asp Leu Val
65 70 75 80
ATA CTC AAG CTT GCT AAT ATA TGT GGT AAA GCT TGT ATG AAC CTA TTG 288
Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala cys Met Lys Leu Leu
85 90 95
GAT AGA TGT AAA GAG ATT ATT CTC AAC TCT AAT AT GTA GAT ATG GTT AGT 336
Asp Arg Cys Lys Glu Island Ile Val Lys Ser Asn Val Asp Met Val Ser
100 105 110
CTT GAA AAC TCA TTG CCG GAA GAG CTT GTT AAA GAG ATA ATT GAT AGA 384
Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Asp Asp Arg
115 120 125
CGT AAA GAG CTT GGT TTG GAG GTA CCT AAA GTA AAA AAA CAT GTC TCG 432
Arg Lys Glu Gly Leu Leu Val Val Lys Val Lys Lys His Val Ser
130 135 140
AAT GTA CAT AAC GCA CTT GAC TCG GAT ATT GAG ATT GTC AAC TTG 480
Asn Val His Lys Ala Leu Asp Asp Asp Asp Glu Island Leu Val Lys Leu 145 150 155 160
CTT TTG AAA GAG GAT CAC ACC AAT CTA GAT GAT CCC TGT GCT CTT CAT 528
Leu Leu Lys Glu Asp His Asp Asn Leu Asp Asp Ala Cys Ala Leu His
165 170 175
TTC GCT GTT GCA TAT TGC AAT GTC ACC AAG ACC GCA ACA GAT CTT TTA AAA 576
Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys
180 185 190
CTT GAT CTT GCC GAT CTC AAC CAT AGG AAT CCG AGG GGA TAT ACG GTG 624
Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val
195 200 205
CTT CAT GTT GCT CCC ATG CGG AAC GAG CCA CAA TTG ATA CTA TCT CTA 672
Leu His Val Ala Ala Met Arg Glu Lys Gln Leu Leu Leu Leu Leu Leu
210 215 220
TTG GAA AAA CGT GCA AGT GCA TCA GAA GCA ACT TTC GAA GCT AGA ACC 720
Leu Glu Lys Gly Ala Ser Ala Ser Ala Thr Glu Glu Gly Arg Thr 225 230 235 240
GCA CTC ATG ATC GCA AAA CAA GCC ACT ATG CCC GTT GAA TGT AAT AAT 768
Ala Leu Met Ala Lys Gln Ala Thr Ala Glu Cys Asn Asn
245 250 255
ATC CCG GAG CAA TGC AAC CAT TCT CTC AAA CCC CGA CTA TGT GTA GAA 816
Ile Pro Glu Gln Cys Lily His Ser Leu Lys Gly Arg Leu Cys Val Glu
260,265,270
ATA CTA GAG CAA GAA GAC AAA CGA GAA CAA ATT CCT AGA GAT GTT CCT 864
Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Pro Island Arg Asp Val Pro Pro
275 280 285
CCC TCT TT GCA GTG GCG GCC GAT GAA TTC AAC ATG ACG CTG CTC GAT 912
Pro Ser Phe Ala Ala Val Ala Asp Glu Leu Lys Met Leu Leu Leu Asp
290,295,300
CTT GAA AAT AGA GTT GCA CTT CCT CAA CGT CTT TT CCA ACG GAA GCA 960
Leu Glu Asn Arg Val Ala Leu Ala Gln Arg Leu Phe Pro Glu Gl Ala 305 310 315 320
CAA GCT GCA ATG GAG ATC GCC GAA ATG AAC GGA ACA TGT GAG TTC ATA 1008 Gin Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr cys Glu Phe Ile
325 330 335
GTG ACT AGC CTC GAG CCT GAC CGT CTC ACT GGT ACG AGA ACA TCA 1056
Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser
340,345,350
CCG GGT GTA AAC ATA GCA CCT TTC AGA ATC CTA GAA GAG CAT CAA AGT 1104
Pro Gly Val Lys Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser
355 360 365
AGA CTA AAA GCG CTT TCT AAA ACC GTG GAA CTC GGG AAA CGA TTC TTC 1152
Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe
370,375,380
CGC CCC TGT TCC GCA GTG CTC GAC CAG ATG ATAC TGT GAG GAC TTG 1200
Pro Arg Cys Ser Ala Val Leu Asp Gln Met Asn Cys Glu Asp Leu 385 390 395 400
ACT CAA CTG GCT TGC GGA GAA GAC GAC ACT GCT GAG AAA CGA CTA CAA 1248
Thr Gln Leu Ala Cys Gly Asp Glu Asp Thr Ala Glu Arg Leu Gln Lys
405 410 415
AAC AAC CAA AGG TAC ATC GAA ATA CAA GAG ACA CTA AAG AAG CCC TTT 1296
Lys Lys Gln Arg Tyr Met Glu Gln Island Glu Thr Leu Lily Lys Ala Phe
420,425,430
AGT GAG GAC AAT TTG GAA TTA GGA AAT TTG TCC CTC ACA GAT TCG ACT 1344
Ser Glu Asn As Leu Leu Leuly As Leu Leu Leu Ser Asp Asp Ser Ser
435 440 445
TCT TCC ACA TBI AAA TCA ACC GGT GGA AAG AGG TCT AAC CGT AAA CTC 1392
Ser Ser Thr Ser Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu
450 455 460
TCT CAT CGT CGT TCA GACTCTTGCC TCTTAGTGTA ATTTTTGCTG 1440
Ser His Arg Arg Arg *
465,470
TACCATATAA TTCTGTTTTC ATGATGACTG TAACTGTTTA TGTCTATCGT TGGCGTCATA 1500
TAGTTTCGCT CTTCGTTTTG CATCCTGTGT ATTATTGCTG CAGGTGTGCT TCAAACAAAT 1560
GTTGTAACAA TTTGAACCAA TGGTATACAG ATTTCTA 1597
(2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 25
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 470 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 25
Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val
1 5 10 15
Arg Pro Pro Pro Lys Gly Val Ser Glucys Ala Asp Glu Asn Cys Cys
20 25 30
His Val Ala cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr
35 40 45
Leu Ala Phe Ile Phe Lys Island Pro Glu Leu Island Thr Leu Tyr Gln Arg
50 55 60
His Leu Leu Asp Asp Val Val Asp Lys Val Val Glu Asp Asp Leu Val
65 70 75 80
Ile Leu Lys Leu Ala Asn Ile cys Gly Lys Ala Cys Met Lys Leu Leu
85 90 95
Asp Arg Cys Lys Glu Island Ile Val Lys Ser Asn Val Asp Met Val Ser
100 105 110
Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Asp Asp Arg
115 120 125
Arg Lys Glu Gly Leu Leu Val Val Lys Val Lys Lys His Val Ser
130 135 140
Asn Val His Lys Ala Leu Asp Asp Asp Asp Glu Island Leu Val Lys Leu 145 150 155 160
Leu Leu Lys Glu Asp His Asp Asn Leu Asp Asp Ala Cys Ala Leu His
165 170 175
Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys
180 185 190
Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val
195 200 205
Leu His Val Ala Ala Met Arg Glu Lys Gln Leu Leu Leu Leu Leu Leu
210 215 220
Leu Glu Lys Gly Ala Ser Ala Ser Ala Thr Glu Glu Gly Arg Thr 225 230 235 240
Ala Leu Met Ala Lys Gln Ala Thr Ala Glu Cys Asn Asn
245 250 255
Ile Pro Glu Gln cys Lily His Ser Leu Lys Gly Arg Leu cys Val Glu
260,265,270
Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Pro Island Arg Asp Val Pro Pro
275 280 285
Pro Ser Phe Ala Val Ala Ala Thr Gln Leu u Ala cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gin
405 410 415 Lilies Gln Arg lilies Tyr Glu Glu Glu Glu Thr Leu Lys Ala Phe
420,425,430
Ser Glu Asn As Leu Leu Leuly As Leu Leu Leu Ser Asp Asp Ser Ser
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 FOR SEQUENCE SEQ ID NO: 26:
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 1608 base pairs
(B) TYPE: nucleic acid
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: cDNA
(ix) PARTICULARITY:
(A) NAME / KEY: CDS
(B) LOCATION: 43..1608
(D) OTHER INFORMATION: / product = = "modified form of u NIM1"
/ note = = "C-terminal deletion with respect to wild type u NIM1"
(xi) DESCRIPTION OF THE SEQUENCE SEQ ID NO: 26 GATCTCTTA ATTTGTGAAT TTCATATAT CGGAACCTGT TG ATC GAC ACC ACC 54
Met Asp Thr Thr
1
ATT GAT GGA TTC GCC GAT TCT GAAT TAT ATC AGC ACT AGT TTC GTC 102
Asp Gly Phe Island Ala Asp Ser Tire Glu Ser Ser Ser Thr Ser Phe Val
5 10 15 20
GCT ACC GAT ACC AAC GAC TCC ATT ATT GTT TAT CTC GCC GCC GAA CAA 150
Ala Thr Asp Asn Thr Asp Ser Ser Val Val Leu Ala Ala Glu Gln
25 30 35
GTA CTC ACC GGA CCT GAT GTA GCT CTG TGA CTG TTG CTC TCC AAC AGC 198
Val Leu Thr Gly Pro Asp Ser Ser Leu Leu Leu Leu Leu Ser Asn Ser
40 45 50
TTC GAA TCC GTC TTT GAC TCG CCG GAT GAT TTC TAC AGC GAC GCT AAC 246
Phe Glu Ser Val Phe Asp Asp Asp Asp Asp Phe Asp Ser Asp Ala Lys
55 60 65
CTT GTT CTC TCC GAC GGC CGC GAA GTT TCT TTC CAC CGC TGC GTT TTC 294
Leu Val Leu Ser Asp Gly Arg Glue Val Ser Phe His Arg Cys Val Leu
70 75 80
TCA CCC AGA AGC TCT TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAC 342
Ser Ala Ser Ser Phe Phe Ser Lys Ser Ala Leu Ser Ala Ala Lys Lys
85 90 95 100
GAG AAA GAC TCC AAC ACC AACC GCC CCC GTG AAG CTC GAG TTC AAC GAG 390
Glu Lys Asp Asn Ser Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu
105 110 115
ATT GCC AAC CAT GAAT TAC GTC GGT TTC GAT TCG GT GTG GT GT TTC 438
Ile Ala Lys Asp Tire Glu Val Gly Phe Asp Ser Val Val Thr Val Leu
120 125 130
GCT TAT GTT AGC TAC AGC AGA CTG AGM CCG CCG CCT AAA GCA GTT TCT 486
Ala Tyr Val Tyr Ser Ser Arg Pro Arg Pro Pro Pro Lys Gly Val Ser Pro
135 140 145
GAA TGC GCA GAC GAG AAT TGC TGC CAC GTG GCT TGC CGC CCG CCC GTG 534
Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val
150 155 160
GAT TTC ATG TTG GTT GTT CTC TAT TTG TT GCT ATC TTC ATC ATC CCT 582
Asp Phe Met Leu Glu Val Leu Tire Leu Ala Phe Ile Phe Lys Ile Pro 165 170 175 180
GAA TTA ATT ACT CTC TAT CAG AGG CAC TTA TTC GAC GTT GTA GAC AAA 630
Glu Leu Thr Leu Tyr Gln Arg His Leu Leu Asp Asp Val Val Asp Lys
185 190 195
GTT GTT ATA GAG GAC ACA TTC GTT ATA CTC AAG CTT GCT AAT ATA TGT 678
Val Val Ile Clu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys
200 205 210
GGT AAA GCT TGT ATG CTA AAC TTG GAT AGT TGT AAA GAG ATT ATT GTC 726
Gly Lys Ala Cys Met Leu Leu Lys Asp Arg Cys Lys Glu Ile Ile Val
215 220 225
AAC TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA GAG 774
Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu
230-235 240
CTT GTT AAA GAG ATA ATT GAT AGC CGT AAA GAG CTT GGT TTG GAG GTA 822
Leu Val Lys Glu Island Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val 245 250 255 260
CCT AAA GTA AAG AAA CAT GTC TCC AAT GTA CAT AAC GCA CTT GAC TCC 870
Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser
265 270 275
GAT GAT ATT GAG TTA GTC AAC TTG TTG CTT AAA GAG GAT CAC ACC AAT 918
Asp Asp Ile Glu Leu Val Leu Leu Leu Leu Lys Glu Asp His Thr Asn
280,285,290
CTA GAT GAT GCG TGT GCT TTC CT TTC GCT GAC GTT TGC AAT GTG 966
Leu Asp Asp Ala cys Ala Leu His Phe Ala Val Ala Tyr cys Asn Val
295 300 305
AAC ACC GCA ACA GAT CTT TTA AAA CTT GAT CTT GCC GAT GTC AAC CAT 1014
Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His
310,315,320
AGG AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT CCC ATG CGG AAC 1062
Arg Asn Pro Arg Gly Tire Thr Val Leu His Val Ala Ala Met Arg Lys 325 330 335 340
GAG CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA 1110
Glu Pro Gln Leu Leu Leu Leu Leu Leu Glu Lys Gly Ser Ala Ser
345 350 355
GAA GCA ACT TTG GAA GGT AGA ACC GCA CTC ATG ATC GCA AAA CAA GCC 1158
Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ala Lys Gln Ala
360 365 370
ACT ATG CCC GTT GAA TGT AAT ATC ATC CCG GAG CAA TGC AAC CAT TCT 1206
Thr Met Ala Val Glu Cys Asn Asn Island Pro Glu Gln Cys Lys His Ser
375,380,385
CTC AAA GGC CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA CGA 1254
Leu Lys C1y Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys Arg
390 395 400
GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TT GCA GTG CCC GCC GAT 1302
Glu Gln Pro Pro Asp Asp Pro Pro Pro Ala Val Ala Ala Ala Asp 405 410 415 420
GAA TTG AAG ATG ACG CTG CTC GAT CTT GAA AAT AGA GTT GCA CTT GCT 1350
Glu Leu Lys Met Leu Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala
425 430 435
CAA CCT CTT TT CCA ACG GAA GCA CAA GCT GCA ATC GAG ATC GCC CAA 1398 Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ala Glu Island
440 445 450
ATC AAC GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC CGT 1446
Met Lys Gly Thr Cys Glu Phe Island Thr Val Ser Ser Glu Pro Asp Asp Arg
455 460 465
CTC ACT GGT ACG AAC AGA ACA TCA CCG GGT GTA AAC ATA CCA TTC TTC 1494
Leu Thr Gly Thr Lys Arg Thr Pro Ser Gly Val Lys Ile Ala Pro Phe
470 475 480
AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA CCC CTT TCT AAA ACC 1542
Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys Thr 485 490 495 500
GTG GAA CTC GGG AAA CCA TTC TTC CCG CGC TGT TCC GCA GTG CTC GAC 1590
Val Glu Leu Gly Lys Arg Phe Phe Arg Pro Cys Ser Ala Val Leu Asp
505 510 515
CAG ATT ATG AAC TGT TCA 1608 Gln Ile Met Asn Cys *
520 (2) INFORMATION FOR SEQUENCE SEQ ID NO: 27
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 522 amino acids
(B) TYPE: nucleic acid
(D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 27
Thr Asp Asp Thr Asp Asp Ply Asp Ala Ser Ser Ser Glu Gl Ser Ser Ser
1 5 10 15
Thr Ser Phe Val Ala Asp Asp Asn Thr Asp Ser Ser Val Val Leu
20 25 30
Ala Ala Glu Gln Leu Val Leu Thr Gly Asp Ser Val Asp Ala Leu Gln Leu
35 40 45
Ser Ser Asn Ser Phe Glu Ser Phe Asp Asp Asp
50 55 60
Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His
65 70 75 80
Arg Cys Val Leu Ser Ala Ser Ser Ser Phe Phe Ser Lys Ser Ala Leu Ala
85 90 95
Ala Ala Lys Lys Glu Lys Asn Asn Thr Asp Ala Ala Val Lys Leu
100 105 110
Glu Leu Lys Glu Ala Lys Asp Asp Tyr Glu Val Gly Phe Asp Ser Val
115 120 125
Val Thr Val Leu Ala Tyr Val Tyr Ser Arg Ser Arg Arg Pro Pro Pro
130 135 140
Lys Gly Val Ser Glu Cys Asp Ala Asp Glu Asn Cys cys His Val Ala Cys 145 150 155 160
Arg Pro Ala Val Asp Phe Met Leu Glu Leu Val Leu Leu Ala Phe Ile
165 170 175
Phe Lys Island Pro Glu Leu Thr Island Tyr Tyr Gln 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
Ala Asn Ile Cys Gly Lys Ala cys Met Lys Leu Leu Asp Arg Cys Lys
210 215 220
Glu Island Val Lys Island Ser Asn Val Asp Val Val Ser Leu Glu Lily Ser 225 230 235 240
Leu Pro Glu Glu Leu Val Lys Glu Ile Asp Asp Arg Arg Lys Glu Leu
245 250 255
Cly Leu Glu Val Pro Lys Val Lys Lily His Val Ser Asn Val His Lys
260,265,270
Ala Leu Asp Asp Asp Asp Glu Ile Leu Val Leu Lys Leu Leu Leu Lys Glu
275 280 285
Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala
290,295,300
Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Leu Leu Leu 305 310 315 320
Asp Val Asn His Arg Asn Pro Arg Gly Tire Thr Val Leu His Val Ala
325 330 335
Ala Met Arg Lys Glu Gln Leu Leu Leu Leu Leu Leu Glu Lys Gly
340,345,350
Ala Ser Ala Ser Ala Thr Glu Glu Gly Arg Thr Ala Leu Met Ile
355 360 365
Ala Lys Gln Ala Thr Ala Val Glu Cys Asn Asn Glu Gln Glu
370,375,380
Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln
385 390 395 400
Glu Asp Lys Arg Glu Gln Pro Island Arg Asp Pro Pro Val Ser Phe Ala
405 410 415
Val Ala Ala Asp Glu Leu Lys Met Leu Leu Leu Asp Leu Glu Asn Arg
420,425,430
Val Ala Leu Ala Gln Arg Leu Phe Pro Glu Ala Gla Gla Ala Ala Met
435 440 445
Clu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu
450 455 460
Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Pro Ser Gly Val Lys
465 470 475 480
Ala Island Pro Phe Arg Island Leu Glu Glu His Gln Ser Arg Leu Lys Ala
485 490 495
Leu Ser Lys Thr Glu Val Leu Gly Lys Arg Phe Pro Phe Arg Cys Ser
500 505 510
Ala Val Leu Asp Gln Island Met Asn Cys *
515 520 (2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 28
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 1194 base pairs
(B) TYPE: nucleic acid
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: cDNA
(ix) PARTICULARITY
(A) NAME / KEY: CDS
(B) LOCATION: 1..1194
(D) OTHER INFORMATION: / product = "modified form of NIM1"
/ note = "N-terminal / C-terminus chimera"
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 28
ATG GAT TCC GTT GTG GTT ACT TTG GCT TAT GTT AGC TAC AGC AGA GTG 48
Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val
1 5 10 15
AGA CCC CCG CCT AAA GGA GTT TCT GAA TGC GCA GAC GAG AAT TGC TGC 96
Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Asp Ala Asp Glu Asn Cys Cys
20 25 30
CTC CTC GCT TGC CGG CCG CCC GTG GAT TTC ATG TTG GAG GTT CTC TAT 144
His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr
35 40 45
TTG GCT TTC ATC TTC AAG ATC CCT GAA TTA ACT ATT CTC TAT CAG AGG 192
Leu Ala Phe Ile Phe Lys Island Pro Glu Leu Island Thr Leu Tyr Gln Arg
50 55 60
CAC TTA TTG GAC GTT GTA GAC AAA GTT GTT GAC ATA GAC ACA TTG GTT 240
His Leu Leu Asp Asp Val Val Asp Lys Val Val Glu Asp Asp Leu Val
65 70 75 80
ATA CTC AAC CTT GCT AAT ATA TGT GGT AAA GCT TGT ATG AAC CTA TTG 288
Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu
85 90 95
GAT AGA TGT AAA GAG AT ATT GTC AAC TCT AAT AT GTA GAT ATG GTT AGT 336
Asp Arg Cys Lys Glu Island Ile Val Lys Ser Asn Val Asp Met Val Ser
100 105 110
CTT GAA AAC TCA TTG CCG GAA GAG CTT GTT AAA GAG ATA ATT GAT AGA 384
Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Asp Asp Arg
115 120 125
CGT AAA GAG CTT GGT TTG GAG GTA CCT AAA GTA AAA AAA CAT GTC TCG 432
Arg Lys Glu Gly Leu Leu Val Val Lys Val Lys Lys His Val Ser
130 135 140
AAT GTA CAT AAC GCA CTT GAC TCC GAT GAT ATT GAG TTA GTC AAC TTG 480
Asn Val His Lys Ala Leu Asp Asp Asp Asp Glu Island Leu Val Lys Leu 145 150 155 160
CTT TTG AAA GAG GAT CAC ACC AAT CTA GAT GAT CCC TGT GCT CTT CAT 528
Leu Leu Lys Glu Asp His Asp Asn Leu Asp Asp Ala Cys Ala Leu His
165 170 175
TTC GCT GTT GCA TAT TOC AAT GTG ACC AAG ACC GCA ACATAT CTT TTA AAA 576
Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys
180 185 190
CTT GAT CTT GCC GAT GTC AAC CAT AGG AAT CCG AGG GCA TAT ACG GTG 624
Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val
195 200 205
CTT CAT GTT GCT CCC ATG CGC AAG GAG CCA CAA TTG ATA CTA TCT CTA 672
Leu His Val Ala Ala Met Arg Glu Lys Gln Leu Leu Leu Leu Leu Leu
210 215 220
TTG GAA AAAA GGT GCA AGT GCA GAA GAA TCA ACT TTG GAA GGT AGA ACC 720
Leu Glu Lys Gly Ala Ser Ala Ser Ala Thr Glu Glu Gly Arg Thr 225 230 235 240
GCA CTC ATG ATC GCA AAA CAA GCC ACT ATG CCC GTT GAA TGT AAT AAT 768
Ala Leu Met Ala Lys Gln Ala Thr Met Ala Gln Cys Asn Asn
245 250 255
ATC CCG GAG CAA TGC AAC CAT TCT CTC AAA GGC CGA CTA TGT GTA GAA 816
Ile Pro Glu Gln Cys Lily His Ser Leu Lys Gly Arg Leu Cys Val Glu
260,265,270
ATA CTA GAG CAA GAA GAC AAA CGA GAA CAA ATT CCT AGA GAT GTT CCT 864
Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Pro Island Arg Asp Val Pro Pro
275 280 285
CCC TCT TT GCA GTG CCC GCC GAAT GAA TTG AAC ATG ACG CTC CTC GAT 912
Pro Ser Phe Ala Ala Val Ala Asp Glu Leu Lys Met Leu Leu Leu Asp
290,295,300
CTT GAA AAT AGA GTT GCA CTT GCT CAA CGT CTT TT CCA ACG GAA GCA 960
Leu Glu Asn Arg Val Ala Leu Ala Gln Arg Leu Phe Pro Glu Gl Ala 305 310 315 320
CAA GCT GCA ATG GAG ATC GA GA GA ATG GGA ACA TGT GAG TTC ATA 1008
Gln Ala Ala Met Glu Ala Glu Island Met Lys Gly Thr Cys Glu Phe Island
325 330 335
GTG ACT AGC CTC GAG CCT GAC CGT CTC ACT GGT ACG AAC AGA ACA TCA 1056
Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser
340,345,350
CCG GGT GTA AAC ATA GCA CCT TTC AGA ATC CTA GAA GAG CAT CAA AGT 1104
Pro Gly Val Lys Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser
355 360 365
AGA CTA AAA GCG CTT TCT AAA ACC GTG GAA CTC GGG AAA CGA TTC TTC .1152
Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe
370,375,380
CGC CCC TGT TCC GCA GTG CTC GAC CAG ATG ATT AAC TGT TGA 1194
Pro Arg Cys Ser Ala Val Leu Asp Gln Met Asn Cys * 385 390 395 (2) INFORMATION FOR SEQUENCE SEQ ID NO: 29
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 398 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF THE SEQUENCE SEQ ID NO: 29
Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val
1 5 10 15
Arg Pro Pro Pro Lys Gly Val Ser Glu cys Asp Ala Asp Glu Asys Cys Cys
20 25 30
His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr
35 40 45
Leu Ala Phe Ile Phe Lys Island Pro Glu Leu Island Thr Leu Tyr Gln Arg
50 55 60
His Leu Leu Asp Asp Val Val Asp Lys Val Val Glu Asp Asp Leu Val
65 70 75 80
Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu
85 90 95
Asp Arg Cys Lys Glu Island Ile Val Lys Ser Asn Val Asp Met Val Ser
100 105 110
Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Asp Asp Arg
115 120 125
Arg Lys Glu Gly Leu Leu Val Val Lys Val Lys Lys His Val Ser
130 135 140
Asn Val His Lys Ala Leu Asp Asp Asp Asp Glu Island Leu Val Lys Leu 145 150 155 160
Leu Leu Lys Glu Asp His Asp Asn Leu Asp Asp Ala Cys Ala Leu His
165 170 175
Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys
180 185 190
Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val
195 200 205
Leu His Val Ala Ala Met Arg Glu Lys Gln Leu Leu Leu Leu Leu Leu
210 215 220
Leu Glu Lys Gly Ala Ser Ala Ser Ala Thr Glu Glu Gly Arg Thr 225 230 235 240
Ala Leu Met Ala Lys Gln Ala Thr Ala Glu Cys Asn Asn
245 250 255 Pro Glu Island Gln Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu
260,265,270
Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Pro Island Arg Asp Val Pro Pro
275 280 285
Pro Ser Phe Ala Ala Val Ala Asp Glu Leu Lys Met Leu Leu Leu Asp
290,295,300
Leu Glu Asn Arg Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala
305 310 315 320
Gln Ala Ala Met Glu Ala Glu Island Met Lys Gly Thr Cys Glu Phe Island
325 330 335
Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser
340,345,350
Pro Gly Val Lys Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser
355 360 365
Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe
370,375,380
Pro Arg cys Ser Ala Leu Val Asp Gln Ile Met Asn Cys *
385 390 395 (2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 30
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 786 base pairs
(B) TYPE: nucleic acid
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: cDNA
(ix) PARTICULARITY
(A) NAME / KEY: CDS
(B) LOCATION: 1.786
(D) OTHER INFORMATION: product = "modified form of NIM1"
/ note = "Ankyrin domains of the NIM1".

(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 30
ATG GAC TCC AAC AAC ACC GCC GCC CTC AAC CTC GAG CTT AAG GAG ATT 48
Met Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu Ile
1 5 10 15
GCC AAG GAT TAC GAA GTC GGT TTC GAT TCC GT GTG ACT GTT TTG GCT 96
Ala Lys Asp Tire Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Ala
20 25 30
TAT GTT AGC TAC AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT GAA 144
Tyr Val Tyr Ser Ser Arg Pro Arg Pro Pro Lys Gly Val Ser Glu
35 40 45
TOC GCA GAC GAG AAT TGC TGC CAC GTG GCT TGC CGG CCG CCC GTG GAT 192
Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp
50 55 60
ATG TTG GAG GTT CTC TAT TTG GCT TTC ATC TTC ATC ATC CCT GAA 240
Phe Met Leu Glu Val Leu Tire Leu Ala Phe Ile Phe Lys Island 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 Gln Arg Leu Leu Leu Asp Asp Val Val Asp Lys Val
85 90 95
GTT ATA GAG GAC ACA TTG GTT ATA CTC AAC CTT GCT AAT ATA TGT GGT 336
Val Glu Island Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Gly
100 105 110
AAA GCT TGT ATG AAC CTA TTG GAT AGT TGT AAA GAG ATT ATT GTC AAG 384
Lys Ala Cys Met Leu Leu Lys Asp Arg Cys Lys Glu Ile Ile Val Lys
115 120 125
TCT AAT GTA GAT ATG GTT AGT CTT GAA AAC TCA TTC CCG GAA GAG CTT 432
Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu
130 135 140
GTT AAA GAG ATA ATT GAT AGM CGT AAA GAG CTT GGT TTG GAG GTA CCT 480
Val Lys Glu Island Asp Asp Arg Arg Lys Glu Leu Gly Leu Glu Pro Val 145 150 155 160
AAA GTA AAG AAA CAT GTC TCG AAT GTA CAT AAG GCA CTT GAC TCG GAT 528
Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser Asp
165 170 175
GAT ATT GAG TTA GTC AAG TTG TTG CTT AAA GAG GAT CAC ACC AAT CTA 576
Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Clu Asp His Thr Asn Leu
180 185 190
CAT GAT GCG TGT GCT CTT CAT TTC GCT GTT GTT TAT TGC AAT GTG AAG 624
Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val Lys
195 200 205
ACC GCA ACA GAT CTT TTA AAA CTT GAT CTT CCC GAT GTC AAC CAT AGG 672
Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His Arg
210 215 220
AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT CCC ATG CGG AAC GAG 720
Asn Pro Arg Gly Tire Thr Val Leu His Val Ala Ala Met Arg Lys Glu 225 230 235 240
CCA CAA TTG ATA CTA TCT CTA TTC GAA AAA GGT GCA AGT GCA TCA GAA 768
Pro Gln Leu Leu Leu Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu
245 250 255
ACTG GCA TTG GAA GGT TGA 786
Ala Thr Leu Glu Gly *
260 (2) INFORMATION FOR SEQUENCE SEQ ID NO: 31
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 262 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 31
Met Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu Ile
1 5 10 15
Ala Lys Asp Tire Glu Val Cly Asp Phe Val Val Thr Val Leu Val Ala
20 25 30
Tyr Val Tyr Ser Ser Arg Pro Arg Pro Pro Lys Gly Val Ser Glu
35 40 45
Cys Ala Asp Glu Asn cys Cys His Val Ala Cys Arg Pro Ala Val Asp
50 55 60
Phe Met Leu Clu Val Leu Tire Leu Ala Phe Ile Phe Lys Ile Pro Glu
65 70 75 80
Leu Ile Thr Leu Tyr Gln Arg Leu Leu Leu Asp Asp Val Val Asp Lys Val
85 90 95
Val Glu Island Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Gly
100 105 110
Lys Ala Cys Met Leu Leu Lys Asp Arg Cys Lys Glu Ile Ile Val Lys
115 120 125
Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu
130 135 140
Val Lys Glu Island Asp Asp Arg Arg Lys Glu Leu Gly Leu Glu Pro Val 145 150 155 160
Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser Asp
165 170 175
Asp Glu Leu Val Leu Leu Leu Leu Glu Asp His Thr Asn Leu
180 185 190
Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr cys Asn Val Lys
195 200 205
Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His Arg
210 215 220
Asn Pro Arg Gly Tire Thr Val Leu His Val Ala Ala Met Arg Lys Glu 225 230 235 240
Pro Gln Leu Leu Leu Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu
245 250 255
Ala Thr Leu Clu Gly *
260 (2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 32
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: / desc = "oligonucleotide"
(xi) DESCRIPTION OF THE SEQUENCE SEQ ID NO: 32 CAACAGCTTC GAAGCCGTCT TTGACGCGCC GGATG 35
(2) INFORMATION FOR SEQUENCE SEQ ID NO: 33
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: / desc = "oligonucleotide
(xi) DESCRIPTION OF THE SEQUENCE SEQ ID NO: 33 CATCCGGCGC GTCAAAGACG GCTTCGAAGC TGTTG 35
(2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 34
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: / desc = "oligonucleotide"
(xi) DESCRIPTION OF THE SEQUENCE SEQ ID NO: 34 GGAATTCAAT GGATTCGGTT GTGACTGTTT TO 32
(2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 35
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: / desc = "oligonucleotide"
(xi) DESCRIPTION OF THE SEQUENCE SEQ ID NO: 35
GGAATTCTAC AAATCTGTAT ACCATTGG 28
(2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 36
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: / desc = "oligonucleotide"
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 36
CGGAATTCGA TCTCTTTAAT TTGTGAATTT C 31
(2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 37
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: / desc = "oligonucleotide"
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 37:
GGAATTCTCA ACAGTTCATA ATCTGGTCG 29
(2) INFORMATION FOR SEQUENCE SEQ ID NO: 38:
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: / desc = "oligonucleotide"
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 38
GGAATTCAAT GGACTCCAAC AACACCGCCG C 31
(2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 39
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) NATURE OF THE STRANDS: single strand
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: / desc = "oligonucleotide"
(xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 39
GGAATTCTCA ACCTTCCAAA GTTGCTTCTG ATG 33

Claims (34)

1. DNA molecule, characterized in that it encodes a modified form of an NIAI protein. 2. DNA molecule according to claim 1, characterized in that it plays the role of a dominant-negative regulator of the signal transduction pathway of the SAR. 3. DNA molecule according to claim 1, characterized in that said modified form of the NIM i protein has alanines instead of serines on the amino acid positions corresponding to positions 55 and 59 of the sequence SEQ ID NO: 3. 4. DNA molecule according to claim 3, characterized in that said modified form of the NIMI protein comprises the amino acid sequence presented in the sequence SEQ ID NO: 23. 5. DNA molecule according to claim 4, characterized in that said DNA molecule comprises the nucleotide sequence shown in the sequence SEQ ID NO: 22 and all of the DNA. 6. DNA molecule according to claim 1, characterized in that the modified form of the NIMI protein is a truncated version of the NIMI gene product. 7. A DNA molecule according to claim 1, characterized in that said modified form of the NIMI protein has an N-terminal truncation of amino acids corresponding approximately to the amino acid positions 1-125 of the sequence SEQ ID NO: 3. 8. DNA molecule according to claim 7, characterized in that said modified form of the NIMI protein comprises the amino acid sequence presented in the sequence SEQ ID NO: 25. 9. DNA molecule according to claim 8, characterized in that said DNA molecule comprises the nucleotide sequence presented in the sequence SEQ ID NO: 24. 10. DNA molecule according to claim 1, characterized in that said modified form of the protein NIM 1 has a C-terminal truncation of amino acids corresponding approximately to the amino acid positions 522-593 of the sequence SEQ ID NO 3.  He. DNA molecule according to claim 10, characterized in that said modified form of the NIMI protein comprises the amino acid sequence presented in the sequence SEQ ID NO: 27. 12. DNA molecule according to claim 11, characterized in that said DNA molecule comprises the nucleotide sequence presented in the sequence SEQ ID NO: 26. 13. DNA molecule according to claim 1, characterized in that said modified form of the NIM I protein has an N-tefminal truncation of amino acids corresponding approximately to the amino acid positions 1 to 125 of the sequence SEQ ID NO And C-terminal truncation of amino acids corresponding approximately to amino acid positions 522-593 of the sequence SEQ ID NO: 3. 14. DNA molecule according to claim 1, characterized in that said modified form of the NIMI protein comprises the amino acid sequence presented in the sequence SEQ ID NO: 29. 15. DNA molecule according to claim 14, characterized in that said DNA molecule comprises the nucleotide sequence presented in the sequence SEQ ID NO: 28. 16. DNA molecule according to claim 1, characterized in that said modified form of the NIM 1 protein consists essentially of ankyrin units corresponding approximately to the amino acid positions 103-362 of the sequence SEQ ID NO: 3. 17. DNA molecule according to claim 16, characterized in that said modified form of the NIM i protein comprises the amino acid sequence presented in the sequence SEQ ID NO: 31. 18. DNA molecule according to claim 1, characterized in that said DNA molecule comprises the nucleotide sequence presented in the sequence SEQ ID NO: 30. 19. A DNA molecule according to claim 1, characterized in that said DNA molecule hybridizes under the following conditions to a nucleotide sequence selected from the group comprising the sequences SEQ ID NO: 22, SEQ ID NO: 24 , SEQ ID NO: 26, SEQ ID NO: 28 and SEQ ID NO: 30: hybridization in 1% BSA 520 mM NaP04, pH 7.2; sodium lauryl sulphate 7%; EDTA 1 mM; 250 mM sodium chloride at 55 ° C for 18 to 24 hours, and wash in 6XSSC for 15 minutes (X3), 3XSSC for 15 minutes (X1) at 55 ° C. 20. Chimeric gene, characterized in that it comprises a promoter having an activity in plants, linked by operational binding to the DNA molecule according to any one of claims 1 to 19. A recombinant vector comprising the chimeric gene of claim 20, characterized in that said vector can be stably transformed into a host cell. 22. A method for activating SAR in a plant, which consists in transforming the plant with a DNA molecule, characterized in that it encodes a modified form of a NIM I protein, characterized in that said modified form of the protein NIM 1 is expressed in said transformed plant and activates the SAR in said plant. 23. A method for conferring resistance to a broad spectrum of diseases to a plant, which consists in transforming the plant with the recombinant vector of claim 21, characterized in that said modified form of the protein NIM 1 is expressed in said transformed plant. and confers plant audit resistance to a broad spectrum of diseases. 24. A method for conferring on a plant a CIM phenotype, which consists of transforming the plant with the recombinant vector of claim 21, characterized in that said modified form of the protein NIM i is expressed in said transformed plant and confers on this plant a CIM phenotype. Host cell, characterized in that it is stably transformed with the vector of claim 21. 26. Host cell according to claim 25, characterized in that it is a plant cell. Plant, plant cells and their descendants, comprising the chimeric gene according to claim 20, characterized in that it has a broad spectrum of disease resistance. 28. Plant, plant cells and their descendants, characterized in that a NIM1 protein involved in the signal transduction cascade leading to the systemic resistance acquired in the plants is expressed in said transformed plant, at levels higher than in a wild type plant. Plant, plant cells and their descendants according to claim 27 or 28, characterized in that said plant is selected from the group consisting of gymnosperms, monocotyledons and dicotyledons. 30. Plant, plant cells and their descendants according to claims 27 or 28, characterized in that said plant is a crop plant. Plant, plant cells and their descendants according to claim 27 or 28, characterized in that said plant is selected from the group consisting of the following plants: rice, wheat, barley, rye, corn, potato, carrot, potato sweet, sugar beet, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, aubergine, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soy, tobacco, tomato, sorghum and sugar cane. 32. A method for conferring a CIM phenotype on a plant cell, a plant and their descendants, which consists in transforming the plant with the recombinant vector comprising the chimeric gene comprising a promoter having an activity in the plants, linked by an operational link to the molecule. DNA encoding a NIMI protein involved in the signal transduction cascade leading to acquired systemic resistance in plants, characterized in that said vector can undergo stable transformations in a host, wherein said NIMI protein is expressed in said plant transformed at higher levels than in a wild type plant. 33. A method for activating systemic resistance acquired in a plant cell, a plant and their descendants, which comprises transforming the plant with the recombinant vector comprising the chimeric gene comprising a promoter having an activity in plants, linked by operational link to the plant. DNA molecule which encodes a NIM1 protein involved in the signal transduction cascade leading to acquired systemic resistance in plants, characterized in that said vector can undergo stable transformations in a host, where said NIMI protein is expressed in said transformed plant at higher levels than in a wild type plant. 34. A method for conferring resistance to a broad spectrum of diseases to a plant cell, a plant and their descendants, which comprises transforming the plant with the recombinant vector comprising the chimeric gene comprising a promoter having an activity in plants, bound by operational binding to the DNA molecule which encodes a NIMI protein involved in the signal transduction cascade leading to acquired systemic resistance in plants, characterized in that said vector can undergo stable transformations in a host, wherein said NIMi protein is expressed in said transformed plant at higher levels than in a wild-type plant. 35. Use of a transgenic plant or its descendants, characterized in that they comprise a chimeric gene according to claim 20 in the context of an agricultural technique. 36. A commercial bag, characterized in that it comprises seeds of a transgenic plant comprising at least one modified form of the protein NIM 1 or of a protein NIM 1 which is expressed in said transformed plant, at higher levels. in a wild-type plant, together with a suitable carrier in an amount sufficient to act as a dominant-negative regulator of the SAR signal transduction pathway, together with corresponding label instructions to their use, to give plants resistance to a broad spectrum of diseases.