NL1007779C2 - Methods of using the NIM1 gene to confer disease resistance to plants. - Google Patents

Description

Methods for rearranging the NIM1 gene to confer disease resistance to plants.

The present invention generally relates to disease resistance in plants with a broad spectrum of activity, including the phenomenon of systemic acquired resistance (SAR). More particularly, the present invention relates to the recombinant expression of wild-type and altered forms of the NIM1 gene, which is involved in the signal transduction cascade leading to SAR, to provide transgenic plants with a resistance to broad disease diseases. spectrum of action. The present invention further relates to high-level expression of the cloned NIM1 gene in transgenic plants that have broad spectrum disease resistance.

Plants are constantly burdened by a wide variety of pathogenic organisms, including viruses, bacteria, fungi and nematodes. Crop plants are particularly vulnerable as they are usually grown as genetically uniform monocultures; when an illness strikes, losses can be serious. Most plants, however, have their own natural defense mechanisms against pathogenic organisms. Natural variation in resistance to plant pathogens has been recognized by plant breeders and pathologists and grown in a wide variety of crop plants. These natural disease resistance genes often provide high levels of resistance to, or immunity to, pathogens.

Systemically acquired resistance (SAR) is a component of the complex system that plants use to defend themselves against pathogens (Hunt and Ryals, Crit. Rev. in Plant Sci. 15, 583-606 (1996), herein in its entirety). incorporated by reference; Ryals et al., Plant Cell 8, 1809-1819 (1996), incorporated herein by reference in its entirety. See also U.S. Patent 5,614,395, incorporated by reference in its entirety). SAR is a particularly important aspect of the reach of plants against pathogens as it provides a pathogenic agent, systemic resistance to a broad spectrum of infectious agents, including viruses, bacteria and fungi. When the SAR signal transduction pathway is blocked, plants become more sensitive to pathogens that usually cause disease, and also become sensitive to some infecting agents that would typically not cause disease (Gaffney et al., 1007779 2

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

10 Seen as a concept, the SAR response can be divided into two stages.

In the initiation phase, an infection with a pathogen is recognized and a signal is passed through the phloem to distant tissues. This systemic signal is detected by target cells, which respond by expression of both SAR genes and disease resistance. The maintenance phase of SAR concerns the time span, ranging from weeks to the entire life of the plant, during which the plant is in a nearly stable state, and the resistance to disease is maintained (Ryals et al., 1996).

Salicylic acid (SA) accumulation appears to be required for SAR signal transduction. Plants that cannot accumulate SA, by treatment with specific inhibitors, epigenetic repression of phenylalanine ammonialyase, or transgenic expression of salicylate hydroxylase, which specifically degrades SA, cannot induce SAR gene expression or disease resistance (Gaffney et al., 1993; Delaney et al., 1994; Mauch-Mani and Slusarenko 1996; Maher et al., Proc. Natl. Acad. Sci. USA 91, 7802-7806 (1994), incorporated herein by reference in its entirety; Pallas et al., Plant J 10,

25 281-293 (1996), incorporated herein by reference). Although it has been suggested that SA

if the systemic signal serves it is currently controversial, and to date it is only known that when SA cannot accumulate, SAR signal transduction is blocked (Pallas et al., 1996; Shulaev et al., 1995, Plant Cell 7, 1691-1701 (1995), incorporated herein by reference in its entirety; Vemooij et al., Plant Cell 6, 30 959-965 (1994), incorporated herein by reference in its entirety).

Recently, Arabidopsis has emerged as 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., Mol. Plant-Microbe Interact 6. 692-698 1007779 3 (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 Plant-Microbe Interact. 7, 378-383 (1994), incorporated herein by reference in its entirety; Dempsey and Klessig, Bulletin de L'Institut Pasteur 93, 167-5 186 (1995), herein in its entirety by reference included). SAR in Arabidopsis has been shown to be activated by both pathogens and chemicals, such as SA, 2,6-dichloroisonicotinic acid (INA) and benzo (1,2,3) thiadiazole-7-carbothionic acid S-methyl ester (BTH) (Uknes et al., 1992; Vemooij et al., Mol. Plant-Microbe Interact. 8, 228-234 (1995), incorporated herein by reference in its entirety; 10 Lawton et al., Plant J. 10, 71-82 (1996 ), incorporated herein by reference in its entirety). Following treatment with INA or BTH or infection with a pathogen, at least three pathogenesis-related (PR) protein genes, namely PR-1, PR-2 and PR-5, are induced simultaneously with the development of resistance (Uknes et al. 1992, 1993). In the tobacco plant, the most well-known species, treatment with a pathogen or an immunizing compound induces the expression of at least nine sets of genes (Ward et al., Plant Cell 3, 1085-1094 (1991), herein in full reference included). Transgenic disease resistant plants have been obtained by transforming plants with different SAR genes (U.S. Patent No. 5,614,395).

A number of Arabidopsis mutants with modified SAR signal transduction (Delaney, 1997) have been isolated. The first of these mutants are the so-called Isd ("lesions stimulating disease") mutants and acdl ("accelerated cell death") mutants (Dietrich et al., Cell 77, 551-563 (1994), hereby incorporated by reference in their entirety Greenberg et al., Cell 77, 551-563 (1994), incorporated by reference in its entirety). These mutants all show some spontaneous necrotic lesion formation on their leaves, increased levels of SA, mRNA accumulation for the SAR genes, and significantly increased disease resistance. At least seven different mutant 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 group of mutants are cim ("constitutive immunity") mutants (Lawton et al., 1993 "The molecular biology of systemic aquired resistance" in "Mechanisms of Defense Responses in Plants", B. Fritig and M. Legrand, eds ( Dordrecht, The Netherlands: Kluwer Academic 1 007779 4

Publishers), pp. 422-432 (1993), incorporated herein by reference in its entirety).

See also International PCT Application WO 94/16077, both of which are incorporated herein by reference in their entirety. Like / sd mutants and acd2, cim mutants show increased SA and SAR gene expression and resistance, but unlike 5 Isd or acd2, they show no detectable lesions on their leaves, cprl ("constitutive expresser of PR genes") may type chn mutant; however, since the presence of microscopic lesions on the leaves of cprl is not excluded, cprl may be a type of Isd mutant (Bowling et al., Plant Cell 6, 1845-1857 (1994), incorporated herein by reference in its entirety).

10 Mutants blocked in the SAR signaling have also been isolated. ndrl ("non-race-specific disease resistance) is a mutant that allows growth of both Pseudomonas syringae containing various avirulence genes, as well as normally avirulent isolates of Peronospora parasitica (Century et al., Proc. Natl. Acad. Sci. USA 92 , 6597-6601 (1995), incorporated herein by reference in its entirety) Apparently, this mutant is blocked early in the SAR signaling.nprl ("nonexpresser of PR genes") is a mutant expressing the SAR signaling pathway. cannot induce after treatment with INA (Cao et al., Plant Cell 6, 1583-1592 (1994), incorporated by reference in its entirety), eds ("enhanced disease susceptibility") mutants have been isolated based on their bacterial potential Support infection following inoculation with a low concentration of bacteria (Glazebrook et al., Genetics 143, 973-982 (1996), incorporated herein by reference in its entirety; Parker et al., Plant Cell 8, 2033-2045 ( 1996), herein in its entirety by far change included). Certain eifc mutants have a phenotype very similar to nprl, and eds5 and eds52 have recently been shown to be allelic to nprl (Glazebrook et al., 1996). niml ("noninducible immunity") is a mutant that supports the growth of P. parasitica (i.e., the cause of downy mildew disease) after treatment with INA (Delaney et al., 1995; International Application WO 94/16077). Although niml can accumulate SA after infection with a pathogen, it cannot induce SAR gene expression or disease resistance, suggesting that the mutation blocks the pathway downstream of SA, niml is also limited in its ability to respond to INA or BTH, suggesting that the blockage is downstream of the action of these chemicals (Delaney et al., 1995; Lawton et al., 1996).

1 n - q 5

Recently, two allelic Arabidopsis genes have been isolated and characterized, the mutants of which are responsible for the niml and nprl phenotypes, respectively (Ryals et al., Plant Cell 9, 425-439 (1997), incorporated herein by reference in its entirety; Cao et al., Cell 88, 57-63 (1997), incorporated herein by reference in its entirety). The wild-type MM7 gene product is involved in the signal transduction cascade leading to both SAR and gene-by-gene disease resistance in Arabidopsis (Ryals et al., 1997). Ryals et al., 1997 also report the isolation of five further alleles of niml exhibiting different phenotypes ranging from slightly limited in chemically induced PR-1 gene expression and resistance to fungi to very strongly blocked. Transformation of the wild-type NPR1 gene into nprl mutants not only complements the mutations, which restores the response of SAR induction to PR gene expression and disease resistance, but also made the transgenic plants more resistant to infection with P. syringae in the absence of SAR induction (Cao et al., 1997).

NF-kB / IkB sienal transduction pathways NF-κΒ / ΙκΒ signaling pathways have been reported as involved in disease resistance responses in a wide variety of organisms ranging from Drosophila to 20 mammals. In mammals, NF-κΒ / ΙκΒ signal transduction can be induced by a wide variety of stimuli, including exposure of the cells to lipopolysaccharide, tumor necrosis factor, interleukin 1 (IL-1), or virus infection (Baeuerle and Baltimore, Cell 87, 13-20 ( 1996); Baldwin, Annu Rev. Immunol. 14, 649-681 (1996)). The activated pathway leads to the synthesis of a number of 25 different factors involved in inflammatory and immune responses, such as IL-2, IL-6, IL-8 and granulocyte / macrophage colony stimulating factor (deMartin et al., Gene 152, 253 255 (1995)). In studies in transgenic mice, disabling NF-κΒ / ΙκΒ signal transduction leads to an impaired immune response, including increased susceptibility to bacterial and viral pathogens (Beg and Baltimore, 30 Science 274, 782-784 (1996); Van Antwerp et al., Science 274, 787-789 (1996); Wang et al., Science 274, 784-787 (1996); Baeuerle and Baltimore (1996)). In Arabidopsis, SAR is functionally analogous to inflammation, in that normal resistance processes are potentiated after SAR activation 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). Furthermore, inactivation of the route leads to increased susceptibility to bacterial, viral and fungal pathogens. It is noteworthy that SA has been reported to block NF-κΒ activation in mammalian cells (Kopp and Ghosh, Science 265, 956-959 (1994)), while SA activates signal transduction in Arabidopsis. Bacterial infection of Drosophila activates a signal transduction cascade leading to the synthesis of a number of fungi-directed proteins such as cercropin B, defensin, diptericin and drosomycin (lp et al., Cell 75, 753-763 (1993); Lemaitre et 10 al., Cell 86, 973-983 (1996)). This induction is dependent on the gene product of dorsal and dif two NF-κΒ homologues, and is suppressed by cactus, an IkB homologue in the fly. Mutants with reduced synthesis of the counterfungi and bacteria targeting bacteria have a greatly reduced resistance to infection.

15 Despite extensive research into and use of high-quality and intensive crop protection measures, including the genetic transformation of plants, disease-related losses still run into billions of dollars annually. There is therefore an ongoing need to develop new crop protection measures based on the ever-increasing understanding of the genetic basis of resistance to plant diseases.

The following definitions will contribute to the understanding of the present invention.

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

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

5 Protoplast: a plant cell without a cell wall.

Progeny plant: a sexually or asexually derived plant of a future generation, which includes, but is not limited to, plants of the offspring.

Transgenic plant: a plant with a stably introduced recombinant DNA into its genome.

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

Recombinant DNA technoloeia: technology that provides 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, transfer of DNA into protoplast (s) or cell (s) in various forms, including, for example, (1) naked DNA in circular, linear or supercoiled forms, (2) DNA that included in nucleosomes or chromosomes or nuclei or parts thereof, (3) DNA complexed or associated with other molecules, (4) DNA included in liposomes, spheroplasts, cells or protoplasts, or (5) DNA transferred from organisms other than the host organism (e.g. Agrobacterium tumefiaciens). These and other different methods of introducing the recombinant DNA into cells are known in the art and can be used to produce the transgenic plant cells or transgenic plants of the present invention.

Recombinant DNA technology also includes the homologous recombination techniques described in Treco et al., WO 94/12650 and Treco et al., WO 95/31560, which can be used to increase peroxidase activity in a monocotile plant. More specifically, regulatory regions (e.g., promoters) can be introduced into the plant genome to enhance expression of the endogenous peroxidase.

Also, the recombinant DNA technology involves introducing a 1007779 8 sequence encoding a peroxidase that does not include selected expression signals into a monocotile plant and examining the transgenic monocotile plant for increased expression of peroxidase as caused by endogenous control sequences in the monocotile plant. This would lead to an increase in the copy number of the peroxidase coding sequences in the plant.

The original insertion of the recombinant DNA into the genome of the R0 plant is not defined as accomplished by traditional plant breeding methods, but rather as engineering methods as described herein. Following the initial insertion, the transgenic descendants can be propagated using essentially traditional culture techniques.

Chimeric gene: a DNA molecule comprising at least two heterologous parts, ie parts derived from pre-existing DNA sequences unrelated in their previous state, these sequences preferably provided by recombinant DNA technology.

Expression cassette: a DNA molecule comprising a promoter and a terminator, between which a coding sequence may be inserted.

Coding sequence: a DNA molecule that, when transcribed and translated, results in the formation of a polypeptide or protein. Gene: a discrete chromosomal region comprising a regulatory DNA sequence responsible for controlling expression, that is, transcription and translation, and a coding sequence that is transcribed and translated to provide a specific polypeptide or protein.

The present invention includes identifying, isolating and characterizing the NIM1 gene encoding a protein involved in the signal transduction cascade in response to biological or chemical inducers that leads to systemically acquired resistance in plants.

The present invention therefore describes an isolated DNA molecule (NIM1 gene) encoding an NIM1 protein involved in the signal transduction cascade leading to systemically acquired resistance in plants.

Within the scope of the present invention, a DNA molecule encoding the NIM1 protein is described which hybridizes to clone BAC-04, ATCC deposit number 97543: 1007779 k 9 hybridization 1% BSA under the following conditions; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 750 mM sodium chloride at 55 ° C for 18-24 hours, and washing in 6XSSC for 15 minutes. (X3) 3XSSC for 15 minutes (XI) at 55 ° C. In a particularly preferred embodiment, the N1M1 gene comprises 5 is included in cosmid D7, ATCC deposit number 97736.

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

Furthermore, an encoded NIM1 protein is described comprising the amino acid sequence shown in SEQ ID NO: 3 10. Also described is the coding sequence of the NIM1 gene, which hybridizes to the coding sequence set forth in SEQ ID NO: 2 under the following conditions: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours, and washing in 6XSSC for 15 minutes (X3) 3XSSC (XI) at 55 ° C. In a particularly preferred embodiment, the NIM1 gene coding sequence comprises the coding sequence listed in SEQ ID NO: 2.

The present invention also describes a chimeric gene comprising a plant-active promoter operably linked to a sequence encoding the NIM1 gene, a recombinant vector comprising such a chimeric gene, wherein the vector is capable of stably transformed into a host, as well as a host stably transformed with such a vector. Preferably, the host is a plant such as one of the important agricultural 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, eggplant, pepper, celery, carrot, pumpkin, especially Cucurbita pepo, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, pineapple, avocado, papaya, mango, banana, soy, tobacco, tomato, sorghum or sugar cane.

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

1007779 • r 10

The present invention is also directed to a method of transferring a CIM phenotype into a plant by transforming the plant with a recombinant vector comprising a chimeric gene which as such comprises a plant-active promoter and which is effective is linked to a coding sequence for an NIMJ gene, wherein the encoded NIM1 protein is expressed at higher levels in the transformed plant than a wild type plant.

The present invention is further directed to a method of activating systemically acquired resistance in a plant by transforming the plant with a recombinant vector comprising a chimeric gene which as such comprises a plant-active promoter that is effective linked to an NIMJ gene coding sequence, wherein the encoded NIM1 protein is expressed at higher levels in the transformed plant than in a wild type plant.

Furthermore, the present invention is directed to a method of transferring broad spectrum disease resistance into a plant by transforming the plant with a recombinant vector comprising a chimeric gene which as such comprises a plant-active promoter that acts on is operably linked to a coding sequence for an NIM] gene, wherein the encoded NIM1 protein is expressed in hotter levels in the transformed plant than in a wild type plant.

Another aspect of the present invention exploits both the understanding that the SAR pathway in plants is similar in function to the NF-kB / 1kB control scheme in mammals and flies, and the discovery that the NIM1 gene product 25 is a structural homologue. of the signal transduction factor IkB subclass α in mammals. Mutations of IkB that act as super-repressors or dominant negatives of the NF-κΒ / ΙκΒ control scheme have been described. The present invention includes altered forms of the wild-type NIM1 gene (SEQ No .: 2) that act as dominant negative regulators of the SAR signal transduction pathway. These altered forms of NIM1 confer on plants transformed therewith the opposite phenotype as the NIM1 mutant; plants, i.e. plants transformed with altered forms of NIM1, exhibit constitutive SAR gene expression and a CIM phenotype.

1 007 779 k 11

Also included by the present invention are DNA molecules that hybridize with a DNA molecule of the present invention as defined above, but preferably with an oligonucleotide test series available from such a DNA molecule and that a contiguous portion of the coding sequence of these modified forms of NIM1 are at least 10 nucleotides in length, under moderately severe conditions.

Factors influencing the stability of hybrids determine the stringency of the hybridization. One such factor is the melting temperature Tm which can be easily calculated according to the formula reported in DNA PROBES, George H. Keller and Mark M. Manak, Macmillan Publishers Ltd., 1993, Section one: Molecular Hybridization Technology; page 8 ff.

The preferred hybridization temperature is in the range of about 25 ° C below the calculated melting temperature Tm and preferably in the range of about 12-15 ° C below the calculated melting temperature Tm, and in the case of an oligonucleotide in the range of about 5-10 ° C below the melting temperature Tm.

According to an embodiment of the present invention, the NIMJ gene is altered such that the encoded product has alanine residues instead of serine residues at the amino acid positions corresponding to positions 55 and 59 of the wild-type Arabidopsis NIM1 amino acid sequence (SEQ ID NO: 3 ). An example of a preferred embodiment of this altered form of the NIM1 gene leading to alteration of these serine residues in analin residues is shown in SEQ ID NO: 22. An exemplary dominant negative form of the NIM1 protein with alanine residues instead of serine residues at amino acid positions 55 and 59 is shown in SEQ ID NO: 23. The present invention also includes modified forms of alleles of NIM1, wherein the coding sequence of such allele hybridizes under moderately severe conditions with the coding sequence which is shown in SEQ ID NO: 22, the following conditions are particularly preferred: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours, and washing with 6XSSC for 15 minutes (X3) 3XSSC for 15 minutes (XI) at 55 ° C. In these embodiments, alleles of NIM1 which hybridize under SEQ ID NO: 22 under the above conditions are modified such that the encoded product has alanine residues instead of 1007779 12 serine residues at the amino acid positions corresponding to positions 55 and 59 of SEQ ID NO: 22.

According to another embodiment of the present invention, the NIMJ gene has been modified such that the encoded product has an N-terminal deleted portion, removing lysine residues that may serve as potential ubiquitination sites in addition to the serine residues at the amino acid positions corresponding to positions 55 and 59 of the wild-type protein. An example of a preferred embodiment of this altered form of the NIM1 gene, encoding a gene product with an N-terminal deletion, is shown in SEQ ID NO: 24. An exemplary dominant negative form of the NIM1 protein with an N-terminal deletion is shown in SEQ ID NO.

25. The present invention further encompasses modified forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under mildly stringent conditions to the coding sequence set forth in SEQ 15 ID NO: 24; particularly preferred are the following conditions: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours, and washing in 6XSSC for 15 minutes (X3) 3XSSC for 15 minutes (XI) at 55 ° C. In these embodiments, alleles of NIM1 that hybridize under the above conditions with SEQ ID NO: 24 are altered such that the encoded product has an N-terminal deletion removing lysine residues which may serve as potential ubiquitination sites in addition to the serine residues at the amino acid positions that correspond to positions 55 and 59 of the wild-type gene product.

In yet another embodiment of the present invention, the ΜΛ / 7 gene is altered such that the encoded product has a C-terminated portion, which is believed to lead to enhanced intrinsic stability by blocking the constitutive phosphorylation of serine. and threonine residues at the C-terminus of the wild-type gene product. An example of a preferred embodiment of this altered form of the N1M1 gene encoding a gene product with a C-terminal deletion is shown in SEQ ID NO: 26. An exemplary dominant negative form of the NIM1 protein with a C-terminal deletion is shown in SEQ ID NO: 27. The present invention also includes modified forms of alleles of NIM1, the coding sequence of such allele hybridizing under moderately severe conditions with the one in SEQ ID NO. : 26 coding sequence shown: particularly preferred are the following conditions: hybridization in 1% 5 BSA; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours, and washing in 6XSSC for 15 minutes (X3) 3XSSC for 15 minutes (XI) at 55 ° C. In these embodiments, alleles of NIM1 that hybridize under the above conditions with SEQ ID NO: 26 are altered such that the encoded product has a C-terminal deletion to remove serine and threonine residues.

In yet another embodiment of the present invention, the NIM1 gene is modified such that the encoded product has both an N-terminal deleted portion and a C-terminal deleted portion, providing the advantages of both embodiments of the invention described above.

A preferred embodiment of the invention is an altered form of the NIM1 gene that has an N-terminated portion of amino acids corresponding to approximately amino acid positions 1-125 of SEQ ID NO: 2 and a C-terminated portion of amino acids corresponding to approximately amino acid positions 522-593 of SEQ ID NO: 3.

An example of a preferred embodiment of this altered form of the NIM1 gene, encoding a gene product having both an N-terminal and a C-terminal deletion, is shown in SEQ ID NO: 28. An exemplary 25 exemplified- negative form of the NIM1 protein with a C-terminal deletion is shown in SEQ ID NO: 29. The present invention also includes altered forms of alleles of NIM1, wherein the coding sequence of such allele hybridizes under moderately severe conditions to the SEQ ID NO: 28 coding sequence shown; particularly preferred are the following conditions: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours, and washing in 6XSSC for 15 minutes (X3) 3XSSC for 15 minutes (XI) at 55 ° C. In the above embodiments, 1007779 14 alleles of NIM1 that hybridize under SEQ ID NO: 28 under the above conditions have been modified such that the encoded product has both an N-terminal deletion, removing lysine residues which may serve as potential ubiquitination sites adjacent to the serine residues at the amino acid positions corresponding to positions 55 and 59 of the wild-type gene product, as well as a C-terminal deletion, thereby removing serine and threonine residues.

In yet another embodiment of the present invention, the ATM / gene has been modified such that the encoded product consists essentially of only the "ankyrin" domains of the wild-type gene product. Preferred is an isolated DNA molecule in which the altered form of the NIM1 protein consists essentially of ankyrin motifs corresponding to approximately amino acid positions 103-362 of SEQ ID NO: 3. An example of a preferred embodiment of this altered form of the ATM / gene encoding the ankyrin domains is shown in SEQ ID NO: 30. An exemplary dominant negative form of the NIM1 protein consists essentially of exclusive ankyrin domains and is shown in SEQ ID NO: 31. The present invention also includes modified forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under moderately severe conditions to the coding sequence shown in SEQ ID NO: 30; particularly preferred are the following conditions: hybridization in 1% BSA; 520 mM NaPO 4, pH 7.2; 7% lauryl sulfate, sodium salt, 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours, and washing in 6XSSC for 15 minutes (X3) 3XSSC for 15 minutes (XI) at 55 ° C. In these embodiments, alleles of NIM1 that hybridize under the above conditions with SEQ ID NO: 30 are modified such that the encoded product consists essentially exclusively of the ankyrin domains of the wild-type gene product.

Therefore, the present invention relates to DNA molecules encoding altered forms of the NIM1 gene, such as those described above, and any DNA-30 molecules that hybridize to them under moderately severe conditions.

The present invention also includes a chimeric gene comprising a plant-active promoter operably linked to any of the above modified forms of the ATM / gene, a recombinant vector comprising a 1007779 such chimeric gene, the vector is capable of being stably transformed into a host cell, as well as a host cell stably transformed with such a vector. Preferably, the host cell is a plant, plant cells, or the descendants thereof, for example, from one of the following 5 important agricultural 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, eggplant, pepper, celery, carrot, pumpkin, in particular Cucurbita pepo, zucchini, cucumber, apple, pear, quince, melon , plum, cherry, peach, nectarine, apricot, strawberry, grape, 10 raspberry, pineapple, avocado, papaya, mango, banana, soy, tobacco, tomato, sorghum or sugar cane.

The present invention is also directed to a method of transferring a CIM phenotype to a plant by transforming the plant with a recombinant vector containing a chimeric gene which as such comprises a plant-active promoter that is active linked to any of the above modified forms of the NIM1 gene, wherein the encoded dominant-negative form of the ΜΛ / 7 gene is expressed in the transformed plant and provides the plant with a CIM phenotype.

The present invention is further directed to a method of activating systemically acquired resistance in a plant by transforming the plant with a recombinant vector comprising a chimeric gene, which as such comprises a plant-active promoter that is effective linked to one or more of the above modified forms of the NIM1 gene, wherein the encoded dominant-negative form of the NIM1 protein is expressed in the transformed plant and activates systemically acquired resistance in the plant.

The present invention is also directed to a method of transferring broad spectrum disease resistance to a plant by transforming the plant with a recombinant vector comprising a chimeric gene, which as such comprises a plant-active promoter is operably linked to any of the above altered forms of the NIMJ gene, expressing the coded dominant-negative form of the NIM1 protein in the transformed plant and providing the plant with a broad spectrum of disease resistance.

In yet another aspect, the present invention is directed to a method of assaying for the presence of a NIM1 gene involved in the signal transduction cascade leading to systemically acquired resistance in a plant, comprising scanning a genomic or cDNA library from the plant to a test sequence of an NIM1 coding sequence which hybridizes to the coding sequence shown in SEQ ID NO: 2 under the following set of conditions: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours, and washing in 6XSSC for 15 minutes (X3) 3XSSC for 15 minutes (XI) at 55 ° C.

Further aspects covered by the invention are:

An isolated DNA molecule of the invention in which the aforementioned modified form of the NIM1 protein comprises alanine residues instead of 15 serine residues at the amino acid positions corresponding to positions 55 and 59 of SEQ ID NO: 3, wherein the DNA molecule is under the the following conditions hybridize to the nucleotide sequence shown in SEQ ID NO: 22: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours, and washing in 6XSSC 20 for 15 minutes (X3) 3XSSC for 15 minutes (XI) at 55 ° C.

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

An isolated DNA molecule of the invention in which the altered form of the NIM1 gene has a C-terminal truncation of amino acids corresponding approximately to amino acid positions 522-593 of SEQ ID NO: 3, wherein the DNA molecule is under the the following conditions hybridize to the nucleotide sequence shown in SEQ ID NO: 26: hybridization in 1% BSA; 520 mM

1007779 17

NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours, and washing in 6XSSC for 15 minutes (X3) 3XSSC for 15 minutes (XI) at 55 ° C.

An isolated DNA molecule of the invention, wherein the altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO: 28, wherein DNA molecule hybridizes with the nucleotide sequence shown in SEQ ID NO: 28 under the following conditions. : hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours, and washing in 6XSSC for 15 minutes (X3) 3XSSC for 15 minutes (XI) at 55 ° C.

An isolated DNA molecule of the invention, wherein the altered form of the NIM1 protein consists essentially of ankyrin motifs corresponding to approximately amino acid positions 103-362 of SEQ ID NO: 3, wherein the DNA molecule under the following conditions nucleotide sequence shown in SEQ ID NO: 30 hybridizes: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours, and washing in 6XSSC for 15 minutes (X3) 3XSSC for 15 minutes (XI) at 55 ° C.

An altered form of the ATM / gene of the invention constructed by mutagenesis.

The use of an isolated DNA molecule according to the invention for activating systemically acquired resistance in a plant cell, in plant or its progeny.

Use of an isolated DNA molecule according to the invention for conferring broad spectrum disease resistance to a plant cell, a plant or its progeny.

The use of an isolated DNA molecule of the invention to confer a CIM phenotype on a plant cell, a plant or its progeny.

The use of resistant plants and their descendants according to the invention for incorporating the property of disease resistance in plant lines by cultivation.

The use of MM / gene variants to confer disease resistance and activate SAR gene expression in plants transformed therewith.

A method of producing an altered form of an NIM1 gene.

A method of producing transgenic progeny from a parent transgenic plant, containing an isolated DNA molecule encoding an altered form of a NIM1 protein of the invention, comprising transforming the parent plant with a recombinant vector molecule of the invention and transferring the trait to the offspring of the parent transgenic plant by known plant breeding techniques.

A method of producing a DNA molecule comprising a DNA portion containing a DNA portion encoding an altered form of a NIM1 protein (a) providing a nucleotide assay capable of specifically hybridizing with an altered form of an N1M1 gene or mRNA, the test sequence comprising a contiguous portion of the altered form of an NIM1 of at least 10 nucleotides in length; (b) examining populations of cloned genomic DNA fragments or cDNA fragments from a selected organism for the presence of other altered forms of an NIM1 coding sequence using the nucleotide assay sequence obtained in step (a); and (c) isolating and multiplying a DNA molecule comprising a DNA portion containing a DNA portion encoding an altered form of a NIM1 protein.

A method of isolating a DNA molecule comprising a DNA portion containing an altered form of an NIM1 sequence comprising (a) providing a nucleotide assay capable of hybridizing specifically with an altered form of an NIM1 gene or mRNA, wherein the test sequence comprises a contiguous portion of the coding sequence of an altered form of a plant NIM1 protein of at least 10 nucleotides in length; (b) examining populations of cloned genomic DNA fragments or cDNA fragments from a selected organism for the presence of other altered forms of NIM1 sequences using the nucleotide test sequence obtained in step (a); and (c) isolating a DNA molecule comprising a DNA portion containing an altered form of a NIM1 gene.

A method of providing transgenic plants expressing larger amounts of the NIMJ gene than the wild type, or expressing functional variants and mutants of the N1M1 gene.

A method of providing transgenic plants expressing larger amounts of the NIM1 gene than the wild type, or expressing functional variants and mutants of the NIM1 gene, wherein the expression of the ΜΛ / 7 gene is at a level at least twice as high as the expression level of the NIM1 gene in wild-type plants.

A method of providing transgenic plants expressing greater amounts of the NIM1 gene than the wild type, or expressing functional variants and mutants of the NJM1 gene, wherein the expression of the NIM1 gene is at a level is at least ten times higher than the expression level of the NIM1 gene in wild-type plants.

The nim mutant phenotype. The present invention relates to mutant plants, as well as genes isolated therefrom, which fail in their normal response to pathogen infections, in that they do not express genes associated with SAR. These mutants are referred to as nwi mutants (for "non-inducible immunity") and are "universal disease susceptible" (UDS) as they are susceptible to many strains and pathotypes of host plant pathogens and also to pathogens commonly found in the host plant do not infect, but normally infect other hosts. Such mutants can be selected by treating seeds or other biological material with mutagenic agents and selecting the progeny plants for the UDS phenotype by treating the progeny plants with known chemical agents that induce SAR (eg INA) and then infect the plants with a known pathogen. Non-inducible mutants develop serious disease symptoms under these conditions, while wild type plants are induced to systemically acquired resistance by the chemical compound 1007779, mm mutants can equally be selected from mutant populations obtained by chemical mutagenesis or by mutagenesis under irradiation. , as well as from populations generated by T-DNA insertion and transposon-induced mutagenesis. Techniques for generating mutant plant lines are well known in the art.

mm mutants provide useful indicia for evaluating disease burden during pathogenesis trials in the field where the natural resistance phenotype of the so-called wild-type (ie the 10 non-mutant) plants may diverge and are therefore not a reliable standard for the provide sensitivity. Furthermore, nim plants can also be used in research into possible transgenes with disease resistance. By using an existing m'm-hn as a transgene receiver, the contribution of the transgene to disease resistance can be directly determined from a baseline level of susceptibility. Furthermore, nim plants can be used as an aid in understanding plant-pathogen interactions, mm host plants do not produce a systemic response after attack by a pathogen, and the pathogen's unhindered development is an ideal system for investigating biological interaction of this with the host.

Since m'm host plants may also be susceptible to pathogens outside the host range they usually fall within, these plants also have substantial utility in the molecular, genetic and biological studies of host-pathogen interactions. Furthermore, the UDS phenotype of mm plants also makes them useful in testing fungicides. Mm mutants selected in a specific host have significant utility in testing fungicides using this host and pathogens for this host. This advantage lies in the mutant UDS phenotype, which overcomes the problems encountered with hosts that are differentially sensitive to different pathogens and pathotypes, or are even resistant to some pathogens or pathotypes.

nim mutants further have utility in testing fungicides against a spectrum of pathogens and pathotypes using a heterologous host, i.e., a host that does not normally fall within the host range of a specific pathogen. For example, the susceptibility of Arabidopsis m'm mutants to pathogens of other species (e.g., crop plants) facilitates effective procedures for testing fungicides against crop plants.

5

The Arabidopsis thaliana niml mutant

An Arabidopsis thaliana mutant called niml ("noninducible immunity") that allows the growth of P. parasitica (the leading cause of downy mildew disease) after treatment with INA is described in 10 Delaney et al., 1995. Although niml SA can accumulate after infection with a pathogen, neither SAR gene expression nor disease resistance can be induced, suggesting that the mutation blocks the pathway downstream of SA, niml is also limited in its ability to respond to INA or BTH, suggesting that the blockage downstream of the action of these chemicals falls (Delaney et al., 15 1995; Lawton et al., 1996). The first Arabidopsis niml mutant (referred to below as niml-1) was isolated from 80,000 plants of a T-DNA tagged Arabidopsis ecotype Issilewskija (Ws-0) population by spraying two week old plants with 0.33 mM INA followed by inoculation with P. parasitica (Delaney et al., 1995). Plants that supported fungicide growth after INA treatment were selected as potential mutants. Five further mutants (referred to below as niml-2, niml-3, niml-4, niml-5, and niml-6) were isolated from 280,000 M2 plants of a Ws-0 population mutagenized with ethyl methanesulfonate (EMS).

To determine whether the mutants were dominant or recessive, Ws-0 plants were used as pollen donors for crossing each of these mutants.

The F plants were classified according to their ability to support fungal growth after treatment with INA. As shown in Table 3 of the examples, all niml-1, niml-2, niml-3, niml-4, and niml-6 F were plants in their wild-type phenotype, indicating a recessive mutation in each line indicates.

30 niml-5 showed the wm phenotype in all 35 F plants, indicating that this specific mutant is dominant. For confirmation, the reverse crossing was performed using niml-5 as the pollen donor to fertilize the Ws-0 plants. In this, all 18 F, plants were phenotypically nim, confirming the 1007779 22 dominance of the niml-5 mutation.

To determine whether the niml-2 through niml-6 mutations were allelic to the previously characterized niml-1 mutation, pollen from niml-1 was used to fertilize niml-2 through niml-6. Since niml-1 carried resistance to kanamycin, the F offspring were identified by antibiotic resistance. In all cases, the kanamycin resistant F, plants were nim, indicating that they are allelic to the niml-1 mutant. Since the niml-5 mutant is dominant and apparently homozygous for the mutation, it was necessary to analyze niml-1 complementation in the F2 generation. If niml-1 and niml-5 are allelic, it would be expected that all F2 plants exhibit an m'm phenotype. If not, 13 of 16 F2 plants would be expected to display a nim phenotype. Of 94 plants, 88 plants clearly supported fungal growth after INA treatment. Six plants showed a related phenotype of black spots on the leaves reminiscent of a lesion-mimicking phenotype and these supported little fungal growth after treatment with INA. Since niml-5 carries a point mutation in the N1M1 gene (see below), it is considered to be a niml allele.

To determine the relative strength of the different niml alleles, each mutant was examined for the growth of P. parasitica under normal growth conditions and after prior treatment with SA, INA or BTH. As shown in Table 1, under normal growth, niml-1, niml-2, niml-3, niml-4, and niml-6 all support approximately the same rate of fungal growth, which was slightly faster than the Ws-0 control. The exception was the niml-5 plants, in which fungal growth was delayed by several days compared to both the other 25 niml mutants and the Ws-0 reference, but eventually all niml-5 plants also perished from the fungus. After treatment with SA, the mutants could be divided into three classes: niml-4 and niml-6 plants showed relatively fast fungal growth; niml-I, niml-2, niml-3 plants showed somewhat less mold growth; and fungal growth in niml-5 plants was even less than in the untreated Ws-0 references. After treatment with INA or BTH, the mutants also appeared to fall into three classes, with niml-4 being most severely limited in its ability to inhibit fungal growth after chemical treatment; niml-1, niml-2, niml-3 and niml-6 were all moderately limited; and niml-5 1 007779 23 was only slightly limited. In these experiments, Ws-0 did not support fungal growth after treatment with INA or BTH. Thus, with respect to the inhibition of fungal growth after chemical treatment, the mutants fall into three classes, with niml-4 being the most limited, niml-1, niml-2, niml-3, and niml-6 a moderate inhibition of the fungus, and niml-5 showed only limited resistance to fungi.

PR-1 mRNA formation was also used as a measure of classifying the different niml alleles. RNA was taken from plants 3 days after treatment with water or chemicals, or 14 days after seeding with a compatible fungus (P. parasitica isolate Emwa). The RNA gel stain in Figure 3 shows that PR-1 mRNA was generated at high levels after treatment of wild-type plants with SA, INA or BTH or infection with P. parasitica. In the niml-1, niml-2 and wW-i plants, the formation of PR-1 mRNA was very much reduced compared to the wild type after chemical treatment. PR-1 mRNA was also reduced after infection with P. parasitica, but there was still some accumulation in these mutants. In the niml-4 and niml-6 plants, the formation of PR-1 mRNA after chemical treatment was reduced even more than in the other alleles (as evidenced by longer exposures) and significantly less PR-1 mRNA was formed after infection with P parasitics, which supports the idea that these could be particularly strong niml alleles. Interestingly, PR-1 mRNA formation had increased in the niml-5 mutant, but was induced only slightly after chemical treatment or infection with P. parasitica. Based on both PR-1 mRNA formation and fungal infection, these mutants fall into three classes: significantly limited alleles (niml-4 and niml-6), moderately limited alleles (niml-1, niml-2, and niml-3) , And a weakly limited allele (niml-5).

Mapping the fine structure of the niml mutation

To determine a rough position for NIM1, 74 F2 nim phenotype plants from a cross between niml-1 (Ws-0) and Landsberg erecta (Ler) were identified by their susceptibility to P. parasitica and lack of accumulation of PR-1 mRNA after treatment with INA. After determining some simple sequence narrow polymorphism (SSLP) markings (Bell and Ecker 1994), 1007779 24 was found to niml at approximately 2.8 centimorgans (cM) of nga 128 and 8.2 cM of ngalll on the lower arm. of chromosome 1. In a subsequent analysis, it was found that niml-1 is between ngal 11 and about 4 cM of the SSLP marker ATHGENEA.

To map the fine structure, 1138 nim plants from an F2 population from cross between niml-1 and LerDP23 were identified based on both their inability to accumulate PR-1 mRNA and their ability to support fungal growth after treatment with ΓΝΑ. DNA was extracted from these plants and examined for zygosity in both ATHGENEA and ngal 11. As shown in Figures 10 5A-5D, between ATHGENEA and niml 93 recombinant chromosomes were identified, representing a genetic distance of about 4.1 cM (93 of 2276) , and between ngal 11 and niml, 239 recombinant chromosomes were identified, indicating a genetic distance of about 10.5 cM (239 from 2276). Informative recombinants in the interval from ATHGENEA to ngal 1115 were further analyzed using amplified fragment length polymorphism (AFLP) analysis (Vos et al., 1995).

Initially, 10 AFLP markers between ATHGENEA and ngal 11 were identified and used to construct a low-resolution map of the area (Figure 5A). The AFLP mark W84.2 (1 cM from niml) and W85.1 (0.6 cM from niml) were used to isolate yeast artificial chromosome (YAC) clones from the CIC (for "Center d'Etude du Polymorphism Humain, INRA and CNRS ") library (Creusot et al., 1995). Two YAC clones, CIC12H07 and CIC12F04, were identified with W84.2 and two YAC clones CIC7E03 and CIC10G07 (results not shown) were identified with the W85.1 marker. However, it was determined that there was an interruption between the two sets of flanking YAC clones. Starting from this point, the bacterial artificial chromosome (BAC) and PI clones overlapping with CIC12H07 and CIC12F04 were isolated and mapped, and three consecutive displacement steps were performed to extend the BAC / P1 contig to N1M1 (Liu et al ., 1995; Chio et al., 1995). At different times during the move, new AFLPs specific for BAC or PI clones were developed and used to determine if the NIM 1 gene had been crossed. Here, it was determined that NIM1 was crossed when BAC and PI clones were isolated showing both AFLP marker L84.6a and AFLP marker L84.8. The AFLP marker L84.6a found on PI clones PI-18, PI-17, and Pl-21 identified three recombinants, and marker L84.8 found on PI clones PI-20, PI-22, PI- 23 and PI-24, and BAC clones, BAC-04, BAC-05, and BAC-5 06 identified one recombinant. Because these clones overlap to form a large contiguous region (> 100 kb) and include AFLP markers flanking niml, the gene was in the contiguous region. The BAC and PI clones enclosing the contiguous region were used to generate eight further AFLP markers, indicating that niml is between L84.Y1 and L84.8, indicating an interruption of about 0.09 cM.

A cosmid library was constructed in the Agrobacterium-compatible T-DNA cosmid vector pCLD04541, using DNA from BAC-06, BAC-04 and PI-18. A cosmid contig was developed using AFLP tags derived from these clones. Physical mapping showed that the physical distance between L84.Y1 and L84.8 was greater than 90 kb, indicating a genetic to physical distance of about 1 megabase per cM. To facilitate the subsequent identification of the NIM1 gene, the DNA sequence of BAC-04 was determined.

20 Isolation of the NIMl Zen

To identify the cosmids containing the NIM1 gene, the 12 cosmids listed in Table 4 of the Examples were transformed into niml-1, after which the transformants were evaluated for their ability to complement the mutant phenotype. Cosmids D5, E1 and D7 were all found to supplement niml-1, as determined by accumulating the ability of transformants PR-1 mRNA after treatment with INA. The ends of these cosmids were sequenced and found to be on the BAC-04 DNA sequence. There were 9,918 base pairs in the DNA region common to D7 and D5 that contained the N1M1 gene. As shown in Figure 5D, four possible gene regions in this 10-kb sequence were identified. Region 1 might encode a 19,105 D protein, region 3 could encode a 44,554 D protein, and region 4 could encode a 52,797 D protein. Region 2 had four open sizes of different sizes close together, suggesting a gene with three introns.

1 0 07 7 7 9 26

Analysis using the NetPlantGene program (Hebsgaard et al., 1996) indicated that there is a high probability that the open reading frame could be spliced together to form one large open reading frame encoding a 66,039 D protein.

To determine which gene region contained the NIM1 gene, gel spots with RNA isolated from Ws-0 leaf tissue and from the various niml mutants after treatment with water or chemicals were examined with DNA test series from each of the four gene regions. In these tests, care was taken to ensure that test runs were marked to a high specificity and that the autoradiographic images were exposed for more than 1 week. According to previous experience, these conditions identify RNA at concentrations of about one copy per cell. The only gene region that produced detectable RNA was gene region 2. As shown in Figure 7, the mRNA identified by the gene region 2 assay series was induced by treatment with BTH of 15 wild-type plants, but not in any of the mutants. Furthermore, the formation of RNA in all plants increased after infection with P. parasitica, indicating that this specific gene is induced after infection with a pathogen.

To further define the gene region encoding NIM1, the DNA sequence of each of the four gene regions was determined for each of the niml alleles and compared with the corresponding gene region for Ws-0. No mutations were observed between Ws-0 and the mutant alleles in gene region 3 or gene region 4 and only a single change was found in gene region 1 in the niml-6 mutant. However, a mutation of a single base pair was found in each of the alleles relative to Ws-0 for region 2. The DNA sequence from gene region 2 is shown in Figure 6. As shown in Table 5 and Figure 6, in niml-1, a single adenosine is inserted at position 3579, causing a reading frame shift, resulting in a seven amino acid change and a 349 amino acid deletion. In niml-2 there is a cytidine-in-thymidine change at position 3763 turning a histidine residue into a tyrosine residue. In niml-30, a single adenosine residue is removed at position 3301, causing a reading frame shift that changes 10 amino acids and removes 412 amino acids from the predicted protein. Interestingly, both niml-4 and niml-5 have a guanosine-to-adenosine change at position 4160 1007779 27, turning an arginine residue into a lysine residue, while in niml-6 there is a cytosine-to-thymidine change that results in a stop codon causing a deletion of 255 amino acids from the predicted protein. Although the mutation in niml-4 and niml-5 changes the consensus donor cleavage site for the mRNA 5, RT-PCR analysis shows that this mutation does not alter the mRNA cleavage (results not shown).

NIM1 homologs

The DNA sequence of the gene region 2 was used in a "Blast" study 10 (Altschul et al., 1990), which has an exact match with the Arabidopsis-expressed sequence marker (EST) T22612 and substantial match with the rice ESTs S2556, S2861, S3060 and S3481 (see Figure 8) were found.

A DNA test series comprising base pairs 2081 to 3266 was used to scan an Arabidopsis cDNA library, and 14 clones corresponding to gene region 2 were isolated. From the cDNA sequence, the position of the exon / intron boundaries shown in Figure 6 could be confirmed. Rapid amplification of cDNA ends by the polymerase chain reaction (RACE) was performed using RNA from INA-treated Ws-0 plants and the probable starting site of transcription was determined at the A at position 2588 in Figure 6.

Using the NIM1 cDNA as a test series, homologs of Arabidopsis NIM1 can be identified and isolated by probing genomic or cDNA libraries from different plants, such as, but not limited to the following crops: rice, wheat, barley, rye , corn, potato, carrot, sweet potato, sugar beet, beans, peas, chicory, lettuce, cabbage, cauliflower, 25 broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, pumpkin, in especially Cucurbita pepo, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, pineapple, avocado, papaya, mango, banana, soy, tobacco, tomato, sorghum or sugar cane. Standard techniques for performing this include hybridization studies from plated cDNA libraries (ie, plaques or colonies; see Sambrook et al., Molecular Cloning, eds., Cold Spring Harbor Laboratory Press. (1989)) and amplification by PCR using oligonucleotide base arrays (see, for example, Innis et al., PCR Protocols, a Guide to Methods and Applications 1007779 28 eds., Academic Press (1990)). Identified homologs are genetically introduced into the expression vectors below and transformed into the above crops. Transformants are assessed for increased disease resistance using appropriate pathogens for the crop to be tested.

For example, NIM1 homologs have been observed in the genomes of cucumber, tomato, tobacco, corn, wheat and barley by using DNA stain analysis. Genomic DNA has been isolated from cucumber, tomato, tobacco, corn, wheat and barley, cut by restriction with the enzymes BamHI, HindlII, Xbal or Sail, separated electrophoretically on 0.8% agarose gels and transferred to nylon membranes by capillary staining. After cross-linking under UV radiation to fix the DNA, the membrane was placed under moderately severe conditions [(1% BSA; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; ImM EDTA, 250 mM sodium chloride) at 55 ° C for 18-24 hours] hybridized to 32 P radiolabeled Arabidopsis thaliana NIMJ cDNA. After hybridization, the stains were washed under moderately severe conditions [6XSSC for 15 min (X3) 3XSSC for 15 min (XI) at 55 ° C; 1XSSC is 0.15M NaCl, 15mM Na Citrate (pH 7.0)] and exposed to X-ray film to reveal the bands corresponding to NIMJ.

Furthermore, expressed sequence markers (EST) found to correspond to the NIMJ gene, such as the rice ESTs described above, can also be used to isolate these homologs. The rice ESTs may be particularly useful for isolating NIMJ homologs from other monocotyledonous plants.

Homologs can also be obtained by PCR. In this method, known homologs (e.g. rice and Arabidopsis) are compared. Areas with a high degree of identity or identity in amino acids and DNA are then used to make basic PCR arrays. When a suitable region has been identified, base strings for this region are made with different substitutions in the third codon position. The PCR reaction is performed with cDNA or genomic DNA under various standard conditions. When a band is obtained, it is cloned and / or sequenced to determine if it is a NIM1 homolog 1 0 07 77 0 29.

High expression of MM / provides disease resistance to plants

The present invention also concerns the production of transgenic plants expressing levels of the MM7 gene that are higher than in the wild type, or expressing functional variants and mutants thereof, thereby exhibiting a broad spectrum of disease resistance. In a preferred embodiment of the invention, the expression of the MM / gene is at a level at least twice above the expression level of the MM / gene in the wild-type plants, and preferably ten times above the expression level of the wild -type. High expression of the MM / gene mimics the influence of inducing compounds in that it leads to plants with a phenotype of constitutive immunity (CIM).

Several methods of producing plants that overexpress the MM / gene and thereby exhibit a CIM phenotype have been described. A first method is to select transformed plants that have a high expression of MM / and therefore have a CIM phenotype caused by an insertion site effect. Table 6 shows the results of testing different transformants for resistance to fungal infection. As can be seen from this Table 20, some of the transformants showed less fungal growth than normal and some showed no visible fungal growth at all. RNA was prepared from pooled samples and analyzed as described in Delaney et al., 1995. Spots were hybridized with the Arabidopsis gene test series PR-1 (Uknes et al., 1992). Three lines showed early induction of PR-1 gene expression, in that 25 PR-1 mRNA could be observed 24 to 48 hours after fungal treatment. These three lines also showed resistance to fungal infection.

Furthermore, methods are described for constructing plant transformation vectors comprising a constitutive plant-active promoter, such as the CaMV 35S promoter, which is operably linked to a coding region encoding an active NIM1 protein. High levels of the active NIM1 protein produce the same disease resistance effect as chemical induction with inducing compounds such as BTH, INA and SA.

1007779 30

The NIMl-een is a homolog of ΙκΒα

The NIM1 gene is a key component of the systemically acquired resistance (SAR) pathway in plants (Ryals et al., 1996). The NIM1 gene is related to the activation of SAR by chemical and biological inducing factors, and is required for SAR and SAR in conjunction with such inducing factors

gene expression. The position of the NIM1 gene was determined by molecular biological analysis of the genome with mutant plants known to carry the mutant niml gene, which gives the host plants extreme sensitivity to a wide variety of pathogens and makes them impossible for them. respond to pathogenic and chemically inducing factors of SAR. The Arabidopsis wild-type NIM1 gene has been mapped and sequenced (SEQ ID NO: 2). The wild-type NIM1 gene product (SEQ ID NO: 3) is involved in the signal tri / induction cascade leading to both SAR and gene-by-gene disease resistance in Arabidopsis (Ryals et al., 1997). Recombinant overexpression of the wild-type form of NIM1 leads to plants with a phenotype of constitutive immunity (CIM) and thereby confers disease resistance to transgenic plants. Increased levels of the active NIM1 protein produce the same disease resistance effect as chemical induction with inducing chemicals such as BTH, 1NA and SA.

The NIM1 gene sequence (SEQ ID NO: 2) was used in BLAST studies, and corresponding sequences were identified based on homology of a rather highly conserved domain in the MM / gene sequence with ankyrin domains found. in a number of proteins such as spectrins, ankyrins, NF-κΒ and IkB (Michaely and Bennett, Trends Cell Biol. 2, 127-129 (1992)). In addition to the ankyrin motif, however, no further strong homologies were observed by conventional computer analysis, including homology with waargenomenκ gebruikelijkeα. Despite the negative results obtained with the computer programs, pairwise visual inspections between the NIM1 protein (SEQ ID NO: 3) and 70 known ankyrin-containing proteins were performed, and surprising similarities with 30 members of the ΙκΒα-class transcription regulators (Baeuerle and Baltimore 1996;

Baldwin 1996) were found. As shown in Figure 1, the NIM1 protein (SEQ ID NO: 3) shows significant homology with ΙκΒα proteins from mouse, rat and pig (SEQ ID NO: 18, 19 and 20, respectively).

1007779 31 NIM1 contains a number of important structural regions of ΙκΒα throughout the length of the protein, including ankyrin domains (indicated by the broken underline in Figure 9), 2-amino terminal serine residues (amino acids 55 and 59 of NIM1), a few lysine residues (amino acids 99 and 100 in 5 NIM1) and an acidic C-terminal portion. Overall, NIM1 and ΙκΝα share 30% of the residues and have conservative replacements in 50% of the residues. Therefore, there is homology between ΙκΒα and NIM1 in the proteins, with an overall agreement of 80%.

One way in which the ΙκΒα proteins act in signal transduction is by binding to the cytosol-containing transcription factor NF-κΒ, preventing it from entering the nucleus and altering the transcription of genes it targets (Baeuerle and Baltimore, 1996 ; Baldwin, 1996). The genes targeted by NF-κΒ control (activate or inhibit) various cellular processes, including antiviral, antimicrobial and cell killing responses (Baeuerle and Baltimore, 1996). When the signal transduction pathway is activated, ΙκΒα is phosphorylated at two serine residues (amino acids 32 and 36 of ΙκΒα in the mouse). This programs ubiquitination at a double lysine residue (amino acids 21 and 22 of mouse ΙκΒα). After ubiquitination, the NF-κΒ / ΙκΒ complex is passed through the proteosome, where ΙκΒα is degraded and NF-κΒ is released to the nucleus.

The phosphorylated serine residues important for ΙκΒα processing are conserved in NIM1 within a large contiguous block of conserved sequence from amino acids 35 to 84 (Figure 9). In contrast to ΙκΒα, where the double lysine residue is located about 15 amino acids towards the N-end of the protein, in NIM1 there is a double lysine residue that is located about 40 amino acids towards the C-end. Furthermore, a high degree of homology exists between NIM1 and ΙκΒα in the serine / threonine-rich carboxyl-terminal region that has been shown to be important in basal conversion rate (Sun et al., Mol. Cell. Biol. 16, 1058-1065 ( 1996)). According to the present invention, based on the analysis of the structural homology and the presence of 30 elements known to be important for the action of ΙκΒα, NIM1 is expected to act in a similar manner to ΙκΒα, and to have analogous effects on the gene regulation in plants.

Plants containing the wild-type NIM1-gcn are expected to exhibit more NIM1 gene product (ΙκΒ homolog) or less phosphorylation of the NIM1 gene product (ΙκΒ homolog) when treated with inducing chemicals . In accordance with this model, the result is that the NF-icB homolog occurring in the plant is kept from the nucleus, and SAR gene expression and resistance reactions can occur. An inactive ATM / gene product is present in the niml mutant plants. Therefore, in accordance with this model, the NF-kB is homologous to enter the nucleus and suppress resistance and SAR gene expression.

In accordance with this idea, animal cells treated with salicylic acid show increased stability / appearance of IkB and a reduction of active NF-κΒ in the nucleus (Kopp and Ghosh, 1994). Mutations of IkB are known to act as super-repressors or dominant negatives (Britta-Mareen Traenckner et al., EMBO 14: 2876-2883 (1995); Brown et al., Science 267: 1485-1488 (1996) Broekman 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-κΒ but are not phosphorylated or ubiquitinated and therefore not degraded. NF-κΒ remains bound to the IkB and therefore cannot enter the core.

20 Modified forms of the A7M / gene

In view of the above, the present invention includes modified forms of ATM / which act as dominant negative regulators of the SAR signal transduction pathway. Plants transformed with these dominant negative forms of ATM / have the opposite phenotype as niml mutant plants, in that the plants transformed with altered forms of NIM1 exhibit constitutive SAR gene expression and therefore have a CIM phenotype. Due to the position of the ATM / gene in the SAR signal transduction pathway, some modifications of the gene, other than specifically described herein, are expected to lead to constitutive expression of SAR genes, and thus to a CIM phenotype. .

Phosphorylation of serine residues in human ΙκΒα is required for stimulus-activated degradation of ΙκΒα and therefore for activation of NF-kB. Mutagenesis of the serine residues (S32 and S36) in human ΙκΒα to alanine residues 1007779 33 leads to stimulus-induced phosphorylation, which blocks proteosome-associated ΙκΒα degradation (Traenckner et al., 1995; Brown et al., 1996; Broekman et al., 1995; Wang et al., 1996). This altered form of ΙκΒα can act as a dominant-negative form in that it retains NF-κΒ in the cytoplasm and therefore blocks downstream of the signal events. Based on a comparison between the amino acid sequences of NIM1 and IkB as shown in Figure 9, serine residues 55 (S55) and 59 (S59) in N1M1 (SEQ ID NO: 3) are homologous to S32 and S36 in human ΙκΒα. To construct dominant negative forms of NIM1, the serine residues at 10 amino acid positions 55 and 59 are mutagenized to alanine residues. Therefore, according to a preferred embodiment of the present invention, the NIM1 gene is altered such that the encoded product has alanine residues instead of serine residues at the amino acid positions corresponding to positions 55 and 59 of the Arabidopsis NIM1 amino acid sequence. The present invention also includes disease-resistant transgenic plants transformed with such an altered form of the N1M1 gene, as well as methods of using this altered form of the NIM1 gene to confer disease resistance and activate SAR gene expression in plants transformed with it.

Removal of amino acids 1-36 (Broekman et al., 1995; Sun et al., 1996) 20 or 1-72 (Sun et al., 1996) from human ΙκΒα, containing ubiquinating lysine residues K21 and K.22 as well as phosphorylation sites S32 and S36 leads to a dominant-negative ΙκΒα phenotype in transfected human cell cultures. N-terminal removal of the first 125 amino acids of the N1M1 gene product removes eight lysine residues that could serve as ubiquinization sites and also as potential phosphorylation sites at S55 and S59 as discussed above. Thus, according to a preferred embodiment of the present invention, the NIM1 gene is altered such that the encoded product lacks approximately the first 125 amino acids compared to the natural Arabidopsis NIM1 amino acid sequence. The present invention also includes disease resistant transgenic plants transformed with such an altered form of the NIM1 gene, as well as methods of using this altered form of the NIM1 gene to confer disease resistance and activate SAR- gene expression in plants transformed with it.

1 007779 34

Removal of amino acids 261-317 from human ΙκΒα can lead to increased intrinsic stability by blocking constitutive phosphorylation of serine and threonine residues in the C-terminal portion. This altered form of ΙκΒα is expected to act as a dominant-negative form. A region rich in serine and threonine residues is located at amino acids 522-593 in the C-terminal portion of NIM1. Therefore, according to a preferred embodiment of the present invention, the NIM1 is modified such that the encoded product lacks approximately its C-terminal portion, including amino acids 522-593, compared to the natural Arabidopsis NIM1 amino acid sequence. The present invention also includes disease resistant transgenic plants transformed with such a form of the NIMI gene, as well as methods of using this altered form of the NIMI gene to confer disease resistance on and activating SAR gene expression plants transformed with it.

According to another embodiment of the present invention, modified forms of the MM7 gene product, resulting from the removal or formation of chimeras at C-terminal and N-terminal portions, are provided. According to yet another embodiment of the present invention, constructs are provided which contain the ankyring regions of the NIMI gene. The present invention also encompasses disease resistant transgenic plants transformed with such a NIM1 chimeric or ankyrin construct, methods for using these variants of the NIMI gene to confer disease resistance, and activating SAR gene expression in , plants that have been transformed with this.

The invention also encompasses methods of activating SAR in plants and conferring on plants a CIM phenotype and broad spectrum disease resistance by transforming the plants with DNA molecules encoding altered forms of the MM / gene product. . The present invention further encompasses plants transformed with an altered form of the NIMI gene.

Disease resistance

The overexpression of the wild-type NIM1 in plants and the expression of altered forms of the NIM1 in plants leads to immunity against a wide variety of plant pathogens, including, but not limited to, viruses and viroids, for example tobacco mosaic virus or cucumber mosaic virus , ring spot or necrosis virus, pelargonium leaf curl virus, red clover spot virus, viruses that restrict the growth of tomatoes, and similar viruses; fungi, for example Phythophthora parasitica and Peronospora tabacina; bacteria, for example Pseudomonas syringae and Pseudomonas tabaci, insects such as aphids, for example Myzus persicae; and lepidoptera, for example Heliothus spp .; and nematodes, for example Meloidogyne incognita. The vectors and methods of the invention are useful against various disease causing organisms including, but not limited to, downy mildew diseases, such as Scleropthora macrospora, Scleropthora rayissiae, Sclerospora graminicola, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora; peronosclerospora; plant rusts such as Puccinia sorphi, Puccinia polysora 15 and Physopella zeae; other 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 impart disease resistance to a wide variety of plants, including flowering plants, monocotyledons and dicotyledons. While disease resistance can be imparted to any plant falling within these broad classes, the invention is particularly useful for use in crop plants important for agriculture, such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato , sugar beet, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, egg plant, pepper, celery, carrot, pumpkin, in particular Cucurbita pepo, zucchini, cucumber, apple , pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, pineapple, avocado, papaya, mango, banana, soy, tobacco, tomato, sorghum or sugar cane. Transformed cells can be regenerated into whole plants such that the gene confers disease resistance to the whole transgenic plants. The expression system can be adjusted so that the disease resistance is expressed continuously or constitutively.

.1 007 7 :: 36

Recombinant DNA technology

The NIM1 DNA molecule or gene fragment that confers disease resistance to plants by enabling the induction of SAR gene expression, or the altered form of the ΜΛ / 7 gene that confers disease resistance in plants by enhancing 5 SAR gene expression, in plants or bacterial cells are taken using conventional recombinant DNA technology. In general, this involves introducing the DNA molecule that falls within SEQ ID NO: 1, a functional variant thereof or a molecule encoding any of the modified forms of NIM1 described above, into an expression system in which the DNA-10 molecule is heterologous (i.e. normally not present). The heterologous DNA molecule is introduced into the expression system or into the vector in an appropriate orientation and proper reading frame. The vector includes the elements required for the transcription and translation of the inserted protein coding sequences. 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 lambda vector systems λgtll, AgtlO and Charon 4; plasmid vectors such as pBI121, pBr322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKClO1, pCDNAII; and similar systems. The NIM1 coding sequence described herein and the modified NIM1 coding sequence can be cloned into the vector using standard cloning techniques in the art, such as described by Maniatis et al., Molecular Cloning: A Laboratory Manual. Cold Spring Laboratory, Cold Spring Harbor, New York (1982).

In order to obtain efficient expression of the gene or gene fragment of the present invention, a promoter leading to an appropriate level of expression or constitutive expression must be present in the expression vector. Typically, RNA polymerase binds to the promoter and initiates transcription of a gene. Promoters differ in their strength, ie, in their ability to act as a promoter for transcription. Depending on the host cell system used, any of a number of suitable promoters can be used. The components of the expression cassette can be modified to enhance expression. For example, truncated sequences, nucleotide substitutions or other modifications can be used. Plant cells transformed with such modified expression systems will overexpress or constitutively express the genes required for SAR activation.

5 A. Construction of plant transformerin vectors

A wide variety of transforming plants for transforming plants are available, and the genes of the invention can be used in combination with such vectors. The choice of the vector will depend on the preferred transformation technique and the species to be transformed. For certain species to be transformed, different markers for selection with antibiotics or herbicides may be preferred. Selection markers commonly used in transformation include the nptll gene conferring resistance to kanamycin and similar antibiotics (Messing & Vierra, Gene 19: 259-268 (1982); Bevan et al., Nature 304: 184-187 (1983)), the bar gene, which confers resistance to herbicide phosphmotricin (White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al., Theor. Appl. Genet 79: 625-631 (1990)), the hph gene , which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol. Cell. Biol. 4: 2929-2931), and the dhfr gene, which confers resistance to methatrexate (Bourouis et al., EMBO J. 2 (7): 1099-1104 (1983)), and the EPSPS gene, which confers glyphosphate resistance (US Patents 4,940,935 and 5,188,642).

1. Vectors for transforming with Agrobacterium.

Numerous vectors for transformation using Agrobacterium tumefaciens are available. These usually carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) and pXYZ. The construction of two typical vectors is described below.

30 a. PCIB200 and pCIB2001:

The binary vectors pcIB200 and pcIB2001 are used for the construction of recombinant vectors for use with Agrobacterium and are constructed in the following manner. pTJS75can be provided by cutting pTJS75 1 0 07 779 38 with Narl (Schmidhauser & Helinski, J. Bacteriol. 164: 446-455 (1985)), allowing the cutting out of a tetracycline resistance gene, after which a whole fragment from pUC4K containing a NPTII bears are inserted (Brass & Vierra,

Gene 19: 259-268 (1982): Bevan et al., Nature 304: 184-187 (1983): McBride et al., 5 Plant Molecular Biology 14: 266-276 (1990)). Xhol coupling sequences are linked to the EcoRV fragment of pCIB7 containing the left and right T-DNA border regions, a plant-selectable nos / nptll chimeric gene, and the pUC polylinking sequence (Rothstein et al., Gene 53: 153-161 (1987) ), and the ΧΛοΖ-cut fragment is cloned into Sa / f-cut pTJS75 jug to provide pCIB200 (see also EP-0.332.104, Example 19). pCIB200 contains the following unique poly coupling restriction sites: EcoRl, SstI, Kpnl, Bglll, Xbal and Sail. pCIB2001 is a derivative of pCIB200 obtained by introducing a number of further restriction sites into the polylinker series. Unique restriction sites in the polylink series of pCIB2001 are EcoRl, SstI, Kpnl, 15 Bglll, Xbal, Sail, Mlul, Bell, Avrll, Apal, Hpal, and Stul. pC! B2001; in addition to these unique restriction sites, it also contains plant and bacterial active kanamycin selection, left and right T-DNA border regions for transformation with Agrobacterium, the RK2-derived trfh activity for mobilization between E. coli and other hosts, and the also of RK2 derived Oril and OrN functions. The 20 pCIB2001 poly coupling series is suitable for cloning plant expression cassettes containing their own control signals.

b. pCIBlO and hygromycin selection derivatives of this:

The binary vector pCIB10 contains a gene encoding kanamycin resistance for selection in plants, T-DNA right and left border sequences, as well as sequences from plasmid pRK525 which has a broad host range, allowing it to multiply in both E. coli and Agrobacterium. Its construction is described by Rothstein et al. (Gene 53: 153-161 (1987)). Several derivatives of pCIB10 containing the hygromycin B phosphotransferase gene, as described by Gritz et al. (Gene 25: 179-188 (1983)), have been constructed. These derivatives allow the selection of transgenic plant cells for hygromycin (pCIB743) or hygromycin and kanamycin (pCIB715, pCIB717) only.

1 OCT "O

39 2. Vectors suitable for transformation other than with Agrobacterium.

Transformation without the use of Agrobacterium tumefaciens avoids the requirement of T-DNA sequences in the chosen transformation vector, so that vectors which do not contain these sequences can also be used, in addition to the vectors described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include particle bombardment transformation, protoplast uptake (e.g. PEG and electroporation) and microinjection. The choice of the vector largely depends on the preferred selection for the species to be transformed.

A. PCIB3064: pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques in combination with selection by the herbicide basta (or phosphotricerine). The plasmid pCIB246 includes the CaMV 35S promoter, which is operably linked to the E. coli GUS gene, and the CaMV 35S transcription terminator, and is described in published International Application WO 93/07278. The 35S promoter of this vector comprises two ATG sequences in the 5 'direction from the starting position. These sites have been mutated using standard PCR techniques such that the ATGs are removed and the restriction sites Sspl and PvuII are obtained. The new restriction sites are 96 and 37 base pairs from the unique Sail site and 101 and 42 base pairs from the actual starting position. The derivative of pCIB246 thus obtained is called pCIB3025. The GUS gene is then excised from pCIB3025 by cutting with Sail and SacI, after which the ends are blunted and coupled again to obtain plasmid pCIB3060. The plasmid pJIT82 is obtained from the John Innes Center, Norwich, and a 400 bp Narrow fragment containing the bar gene of Streptomyces viridochromogenes is excised and placed in the Hpal site of pCIB3060 (Thompson et al., EMBO J 6: 2519-2523 ( 1987)). Thus, pCIB3064 containing the bar gene 30 was obtained under the control of the CaMV 35S promoter and herbicide selection terminator, an ampicillin resistance gene (for selection in E. coli), and a polylinker sequence with unique sites Sphl, PstI, HindIII and BamHI. This vector is suitable for cloning plant expression cassettes containing its own 1007779 40 control signals.

b. pSOG19 and pSOG35: pSOG35 is a transformation vector using the E. coli gene dihydrofolate reductase 5 (DFR), which confers resistance to methotrexate, as a selectable marker. PCR is used to amplify a 35S promoter (-800 bp), intron 6 from the maize Adhl gene (-550 bp) and 18 bp of the GUS untranslated leader sequence of pSOG10. A 250-bp fragment encoding the E. coli dihydrofolate reductase type II gene is also amplified by PCR and these two PCR fragments are combined with a SacI-Pstl fragment from pB1221 (Clontech) which is the backbone of the pUC19 vector and contains the nopaline synthase terminator. Merging these fragments gives pSOG19 containing the 35S promoter, joined with the intron 6 sequence, the GUS leading sequence, the DHFR gene and the nopaline synthase terminator. Replacing the GUS leading sequence in pSOG19 with the leading sequence from "Maize Chlorotic Mottle Virus" (MCMV) gives the vector pSOG35. pSOG19 and pSOG35 carry the pUC gene for resistance to ampicillin and have HindlII, Sphl, PstI and EcoRl sites available for foreign substance cloning.

20 B. Requirements for the Construction of Plant Expression Cassettes

Gene sequences intended for expression in transgenic plants are first pooled in expression cassettes behind an appropriate high level expression promoter and upstream of an appropriate transcription terminator. These expression cassettes can then be easily introduced into the above-described plant transformation vectors.

1. Choice of the promoter

The choice of the promoter used in expression cassettes will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Certain promoters will express transgenes in specific cell types (such as leaf epidermis cells, mesophyll cells, root cortex cells) or in specific tissues or organs (e.g. roots, leaves or flowers) and their choice will correspond to the target site for the accumulation 1007779 41 of the MM7 gene product or the modified NIM1 gene product. Alternatively, the selected promoter may provide expression of the gene under a light-induced or other time-regulated promoter.

5 a. Constitutive expression, the CaMV 35S promoter:

Construction of the plasmid pCGN1761 is described in published patent application EP-0.392.225 (Example 23) which is incorporated herein by reference. pCGN1761 contains the "double" 35S promoter and the tml transcription terminator with a unique EcoR1 site between the promoter and the terminator and has a pUC type base chain. A derivative of pCGN1761 is constructed with a modified polylinker site which contains adjacent to the existing EcoRJ sites Notl and A7O / sites. The derivative is designated as pCGN1761ENX. pCGN1761ENX can be used to clone cDNA sequences or gene sequences (including microbial ORF sequences) within its polylinker sequence for expression under the control of the 35S promoter in transgenic plants. The entire 35S promoter gene sequence / m / terminator cassette of such construction can be excised by HindIII, Sphl, Sail and ATW sites 5 'from the promoter and Xbal,

BamHl and Bgll position 3 'to the terminator for transfer to 20 transformation vectors such as the vectors described above. Furthermore, the double 35S promoter fragment can be removed by 5 'excision with Hindlll, Sphl, Sail, Xbal or Pstl, and 3' excision at each of the restriction sites of the polylinker series (EcoRl, Notl or Xhol) before replacing with another promoter.

25 b. Modification of pCGN1761ENX by optimizing the translation initiation site.

For any of the constructs described herein, modifications can be made around the cloning sites by introducing sequences that can enhance translation. This is especially useful when overexpression is desirable.

pCGN1761ENX is cleaved with Sphl, treated with T4 DNA polymerase and coupled again, removing the SpAZ site located 5 'to the double 35S promoter. This gives vector 1007779 42 pCGN1761ENX / Sph-. pCGN1761ENX / Sph- is cleaved with EcoRI, and linked to a two-stranded molecular adapter with the sequence 5'-AATTCTAAAGCATGCCGATCGG-3 75 AATTCCGATCGGC ATGCTTTA-3 '(SEQ ID NOS: 12 and 13). This gives the vector pCGNSENX, which contains the quasi-5 optimized plant-active translation initiation site TAAA-C in addition to the ATG which itself is part of a Sphl site suitable for cloning heterologous genes at their initiating methionine residue. Downstream of the Sphl site, the EcoRI, Notl and jftoZ sites are retained.

An alternative vector is constructed using an NcoI site at the initiating ATG. This vector, designated pCGN1761ENX, is provided by introducing a two-strand molecular adapter of the sequence 5'-AATTCT AAACCAT GGCGATCGG-375 '-ATATCCG ATCGCCATGGTTT A-3' (SEQ ID NOS: 14 and 15) to the £ co. /? / - place of pCGN1761ENX. The vector therefore includes the quasi-optimized sequence TAAACC in addition to the initiating ATG located within the Ncol site. Downstream sites are EcoRI, Notl and Xhol. However, prior to this operation, the two TVcoZ sites in the pCGN1761ENX vector (at positions upstream of the 5 '35S promoter unit) are removed using similar techniques as described above for Sphl or alternatively using 20 "inside-out" PCR (Innes et al. PCR protocols: A guide to methods and applications, Academic Press, New York (1990)). It can be investigated whether this operation has any potential adverse effect on expression by introducing planetary cDNA or a reporter gene sequence into the cloning site, followed by routine expression analysis in plants.

C. Expression under a chemically controllable or a pathogen controllable promoter:

The double 35S promoter in pCGN1761ENX can be replaced by any other desired promoter that leads to suitably high expression levels. For example, a chemically regulated PR-1 promoter, which is described in U.S. Patent No. 5,614,395, incorporated by reference in its entirety, can replace the double 35S promoter. Preferably, the selected promoter is cut from its source using restriction enzymes, but 1007779 43 can alternatively be amplified by PCR using base series containing suitable terminal restriction sites. When PCR amplification is performed, the promoter must be sequenced to check for amplification errors after cloning the amplified vector into the target vector. The chemical / pathogen-controllable tobacco PR-1a promoter is cleaved from plasmid pCIB1004 (see EP-0.332.104, Example 21 for its construction, which is hereby incorporated by reference) and transferred to plasmid pCGN1761ENX (Uknes et al. 1992) . pCIB1004 is cleaved with Ncol and the 3'-protruding portion of the linearized fragment thus obtained is blunted by treatment with T4 DNA polymerase. The fragment is then cleaved with Hind III and the thus obtained PR-1a promoter containing fragment is gel purified and cloned in pCGN! 761ENX with the double 35S promoter removed. This is done by cutting with Xhol and filling with T4 polymerase, followed by cutting with Hind III and isolation of the larger vector terminator containing fragment into which the pCIB1004 promoter is cloned. This gives a pCGN1761ENX derivative with the PR-1a promoter and the tml terminator and an intermediate polylinker sequence with unique EcoRI and Notl sites. Selected NI M1 genes can be introduced into this vector, and the fusion products (ie, promoter gene terminator) can then be transferred to any selected transformation vector, including the vectors described in this application.

Various chemical control agents can be used to induce the expression of the NIM1 coding sequence in the plants transformed according to the present invention. In the context of the present specification, the term "chemical control agents" includes chemicals known to be inducers for the PR-1 promoter in plants, or closely related derivatives thereof. A preferred group of PR-1 promoter control agents 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-thiadiazole carboxylic acid, benzo-1,2,3-thiadiazole thiocarboxylic acid, cyanobenzo-1,2,3-thiadiazole, benzo-1,2,3-thiadiazole carboxylic acid amide, benzo-1,2,3-thiadiazole carboxylic acid hydrazide, benzo-1,2, 3-thiadiazole-7-carboxylic acid, benzo-1,2,3-1007 779 44 thiadiazole-7-thiocarboxylic acid, 7-cyanobenzo-1,2,3-thiadiazole, benzo-1,2,3-thiadiazole carboxylate in which the alkyl group one to six carbon atoms, methylbenzo-1,2,3-thiadiazole-7-carboxylate, n-propylbenzo-1,2,3-thiadiazole-7-carboxylate, benzylbenzo-1,2,3-thiadiazole-7-carboxylate, benzo-1,2,3-thiadiazole-7-5 carboxylic acid sec-butylhydrazide, and suitable derivatives thereof. Other chemical inducers include, for example, benzoic acid, salicylic acid (SA), polyacrylic acid, and substituted derivatives thereof; wherein suitable substituents include lower alkyl groups, lower alkoxy groups, lower alkylthio groups and halogen. Yet another group of control agents for the chemical inducible DNA-10 sequences of the present invention is based on the pyridine carboxylic acid structure, such as the isonicotinic acid structure and preferably the haloisonicotinic acid structure. Preference is given to dichloroisonicotinic acids and derivatives thereof, for example the lower alkyl esters. Suitable advantages of this series of control compounds are, for example, 2,6-dichloroisonicotinic acid (INA), and the lower alkyl esters thereof, especially the methyl ester.

d. Constitutive expression, the actin promoter.

Different isoforms of actin are known to be expressed in most cell types, making the actin promoter a good choice for a constitutive promoter. In particular, the promoter of the rice-derived Act1 gene has been cloned and characterized (McElroy et al. Plant Cell 2: 163-171 (1990)). A 1.3kb fragment of the promoter was found to contain all the control elements required for expression in rice protoplasts. Furthermore, various expression vectors based on the Act1 promoter have been constructed, particularly for use in monocotyledonous plants (McElroy et al. Mol. Gen. Genet. 231: 150-160 (1991)). These include the .4c // intron 1, Adhl 5 'flanking sequence and Adhl intron 1 (from the corn alcohol dehydrogenase gene) and the sequence of the CaMV 35S promoter. Vectors expressing the highest expression were fusions of 35S and the Actl intron or the Actl 5 flanking sequence and the Actl intron. Optimization of the sequences around the initiating ATG (of the GUS reporter gene) also gave increased expression. The promoter expression cassettes described by McElroy et al. (Mol. Gen. Genet. 231: 150-160 (1991)) are readily adaptable for the expression of cellulase genes and are particularly suitable for use in monocotyledonous hosts. For example, promoter-containing fragments can be removed from the McElroy constructs and used to replace the double 35S promoter in pCGN1761ENX, which is then available for insertion of specific gene sequences. The fusion genes thus constructed can then be transferred to suitable transformation vectors. A separate report found that the rice-derived ActI-ρτοταοΧοτ with its first intron gives high expression in cultured barley cells (Chibbar et al. Plant Cell Rep. 12: 506-509 (1993)).

10 e. Constitutive expression, the ubiquitin promoter:

Ubiquitin is another gene product known to form in many cell types, and its promoter has been cloned from several species for use in transgenic plants (e.g., sunflower - Binet et al. Plant Science 79: 87-94 (1991) and corn - Christensen et al. Plant Molec Biol. 12: 619-632 (1989)). The maize ubiquitin promoter has been developed for transgenic monocotyledon systems and its sequence as well as vectors for transformation of monocotyledonous plants are described in patent application EP-0.342.926 (from Lubrizol) which is incorporated herein by reference. Taylor et al. (Plant Cell Rep. 12: 491-495 (1993)) describe a vector (pACH25) containing the maize ubiquitin promoter and its first intron, as well as its high activity in cell suspensions of various monocotyledonous plants when inserted by micro-projectile bombardment. The ubiquitin promoter is suitable for the expression of cellulase genes in transgenic plants, especially monocotyledonous plants. Suitable vectors are derivatives of pACH25 or any of the transformation vectors described in this application, adapted by introducing the appropriate ubiquitin promoter and / or intron sequences.

f. Specific expression in the root:

A further pattern of expression for the NIM1 gene of the present invention is root expression. A suitable promoter for root expression has been described by the Framond (FEBs 290: 103-106 (1991)), as well as in published patent application EP-0.452.269 (from Ciba-Geigy) which is incorporated herein by reference. This promoter is transferred to a suitable 1007779 46 vector such as pCGN1761ENX for insertion of a cellulase gene and subsequent transfer of the entire promoter gene terminator cassette to a target transformation vector.

5 g. Promoters inducible by injury:

Injury-inducible promoters can also be suitably used in the expression of NIM1 genes of the invention. Several such promoters have been described (eg, Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989), Rohrmeier & Lehle, 10 Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), Warner et al. Plant J. 3: 191-201 (1993)) and these are all suitable for use in the present invention. Logemann et al. Describe the 5 'upstream sequences of the dicotyle potato wunl gene. Xu et al. Show that an injury-inducible promoter of the dicotyledonous potato plant (pinl) is active in the monocotyledonous rice plant. Describe further

Rohmeier & Lehle cloning of the maize-derived Wipl cDNA which is induced by injury and which can be used by standard techniques to isolate the related promoter. Similarly, Firek et al. And Warner et al. Have described an injury-inducible gene from the monocotyledonous plant Asparagus officinalis, which gene is expressed at local injuries and pathogen intrusion sites. Using cloning techniques well known in the art, these promoters can be transferred to suitable vectors, linked to the NIM1 genes of the present invention, and used to express these genes in sites where plants are injured.

h. Pith preference expression:

Patent application WO 93/07278 (from Ciba-Geigy), which is incorporated herein by reference, describes the isolation of the maize-derived trpA gene which is preferably expressed in pith cells. The gene sequence and promoter up to -1726 base pairs from the start of transcription are described. Using standard molecular biology techniques, this promoter, or 1007779 47 parts thereof, can be transferred to a vector such as pCGN1761, in which it can replace the 35S promoter and be used to control expression of a foreign gene, in preferred in pith cells. In fact, fragments containing the pith preferred promoter or parts thereof can be transferred to any vector and adapted for use in transgenic plants.

i. Leaf specific expression:

A maize gene encoding phosphoenol carboxylase (PEPC) has been described by Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)). Using standard molecular biology techniques, the promoter for this gene can be used to control the specific expression of any gene in the leaves of transgenic plants.

15 j. Chloroplast Expression:

Chen & Jagendorf (J. Biol. Chem. 268: 2363-2367 (1993) have described the successful use of a chloroplast-derived transit peptide for the insertion of a heterologous transgene. The peptide used is the transit peptide of the Nicotiana rbsS gene. plumbaginifolia (Poulsen et al. Mol.

20 Gen. Genet. 205: 193-200 (1986)). Using the restriction enzymes Dral and Sphl.pr 7jp509I and Sphl, the DNA sequence encoding this transit peptide can be excised from the plasmid prbcS-8B and processed for use with any of the constructs described above. The Dral-Sphl fragment extends from -58 relative to the initiating rbcS ATG to, and including, the first amino acid (also a methionine) of the full peptide immediately after the import cleavage site, while the Tsp5091-Sphl fragment ranges from -8 relative to the initiating rbcS ATG to, and including, the first amino acid of the full-fledged peptide.

These fragments can therefore be suitably inserted into the polylinker sequence of any selected expression cassette to obtain a transcription fusion to the untranslated leader sequence of the selected promoter (ie, 35S, PR-1a, actin, ubiquitin, etc.), while simultaneously allows insertion of a NIM1 gene into a correct fusion downstream of the 1007779 48 transit peptide. Constructions of this type are common in the art. For example, where the Dral end is already blunt, the 5 'Tsp509I site can be blunted by treatment with T4 polymerase, or alternatively linked to a coupling sequence or adapter sequence that facilitates its fusion with the selected promoter. The 3 'Sphl site can be maintained as such, or alternatively can be linked to adapter or coupling sequences to facilitate its insertion into the selected vector in such a way as to provide suitable restriction sites for the subsequent insertion of a selected NIM1. -gene. Particularly preferred, the Sphl site ATG is retained and forms the first ATG of the target NIM1 gene. Chen & Jagendorf provide consensus sequences for ideal cleavage of the chloroplast imports, and in all cases a methionine is preferred in the first place of the whole protein. In successive places there is more variation and the amino acid cannot be so critical. In any case, fusion constructs can be assessed in vivo for insertion efficiency using the method described by Bartlett et al. (In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier biz. 1081- 1091 (1982)) and Wasmann et al. (Mol. Gen. Genet. 205: 446-453 (1986)). Typically, the best approach is to generate fusions using the chosen NIM1 gene or the modified form of the NIMl gene without modifications at the amino terminus, and only include modifications when it is clear that such fusions are not are incorporated into the chloroplasts with high efficiency, in which case modifications can be made in accordance with the known literature (Chen &Jagendorf; Wasman et al .; Ko & Ko. J. Biol. Chem. 267; 13910-13916 (1992) ).

A preferred vector is constructed by transferring the Dral-Sphl transit peptide encoding a fragment of prbcS-SB to the cloning vector pCGN 1761 ENXJSph-. This plasmid is cut with EcoRI and blunt ended by treatment with T4 DNA polymerase. 30 Plasmid prbcS-SB is cut with Sphl and linked to a two-stranded molecular adapter with the sequence 5'-CCAGCTGGAATTCCG-375'-CGGAATTCCAGCTGGCATG-3 '(SEQ ID NOS: 16 and 17). The product thus obtained is phosphorylated 5'-end by treatment with T4 kinase. Subsequent splitting with Dral releases the fragment encoding the transit peptide, after which the fragment is linked to the blunted EcoR1 sites of the modified vector described above. Clones oriented with the 5 'end of the introduced portion to the 3' end of the 35S promoter are identified by sequencing. These clones carry a DNA fusion of the 35S leader sequence to the ri> cS-8A promoter transit protein sequence ranging from -58 relative to the rbcS ATG to the ATG of the mature protein, and have a unique 5ρΛ in this region. / site and a newly formed EcoRl site, as well as the existing Notl and ^ 7io / sites of 10 pCGN1761ENX. This new vector is referred to as pCGN1761 / CT. DNA sequences are transferred to pCGN1761 / CT in the appropriate reading frame by amplification using PCR techniques and incorporation of a Sphl, NSphl, or Ma /// site at the amplified ATG, which after cleavage using the appropriate restriction enzyme is coupled to the SpAZ-cleaved pCGN1761 / CT. To facilitate construction, it may be necessary to change two amino acids of the product of the cloned gene; however, in almost all cases, the use of PCR together with standard site-directed mutagenesis will allow the construction of any desired sequence around the cleavage site and the first methionine residue of the mature protein.

A further preferred vector is constructed by replacing a double 35S promoter of pCGN1761ENX with the BamHI-Sphl fragment of prbcS-ÜA which is the full light-regulated rbcS-ÜA promoter from -1038 (relative to the starting site). from the transcription) to the first methionine residue of the mature protein. The modified pCGN1761 with the destroyed Sphl site is cleaved with PstI and EcoRl and treated with T4 DNA polymerase to blunt the ends. prbcS-SA is cleaved with Sphl and linked to the two-stranded molecular adapter of the sequence described above. The product thus obtained is phosphorylated 5'-end by treatment with T4 kinase. Subsequent cleavage with BamHI 30 releases the fragment comprising the promoter transit peptide, which fragment is treated with T4 DNA polymerase to blunt the 5am /// ends. The promoter transit peptide fragment thus obtained is cloned into the prepared pCGN1761ENX vector, obtaining a construction containing the rbcS-8A-1 007779 50 promoter and the transit protein with an iSpW site located at the heterologous gene insertion site. located. Furthermore, downstream of the Sphl site EcoRl (reformed), Notl and Xhoï cloning sites are located. The construct is referred to as pCGN1761rbcS / CT.

Similar operations can be performed using sequences encoding GS2 chloroplast transit peptide from other sources (from monocotyledonous and dicotyledonous plants) or from other genes. Furthermore, similar procedures can be followed to target other subcellular compartments, such as mitochondria.

10 2. Transcription Terminators.

Various transcription terminators for use in expression cassettes are available. They ensure termination of the transcription after the transgene and its proper polyadenylation. Suitable transcription terminators are those known to be active in plants and include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, and the pea-derived rbcS E9 terminator. These can be used in monocotyledonous plants as well as in dicotyledonous plants.

3. Sequences for enhancing or controlling expression.

Several sequences have been found to enhance gene expression from within the transcription unit and these sequences in conjunction with the genes of the present invention can be used to enhance their expression in transgenic plants.

Several intron sequences have been shown to enhance expression, especially in cells of monocotyledonous plants. For example, it has been found that the introns of the maize-derived Adhl gene significantly enhance the expression of the wild type gene under its natural promoter when introduced into maize cells. Intron 1 appears to be particularly effective and enhances expression in fusion constructs with the chloramphenicol acetyl transferase gene (Callis et al., Genes Develop. 1: 1183-1200 (1987)). In the same experimental system, the intron of the maize-derived bronze 1 gene had a similar effect in enhancing expression. Intron sequences have often been included in 1007779 51 plant transforming vectors, usually with the untranslated lead sequence.

A number of virus-derived untranslated leader sequences are also known to enhance expression, and are particularly active in cells of dicotyledonous plants. In particular, the tobacco mosaic mosaic virus (TMV, the "W sequence"), Maize Chlorotic Mottle Virus (MCMV) and alpha mosaic mosaic virus (AMV) have been shown to be effective in enhancing expression (see, for example, Gallie et al Nucl Acids Res 15: 8693-8711 (1987) Skuzeski et al Plant Molec Biol 15: 65-79 (1990)).

10 4. Targeting the gene product within the cell.

It is known that various mechanisms for targeting gene products are present in plants and the sequences that control the action of these mechanisms have been characterized to some degree. For example, the targeting of gene products to the chloroplast is controlled by a signal sequence located at the amino-terminal end of various proteins, which is cleaved during introduction into the chloroplast to form the full-fledged protein (eg, Comai et al. J. Biol Chem 263: 15104-15109 (1988)). These signal sequences can be linked to heterologous gene products so that the heterologous gene product is introduced into the chloroplast (van den Broeck et al. Nature 313: 358-363 (1985)). DNA encoding the appropriate signal sequences can be isolated from the 5 'end of the cDNAs encoding the RUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2 protein and many other proteins known to 25 are located in the chloroplast.

Other gene products are located in other organelles, such as the mitochondrium and peroxisome (see, for example, Unger et al. Plant Molec. Biol.

13: 411-418 (1989)). The cDNAs encoding these products can also be manipulated to achieve targeting of the heterologous gene products to these organelles. Examples of such sequences are the nuclear-encoded ATP ases and specific aspartate aminotransferase isoforms for mitochondria. Targeting of cellular protein bodies has been described by Rogers et al. (Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).

1007779 52

Furthermore, sequences have been characterized that determine the targeting of gene products to other cell compartments. Amino-terminal sequences are responsible for targeting the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)). Furthermore, amino-5 terminal sequences in conjunction with carboxyl terminal sequences are responsible for targeting gene products to the vacuole (Shinshi et al.

Plant Molec. Biol. 14: 357-368 (1990)).

By linking appropriate targeting sequences described above to the target transgenic sequences, it is possible to target the transgenic product to any organelle or cell compartment. For example, to target the chloroplast, the chloroplast signal sequence of the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene must be linked in the appropriate reading frame to the amino terminal ATG of the transgene. The selected signal sequence must also include the known cleavage site, and the engineered fusion must also contain the 15 amino acids after the cleavage site required for the cleavage. In some cases, this requirement can be met by adding a small number of amino acids between the cleavage site and the ATG of the transgene, or, alternatively, replacing a number of amino acids in the transgene sequence.

Fusions constructed for input into the chloroplast can be tested for their efficacy in the incorporation into the chloroplast by in vitro translation or in vitro transcription constructs followed by in vitro incorporation into chloroplasts, using the techniques that are described by Bartlett et al. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier biz. 1081-1091 (1982) and Wasmann et al. Mol. Gene. Genet. 205: 25 446-453 (1986). These construction techniques are well known in the art and can also be applied to mitochondria and peroxisomes.

The above-described cell targeting mechanisms can be used not only in conjunction with the related promoters, but against in conjunction with heterologous promoters, to achieve a specific targeted targeting within the cell under the control of transcription by a promoter that has an expression pattern different from the promoter from which the direction signal is derived.

1007779 53 C. Transformation.

After the NIM1 coding sequence 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 present system can be used in any plant that can be transformed and regenerated. Such transformation and regeneration techniques are well known in the art. Techniques for constructing plant-active expression cassettes as well as introducing foreign DNA into plants have been widely described in the art. Generally, for incorporating foreign DNA into plants, Ti plasmid vectors are used to introduce foreign DNA. Direct DNA uptake, liposomes, electroporation, microinjection and microprojectiles are also suitable for insertion. Such techniques have been described in the art. See, e.g., Bilang et al. (1991) Gene 100: 247-250; Scheid et al., (1991) Mug Gem Genet. 228: 15 104-112: Guerche et al., 1987) Plant Science 52: 111-116; Neuhause et al., (1987)

Theor. Appl. Genet. 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 Weissbach and Weisbach, eds.) Academic Press, Inc. (1988); and Methods in Plant 20 Molecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc. (1989). See also U.S. Patent No. 5,625,136, which is incorporated herein in its entirety. It is to be understood that the transformation technique will depend on the plant cell to be transformed. Transformation of tobacco, tomato, potato, and Arabidopsis thaliana using a binary Ti vector system. Plant Physiol. 81: 301-305, 1986; Fry, J., Bamason, A., and Horsch, R.B. Transformation of Brassica napus with

Agrobacterium tumefaciens based vectors. Pl. Cell Rep. 6: 321-325, 1987; Block, M.d. Genotype independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens. Theor. appl. netted. 76: 767-774, 1988; Deblock, M., Brouwer, D.D., and Terming, P. Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the Expression of the bar and neo genes in the transgenic plants. Plant Physiol. 91: 694-701, 1989; Baribault, T.J., Skene, K.G.M., Cain, P.A., and Scott, N.S. Transgenic grapevines: regeneration of shoots expressing beta-glucuronidase. Pl. Cell Rep. 41: 1045-1049, 1990; Hinchee, M.A.W., Newell, 1 007 779 54 C.A., ConnorWard, D.V., Armstrong, T.A., Deaton, W.R., Sato, S.S., and Rozman, R.J. Transformation and regeneration of non-solanaceous crop plants. Stadler.Genet.Symp. 203212,203-212, 1990; Barfield, D.G. and Pua, E.C. Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated 5 transformation. Pl. Cell Rep. 10: 308-314, 1991; Cousins, Y.L., Lyon, B.R., and

Llewellyn, D.J. Transformation of an Australian cotton cultivar: prospects for cotton improvement through genetic engineering. Aust. J. Plant Physiol. 18: 481-494. 1991; Chee, P.P. and Slightom, J.L. Transformation of Cucumber Tissues by Microprojectile Bombardment Identification of Plants Containing Functional and Nonfunctional 10 Transferred Genes. GENE 118: 255-260,1992; Christou, P., Ford, T.L., and Kofron, M. The development of a variety-independent gene-transfer method for rice. Trends. Biotechnol. 10: 239-246, 1992; Halluin, K., Bossut, M., Bonne, E., Mazur, B., Leemanr, J., and Botterman, J. Transformation of sugarbeet (Beta vulgaris L.) and evaluation of herbicide resistance in transgenic plants. Bio / Technol. 10: 309-314.

1992; Dhir, S.K., Dhir, S., Savka, M.A., Belanger, F., Kriz, A.L., Farrand, S.K. and

Widholm, J.M. Regeneration of Transgenic Soybean (Glycine Max) Plants from Electroporated Protoplasts. Plant Physiol 99: 81-88,1992; Ha, S.B., Wu, F.S., and Thome, T.K. Transgenic peat-type tall fescue (Festuca arundinacea Schreb.) Plants regenerated from protoplasts. PI. Cell Rep. 11: 601-604, 1992; Blechl, A.E. Genetic 20 Transformation The New Tool for Wheat Improvement 78th Annual Meeting

Keynote Address. Cereal Food World 38: 846-847, 1993; Casas, A.M., Kononowicz, A.K., Zehr, U.B., Tomes, D.T., Axtell, J.D., Butler, L.G., Bressan, R.A., and Hasegawa, P.W. Transgenic Sorghum Plants via Microprojectile Bombardment. Proc Natacad Sci USA 90: 11212-11216, 1993; Christou, P. Philosphy and Practice of 25 Variety Independent Gene Transfer into Recalcitrant Crops. 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 Through Agrobacterium Rhizogenes Transformation. Transgenic Res 2: 330-335,1993; Davies, D.R., Hamilton, J., and Mullineaux, P. Transformation of Peas. Pl. Cell Rep. 12: 180-183,30 1993; Dong, J.Z. and Mchughen, A. Transgenic Flax Plants from Agrobacterium

Mediated Transformation Incidence of Chimeric Regenerants and Inheritance of Transgenic Plants. Plant Sci 91: 139-148,1993; Fitch, M.M.M., Manshardt, R.M., Gonsalves, D., and Slightom, J.L. Transgenic Papaya Plants from Agrobacterium 1 0 07 7 7 9 55

Mediated Transformation of Somatic Embryos. Pl. Cell Rep. 12: 245-249, 1993; Franklin, C.I. and Trieu, T.N. Transformation of the Forage Grass Caucasian Bluestem via Biolistic Bombardment Mediated DNA Transfer. Plant Physiol 102: 167, 1993; Golovkin, M.V., Abraham, M., Morocz, S., Bottka, S., Feher, A., and Dudits, 5 D. Production of Transgenic Maize Plants by Direct DNA Uptake into Embryogenic Protoplasts. Plant Sci 90: 41-52, 1993; Guo, G.Q., Xu, Z.H., Wei, Z.M., and Chen, H.M.Transgenic Plants Obtained from Wheat Protoplasts Transformed by Peg Mediated Direct Gene Transfer. Chin Sci Bull 38: 2072-2078. 1993; Asano, Y. and Ugaki, M. Transgenic plants of Agrostis alba obtained by electroporation mediated 10 direct gene transfer into protoplasts. PI.Cell.Rep. 13, 1994; Ayres, N.M. and Park, W.D. Genetic Transformation of Rice. Crit Rev Plant Sci 13: 219-239, 1994; Barcelo, P., Hagel, C., Becker, D., Martin, A., and Lorz, H. Transgenic Cereal (Tritordeum) Plants Obtained at High Efficiency by Microprojectile Bombardment of Inflorescence Tissue. PLANT J 5: 583-592, 1994; Becker, D., Brettschneider, R., and Lorz, H.

15 Fertile Transgenic Wheat from Microprojectile Bombardment of Scutellar Tissue. Plant J 5: 299-307. 1994; Biswas, G.C.G., Iglesias, V.A., Datta, S.K., and Potrykus, 1. Transgenic Indica Rice (Oryza Sativa L) Plants Obtained by Direct Gene Transfer to Protoplasts. J Biotechnol 32: 1-10, 1994; Borkowska, M., Kleczkowski, K., Klos, B., Jikubiec, J., and Wielgat, B. Transformation of Diploid Potato with an 20 Agrobacterium Tumefaciens Binary Vector System. 1. Methodological Approach.

Acta Physiol Plant 16: 225-230, 1994; Brar, G.S., Cohen, B.A., Vick, C.L., and Johnson, G.W. Recovery of Transfgenic Peanut (Arachis Hypogaea L) Plants from Elite Cultivars Utilizing Accell (R) Technology. Plant J 5: 745-753, 1994; Christou, P. Genetic Engineering of Crop Legumes and Cereals Current Status and Recent 25 Advances. Agro Food Ind Hi Tech 5: 17-27, 1994; Chupeau, M.C., Pautot, V., and Chupeau, Y. Recovery of Transgenic Trees after Electroporation of Poplar Protoplasts. Transgenic Res 3: 13-19, 1994; Eapen, S. and George, L. Agrobacterium Tumefaciens Mediated Gene Transfer in Peanut (Arachis Hypogaea L). PI. Cell Rep. 13: 582-586, 1994; Hartman, C.L., Lee, L., Day, P.R., and Turner, N.E. Herbicide 30 Resistant Peat Grass (Agrosis Palustris Huds) by Biolistic Transformation. Bid-Technology 12: 919923, 1994; Howe, G.T., Goldfarb, B., and Strauss, S.H. Agrobacterium Mediated Transformation of Hybrid Poplar Suspension Cultures and Regeneration of Transformed Plants. Plant Cell Tissue & Orgart Culture 36: 59-71, 1 0 0 ~, 7'q 56 1994; Konwar, B.K. Agrobacterium Tumefaciens Mediated Genetic Transformation of Sugar Beet (Beta Vulgaris L). J Plant Biochem Biotechnol 3: 37-41, 1994; Ritala, A., Aspegren, K., Kuiten, U., Salmenkalliomarttila, M., Mannonen, L., Hannus, R., Kauppinen, V., Teeri, T.H., and Enari, T.M. Fertile Transgenic Barley by Particle 5 Bombardment of Immature Embryos. Plant Mol Biol 24: 317-325, 1994; Scorza, R., Cordts, J.M., Ramming, D.W., and Emershad, R.L. Transformation of Grape (Vitis Vinifera L) Somatic Embryos and Regeneration of Transgenic Plants. J Cell Biochem: 102, 1994; Shimamoto, K. Gene Expression in Transgenic Monocots. Curr Opinbiotechnol 5: 158-162, 1994; Spangenberg, G., Wang, Z.Y., Nagel, J., and 10 Potrykus, I. Protoplast Culture and Generation of Transgenic Plants in Red Fescue (Festuca Rubra L). Plant Sci 97: 83-94, 1994; Spangenberg, G., Wang, Z.Y., Nagel, J., and Potrykus, I. Gene Transfer and Regeneration of Transgenic Plants in Forage Grasses. J Cell Biochem: 102, 1994; Wan, Y.C. and Lemaux, P.G. Generation of Large Numers of Independently Transformed Fertile Barley Plants. Plant Physiol 15 104: 3748, 1994; Weeks, J.T., Anderson, O.D., and Blechl, A.E. Stable

Transformation of Wheat (Triticum Aestivum L) by Microprojectile Bombardment. J Cell Biochem: 104, 1994; Ye, X.J., Brown, S.K., Scorza, R., Cordts, J., and Sanford, J.C. Genetic Transformation of Peach Tissues by Particle Bombardment. Jamer Sochortsci 199: 367-373, 1994; Spangenberg, G., Wang, Z.Y., Nagel, J., and 20 Potrykus, I. Protoplast Culture And Generation 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 (i.e. strains LBA4404 or EHA105) is particularly suitable because of its well known ability to transform plants.

1. Transformation of dicotyledonous plants.

Transformation techniques for dicotyledonous plants are well known in the art and include techniques based on Agrobacterium and techniques that do not require the use of Agrobacterium. Techniques without the use of Agrobacterium include the direct uptake of exogenous genetic material by 1007779 57 protoplasts or cells. This can be achieved by PEG or electroporation transfer, particle bombardment, or microinjection. Examples of these techniques are described by Paszkowski et al., EMBO J 3: 2717-2722 (1984), Potrykus et al., Mol. Gene. Genet. 199: 169-177 (1985), Reich 5 et al., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327: 70-73 (1987). In all these cases, the transformed cells are regenerated into whole plants using standard techniques known in the art.

Transformation Using Agrobacterium is a preferred technique for transforming dicotyledonous plants because of its high transformation efficiency and broad applicability to a wide variety of species. Different crop species include alfalfa and poplar (EP-0.317.511), cotton, EP-0.249.432 (tomato, from Calgene), WO 87/07299 (Brassica, from Calgene), US-A-4,795,855 (poplar )). Agrobacterium transformation typically involves transferring the binary vector carrying the target foreign DNA (eg, pCIB200 or pCIB2001) to an appropriate Agrobacterium strain, which may depend on the genes present which are used by the host Agrobacterium. strain on a co-resident Ti plasmid or carried chromosomally (eg strain CIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)). Transfer of the recombinant binary vector to Agrobacterium is performed by a triparenteral crossing procedure using an E. coli carrying the recombinant binary vector, as well as a helping E. coli strain carrying a plasmid such as pRK2013 capable of mobilizing the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vetor can be transferred to Agrobacterium by DNA transformation (Höfgen & Willmitzer, Nucl. Acids Res . 16: 9877 (1988)).

Transformation of the target plant species by recombinant Agrobacterium typically involves co-cultivating the Agrobacterium with plant tissues and follows protocols well known in the art. Transformed tissue is regenerated on selectable medium containing the antibiotic or herbicide resistance marker present between the T-DNA border regions of the binary plasmid.

Another approach to transforming plant cells with a gene involves bombarding plant tissues and cells with inert or biologically active particles. This technique is described in U.S. Patent Nos. 4,945,050; 5,036,006 and 5,100,792, all from Sanford et al. In general, this procedure involves the inerting of inert or biologically active particles at the cells under conditions suitable for penetrating the outer surface of the cell such that the particles in the interior thereof are taken up. When inert particles are used, the vector can be introduced into the cell by coating the particles with the vector carrying the desired gene. Alternatively, the target cell may be surrounded by the vector such that the vector enters the cell in the wake of the particle. Biologically active particles (e.g., dried yeast cells, dried bacteria or a bacteriophage, each containing the DNA to be introduced) can also be shot into the plant cell tissue.

2. Transformation of monocotyledonous plants.

15 Transforming most types of monocotyledonous plants has also now become routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and bombardment of callus tissue with particles. Transformations can be performed with a single DNA species or with multiple DNA species (ie co-transformation) and both of these techniques are suitable for use in the present invention. Co-transformation can have the advantage of avoiding the construction of complete vectors and can lead to transgenic plants with unlinked sites for the gene of interest and the selectable marker, which allows to remove the selectable marker in later generations, when deemed desirable. However, a drawback of the use of co-transformation is the frequency of less than 100% with which the individual DNA species are introduced into the genome (Schocher et al. Biotechnology 4: 1093-1096 (1986)).

Patent applications EP-0.292.435 ([1280/1281] from Ciba-Geigy), EP-0.392.225 (from Ciba-Geigy) and WO 93/07278 (from Ciba-Geigy) describe techniques for preparing callus and protoplasts from a high quality inbred maize line, transformation of protoplasts using PEG or electroporation, and regeneration of the maize plant from transformed protoplasts. Gordon-Kamm et al.

1007779 59 (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 using particle bombardment. Furthermore, patent application WO 93/07278 (to Ciba-Geigy) and Koziel et al. (Biotechnology Π .: 5 194-200 (1993)) describe techniques for transforming high quality inbred maize lines by particle bombardment. This technique utilizes 1.5-2.5 mm immature maize embryos cut from a maize ear 14-15 days after pollination, and a PDS-1000He Bolistics bombardment device.

Rice transformation can also be performed by direct gene transfer techniques and the use of protoplasts or particle bombardment. Transformation using protoplasts has been described for Japonica types and Indica types (Zhang et al. Plant Cell Rep 7: 379-384 (1988); Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology 8: 736-740 15 (1990)). Both types can also be routinely transformed by particle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)).

Patent application EP-0.332.581 (from Ciba-Geigy) describes techniques for generating, transforming and regenerating Pimpea protoplasts. These techniques allow the transformation of Dactylis and wheat. Furthermore, transforming wheat by firing particles into cells of long-term regenerable callus type C has been described by Vasil et al. (Biotechnology J_0: 667-674 (1992)), and by firing particles on immature embryos and immature embryos obtained from embryos also by Vasil et al. (Biotechnology IT1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102: 1077-1084 (1993)).

However, a preferred technique of transforming wheat involves transforming wheat by bombarding immature embryo particles and involves a step at a high sucrose or high maltose content prior to gene insertion. Prior to bombardment, any number of embryos (0.75-1 mm in length) are plated on MS medium with 3% sucrose 30 (Murashiga & Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg / 1 2,4-D for inducing somatic embryos, which is performed in the dark. On the day of bombardment, the embryos are removed from the induction medium and placed on the osmotic medium (i.e. induction medium to which sucrose or maltose at 1 007779 60 the desired concentration has been added, usually 15%). The embryos are subjected to plasmolysis for 2-3 hours and then bombarded. Twenty embryos per target plate is common, but not critical. A suitable gene-carrying plasmid (such as pCIB3064 or pSG35) is deposited on micrometer size gold particles using standard procedures. Each embryo-containing plate is bombarded with the DuPont Biolistics® helium device at a firing pressure of 1000 psi using a standard 80 mesh screen. After bombardment, the embryos are put back in the dark so that they can recover for about 24 hours (still on osmolitic). After 24 hours, particles are removed from the osmolitic and placed back into the induction medium where they reside for about a month before regeneration. About a month later, the embryo explants with developing embryogenic callus are transferred to regeneration medium (MS + 1 mg / 1 NAA, 5 mg / 1 GA) which further contains the appropriate selection agent (10 mg / 1 basta in the case of pCIB3064 and 2 mg / l methotrexate in the case of pSOG35). After about a month, the developing shoots are transferred to larger sterile containers known as "GA7s" containing half the strength of MS, 2% sucrose, and the same concentration of selection agent. Patent application 08 / 147,161 describes techniques for transforming wheat and is incorporated herein by reference 20.

Recently, the transformation of monocotyledonous plants using Agrobacterium has been described. See, for example, WO 94/00977 and U.S. Patent 5,591,616, both of which are incorporated herein by reference.

25 Growing

The isolated gene fragment of the present invention or modified forms of the M / W gene can be used to transmit disease resistance to a wide variety of plant cells, including those of flowering plants, monocotyledons and dicotyledons. While the gene can be introduced into any plant cell that falls within these broad classes, it is particularly suitable for use in crop plant cells, such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, brinjal, 1 007779 61 pepper, celery, carrot, pumpkin, in particular Cucurbita pepo, zucchini, cucumber, apple , pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, pineapple, avocado, papaya, mango, banana, soy, tobacco, tomato, sorghum or sugar cane.

The high-level expression of the NIM1 gene and its mutants necessary for constitutive expression of SAR genes, in combination with other traits important for production and quality, can be incorporated into plant lines by cultivation. Thus, further embodiments of the present invention include a method of producing transgenic progeny from a parent transgenic plant containing an isolated DNA molecule encoding an altered form of a NIM1 protein of the present invention, which method comprises transforming the parent plant with a recombinant vector molecule of the invention and the transfer of the trait to the progeny of transgenic parent plant using known plant breeding techniques. Methodologies and techniques for cultivation are known in the art. See, for example, Welsh, J.R., Fundamentals of Plant Genetics and Breeding. John Wiley & Sons, NY (1981); Crop Breeding. Wood, D.R. (Ed.) American Society of Agronomy Madison, Wisconsin (1983); Mayo 0., The Theory of Plant Breeding. Second Edition, Clarendon Press, Oxford (1987); Singh,

20 D.P., Breeding for Resistance to Diseases and Insect Pests. Springer-Verlag, NY

(1986); and Wricke and Weber, Quantitative Genetics and Selection Plant Breeding. Walter de Gruyter and Co., Berlin (1986).

Multiplying the genetic traits conferred on the transgenic seeds and plants and maintaining them in the offspring.

The genetic traits conferred on the transgenic seeds and plants described above are transmitted by sexual reproduction or vegetative growth and can therefore be retained and propagated in the offspring. Generally, this conservation and propagation uses known agricultural techniques developed for specific purposes, such as cultivation, sowing or harvesting. Specialized processes such as hydroponic techniques or techniques under glass can also be used. If the growing crop is vulnerable to attack and damage from insects or infections as well as from 1007779 62 for competition from weeds, measures are taken to control weeds, plant diseases, insects, nematodes, and other adverse conditions to improve yield. These include mechanical techniques such as soil treatment or weed removal and infected plants, as well as the use of agrochemicals such as herbicides, fungicides, gametocides, nematicides, growth regulators, ripeners and insecticides. The beneficial genetic properties of the transgenic plants and seeds of the invention can also be used in plant cultivation, the purpose of which is to develop plants with improved properties such as tolerance to pests, herbicides or stress, improved nutritional value, improved yield or improved structure leading to less loss due to disturbance or damage. The different breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing the pollination of the parent lines or selecting suitable offspring. Different cultivation measures can be taken depending on the desired properties. The relevant techniques are well known in the art and include, but are not limited to, hybridization, inbreeding, backcross, multi-line, variety mixing, interspecific hybridization, aneuploid techniques, and the like.

Hybridization techniques also include sterilizing plants to provide male or female sterile plants by mechanical, chemical or biochemical techniques. Cross-pollination of a male sterile plant with pollen from another line ensures that the genome of the male sterile, but female fertile plant uniformly acquires the properties of both parent lines. For example, the transgenic seeds and plants of the invention can be used to cultivate improved plant lines that increase, for example, the effectiveness of conventional methods such as herbicide or pesticide treatment or allow the abandonment of such methods because of their modified genetic properties. Alternatively, new crops with improved stress tolerance can be obtained which, due to their improved genetic "ability", yield a better quality harvest product than those unable to withstand similar adverse conditions during development.

1007779 63

In seed production, germination quality and seed uniformity are essential product properties, while germination quality and seed uniformity harvested and sold by the farmer is not important. Since it is difficult to keep a crop free from seeds of other crops and from weeds, to combat seed-borne diseases and to produce seed with good germination, seed producers all experienced in the art of reproduction , conditioning and marketing pure seed, developed very extensive and well-defined seed production methods. It is therefore common for the farmer to buy 10 certified seed that meets certain specific quality requirements, rather than seed he uses seed harvested from his own crops. Seed propagating material is usually treated with a protective coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures thereof. Commonly used protective coatings include compounds such as captan, carboxin, thiram (TMTD®), methalaxyl (Apron®), and pirimiphos-methyl (Actellic®). If desired, these compounds are formulated together with further carriers, surfactants or use-enhancing adjuvants as commonly used in the formulation art to provide protection against damage caused by harmful bacteria, fungi or animals. The protective coatings can be imparted by impregnating the propagation material with a liquid formulation or coating with a combined wet or dry formulation. Other commissioning techniques are also possible, such as a treatment that targets the buds or fruit.

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

The seeds can be provided in a bag, container or vessel, which includes a suitable packaging material, the bag or container being closable for holding the seeds. The bag, container or vessel may be designed for short or long term storage, or both, of the seed. Examples of suitable packaging materials are paper, such as kraft paper, rigid or bendable plastic, or another 1007 779 64 polymer material, glass or metal. Preferably, the bag, container or vessel consists of several layers of packaging material, of the same or different types. According to an embodiment, the bag, container or vessel is provided such that the contact of the seed with water and moisture is limited or excluded. In an example, the bag, container or vessel is closed, for example closed by heating, so that moisture or water is prevented from entering. In another embodiment, water absorbent materials are placed between or adjacent to the layers of the packaging material. According to yet another embodiment, the bag, container or vessel, or the packaging material from which it is made, is treated to limit, suppress or prevent disease, contamination or other adverse effects on the storage or transportation of the seed. An example of such a treatment is sterilization, for example by chemical means or by exposure to radiation. Included within the present invention is a commercial bag comprising transgenic plant seed comprising at least an altered form of an NIM1 protein, or an NIM1 protein expressed in the transformed plant at levels greater than in a wild-type plant, together with a suitable carrier, and together with a package insert for their use to transmit disease resistance with a broad spectrum of plants.

20

Disease resistance

Disease resistance assessment is performed by methods known in the art. See, e.g., Uknes et al. (1993) Molecular Plant Microbe Interactions 6: 680-685; Gorlach et al., (1996) Plant Cell 8: 629-643; Alexander et 25 al., Proc. Natl. Acad. Sci. USA 90: 7327-7331.

A. Assay for resistance to Phvtophthora parasitica (Black roti.

Assays for resistance to Phytophthora parasitica, the organism that causes black rot, is performed on six week old plants grown as described in Alexander et al., Proc. Natl. Acad. Sci. USA 90: 7327-7331. The plants are watered, then the water is drained well, and then seeded by applying 10 ml of a sporangium suspension (300 sporangia / ml) to the soil. The grafted plants are kept in a greenhouse, at a day temperature of 23-25 ° C and a night temperature of 20-22 ° C. The impairment index used for the assay is as follows: 0 = no symptoms; 1 = no symptoms; 1 = some signs of deterioration, with reduced bloating; 2 = clear symptoms of deterioration, but no decay or limitation of growth; 3 = clear symptoms of damage with growth restriction, but no rotting of the trunk; 4 = significant damage, with visible trunk rot and some damage to the root system; 5 = like 4, but with the plants dead or nearly dead, and with significant damage to the root system. All assays are blindly assessed with plants arranged in any design.

10 B. Assay for resistance to Pseudomonas svringae.

Pseudomonas syringae pv. tobacco strain # 551 is injected into the two lower leaves of different 6-7 week old plants at a concentration of 106 or 3 x 106 per ml in H 2 O. Six separate plants are assessed at each time.

The plants infected with Pseudomonas tabaci are classified according to a 5-point scale for the severity of the condition, 5 = 100% dead tissue, 0 = no symptoms. A T-test (LSD) is performed on the results of each day and the groupings are indicated after the mean disease assessment value. Values followed by the same letter on the same 20 day of assessment are not statistically significantly different.

C. Assay for resistance to Cercosttora nicotianae.

A spore suspension of Cercospora nicotianae (ATCC # 18366) (100,000-150,000 spores per ml) is sprayed onto the surface of the leaves to the point of dripping. The plants are kept in 100% humidity for 5 days. Then the plants are sprayed with water 5-10 times a day. Six separate plants are evaluated at each time point. Cercospora nicotianae is judged based on the percentage of the leaf area showing disease symptoms. A T-test (LSD) is performed on each day's assessments and the groupings are indicated after the mean disease assessment value. Values followed by the same letter on assessment day are not statistically significantly different.

1 007779 66 D. Assay for resistance to Peronosoora parasitica

Assays for resistance to Peronospora parasitica are performed on plants as described by Uknes et al. (1993). The plants are inoculated with a controllable isolate of P. parasitica by spraying it with a suspension of 5 conidia (about 5 x 104 spores per milliliter). The inoculated plants are incubated in a growth chamber with a cycle of 14 hours day / 10 hours night under humid conditions at 17 ° C. The plants are examined for the presence of conidiophores 3-14 days, preferably 7-12 days after seeding. Furthermore, different plants from each treatment are randomly selected and stained with lactophenol-trypan blue (Keogh et al., Trans. Br. Mycol. Soc. 74: 329-333 (1980)) for microscopic examination.

Brief description of the figures

Figure 1 shows the influence of chemical inducing factors on the induction of SAR gene expression in wild-type and «/« // plants. Chemical induction of SAR genes is reduced in plants. Water, SA, INA or BTH is assigned on wild type (WT) and niml plants. After 3 days, RNA is prepared from these plants and examined for expression of PR-1, PR-2 and PR-5.

Figure 2 shows PR-1 gene expression in pathogen-infected Ws-0 and 20 plants. PR-1 induction by pathogen is reduced in plants. Wild-type (WT) and plants were inoculated by spraying with the Emwa variety of P. parasitica. On days 0, 1, 2, 4 and 6, samples were taken and the RNA was analyzed by blot hybridization with an A.thaliana PR-1 cDNA assay series to measure PR-1 mRNA formation.

Figure 3 shows the formation of PR-1 mRNA in niml mutants and wild type plants after infection with a pathogen or after chemical treatment. Plants containing the niml alleles niml-1, -2, -3, -4, -5 and -6 and Ws-0 (Ws) were treated with water (C), SA, INA 3 days before the RNA was isolated. or BTH. The Emwa sample consists of RNA that is 14 days after inoculation of the Emwa isolate with P.

30 parasitics from plants were isolated. Spots were hybridized using an Arabidopsis PR-1 cDNA as a test series (Uknes et al., 1992).

Figure 4 shows the levels of SA formation in Ws-0 and «/« // plants infected with P. syringae. niml plants form SNA after exposure to a 1007779 67 pathogen. Leaves of wild type and niml plants are infiltrated with Pst DC3000 (avrRpt2) or alone with support medium (10 mM MgCl2). After 2 days, samples were taken from the untreated, the MgCl2-treated and the DC3000 (avrR /? / 2) -treated plants. The bacteria treated samples 5 were separated into primary (infiltrated) and secondary (non-infiltrated) leaves. Free SA and total SA after hydrolysis with β-glucosidase were determined by HPLC. Error bars indicate the SD of three replicated samples.

Figures 5A-D show a general map with increasing resolution of the chromosomal region around NIM1 with the indicated recombinants, including 10 BACs, YACs and cosmids in the ΜΛ / 7 region.

(A) Map position of NIM1 on chromosome 1. The total number of gametes examined is 2276.

(B) Yeast artificial chromosome (streaked) bacterial artificial chromosome (BAC) and PI clones used to clone N1M1.

(C) Cosmid clones that include the NIM1 site. The three cosmids that complement tiiml-1 are shown as thicker lines.

(D) The four putative gene regions on the narrowest fragment of the complementing genomic DNA. The four open reading frames that make up the NIMl gene are indicated by the open stripes. The arrows indicate the direction of the transcription. The numbering is relative to the first base of the Arabidopsis genomic DNA present in cosmid D7.

Figure 6 shows the nucleic acid sequence of the NIM1 gene and the amino acid sequence of the MM / gene product, including changes in the 25 alleles. This nucleic acid sequence, which lies on the opposite strand as the 9.9 kb sequence shown in SEQ ID NO: 1, is also present in SEQ ID NO: 2, and the amino acid sequence of the MM / gene product is also shown in SEQ ID NO: 3.

Figure 7 shows the formation of NIM1 as induced by treatment with INA, BTH, SA and pathogen in wild type plants and in mutant alleles of niml. The RNA gel spots in Figure 3 were examined for expression of RNA using a test series derived from 2081-3266 in the sequence shown in Figure 6.

1 007779 68

Figure 8 is an amino acid sequence comparison of the Expressed Sequence Tag regions of the NIM1 protein and cDNA protein products of 4 rice gene sequences (SEQ ID NOS: 4-11); the numbers correspond to the amino acid positions in SEQ ID NO: 3).

Figure 9 depicts a comparison of the NIM1 protein sequence sequences with ΙκΒα (mouse, rat and pig). Vertical bars (|) above the sequences indicate that the amino acids in the sequences of NIM1 and ΙκΒα are identical (matrix score equal to 1.5); colons (:) above the sequences indicate a match score of> 0.5; some dots (.) above the sequences indicate a score corresponding to <0.5 but> 0.0; and a score of <0.0 indicates no similarity and no markings above the sequences (see the examples). The positions of the mammalian anκΒα ankyrin domains were identified according to the Martin et al., Gene 152, 253-255 G995). The points within a sequence indicate breaks between the NIM1 and ΙκΒα proteins. The five ankyrin repeating regions in ΙκΒα are indicated by the dotted line below the sequence.

Amino acids are numbered relative to the NIM1 protein, with breaks indicated where required. Plus signs (+) are placed above the sequences after every 10 amino acids.

20 Deponerines

The following vector molecules have been deposited with the American Type Culture Collection 12301 Parklawn Drive Rockville, MD 20852, USA, on the dates indicated below:

Plasmid BAC-04 was deposited with the ATCC on May 8, 1996 as ATCC 97543. Plasmid PI-18 was deposited with the ATCC on June 13, 1996 as ATCC 97606. Cosmid D7 was deposited with the ATCC on September 25, 1996 as ATCC 97736.

Brief description of the sequences in the Hist of sequences 30 SEQ ID NO: 1 - the 9919-bp genomic sequence of the MM / gene region 2 in

Figure 5D.

SEQ ID NO: 2 - the 5655-bp genomic sequence in Figure 6 (opposite strand of SEQ ID NO: 1), which includes the coding region of the Arabidopsis thaliana 1007779 69 NIM1 gene.

SEQ ID NO: 3 - the amino acid sequence of wild-type NIM1 protein as encoded by the sequences of SEQ ID NO: 2.

SEQ ID NO: 4 - Rice-1 amino acid sequence 33-155 from Figure 8.

5 SEQ ID NO: 5 - Rice-1 amino acid sequence 215-328 from Figure 8.

SEQ ID NO: 6 - Rice-2 amino acid sequence 33-155 from Figure 8.

SEQ ID NO: 7 - Rice-2 amino acid sequence 208-288 from Figure 8.

SEQ ID NO: 8 - Rice-3 amino acid sequence 33-155 from Figure 8.

SEQ ID NO: 9 - Rice-3 amino acid sequence 208-288 from Figure 8 10 SEQ ID NO: 10 - Rice-4 amino acid sequence 33-155 from Figure 8.

SEQ ID NO: 11 - Rice-4 amino acid sequence 215-271 from Figure 8.

SEQ ID NO: 12 - Oligonucleotide.

SEQ ID NO: 13 - Oligonucleotide.

SEQ ID NO: 14 - Oligonucleotide.

15 SEQ ID NO: 15 - Oligonucleotide.

SEQ ID NO: 16 - Oligonucleotide.

SEQ ID NO: 17 - Oligonucleotide.

SEQ ID NO: 18 is the mouse ΙκΒα amino acid sequence of Figure 8.

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

20 SEQ ID NO: 20 is the porcine ΙκΒα amino acid sequence of Figure 8.

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

SEQ ID NOS: 22 and 23 are, respectively, the DNA coding sequence and the encoded amino acid sequence of a dominant negative form of the NIM1 protein with alanine residues instead of serine residues at amino acid positions 55 and 59.

SEQ ID NOS: 24 and 25 are the DNA coding sequence and the encoded amino acid sequence of a dominant negative form of the NIM1 protein with an N-terminal deletion, respectively.

SEQ ID NOS: 26 and 27 are the DNA coding sequence and the encoded amino acid sequence of a dominant negative form of the NIM1-30 protein with a C-terminal deletion, respectively.

SEQ ID NOS: 28 and 29 are the DNA coding sequence and the encoded amino acid sequence of an altered form of the NJM1 gene with both N-terminal and C-terminal amino acid deletions, respectively.

1007779 70 SEQ ID NOS: 30 and 31 are the DNA coding sequence and the encoded amino acid sequence of the ankyrin domain of NIM1, respectively.

SEQ ID NOS: 32 to 39 are oligonucleotide base strings.

5 Definitions acd: accelerated cell death ("accelerated cell death") mutant plant AFLP: amplified fragment length polymorphism avrRpt2: avirulence gene Rpt2, isolated from Pseudomonas syringae BAC: bacterial artificial chromosome 10 BTH: benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester CIM: phenotype of constitutive immunity (SAR is constitutively activated) cim: mutant plant with constitutive immunity cM: centimorgans cpr \: mutant plant expressing constitutive PR gene

Col-0: Arabidopsis ecotype Columbia ECs: enzyme combinations

Emwa: Peronospora parasitica isolate compatible with the Ws-0 ecotype of Arabidopsis 20 EMS: ethyl methyl sulfonate INA: 2,6-dichloroisonicotinic acid

Ler: Arabidopsis ecotype Landsberg erecta

Isd: mutant plant with lesions simulating disease nahG: salicylate hydroxylase Pseudomonas putida that converts salicylic acid into catechol 25

NaGh \ ArabidopsisA \) x \ transformed with the «a / iG gene ndr: mutant plant with non-breed specific disease resistance nim: mutant plant with non-inducible immunity N1M1: the wild type gene involved in the SAR * signal transduction cascade NIM1: the protein encoded by the wild-type ΜΛ / 7 gene nim} ·. mutant alleles of NIM1, which confer susceptibility to disease on the plant; also refers to mutant Arabidopsis thaliana plants containing the niml mutant allele of NIM1 1007779 71

Noco: Peronospora parasitica isolate compatible with Arabidopsis Col-O ecotype ORF: open reading frame PCs: test series combinations 5 PR: pathogenesis related SA: salicylic acid SAR: systemic acquired resistance SSLP: simple sequence length polymorphism UDS: universal disease susceptibility phenotype 10 Wela: Peronospora parasitica isolate compatible in the Weiningen Arabidopsis ecotype Ws-0: Arabidopsis ecotype Issilewskija WT: wild type YAC: yeast artifidal chromosome 15

Examples

The invention is further illustrated by the following detailed procedures, preparations and examples. The examples are illustrative only, and should not be construed as limiting the scope of the invention.

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

Enquist, Experiments with Gene Fusions. Cold Spring Harbor Laboratory, Cold 25 Spring Harbor, NY (1984) and by Ausubel, F.M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc, and Wiley-Interscience (1987).

A. Characterization of the mm7 mutants 30

Example 1: Plant lines and fungal strains

Arabidopsis thaliana ecotype Isilewskija (Ws-0; strain number CS 2360) and fourth generation (T4) seeds from T-DNA transformed lines were obtained 1007779 72 from the Ohio State University Arabidopsis Biological Resource Center (Columbus, OH). Second generation (M2) seeds of Ws-O plants mutagenized with ethyl methanesulfonate (EMS) were obtained from Lehle Seeds (Round Rock, TX).

5 Pseudomonas syringae pv. Tomato (Pst) strain DC3000 containing the cloned avrRpt2 gene [DC3000 (avrRpi2)] was obtained from B. Staskawicz, University of California, Berkeley. P. parasitica pathovars and their sources were as follows: Emwa from E. Holub and I.R. Crute, Horticultural Research Station, East Mailing, Kent; Wela from A. Slusarenko and B. Mauch-Mani, Institut fur Pflanzenbiologie, 10 Zurich, Switzerland; and Noco from J. Parker, Sainsbury Laboratory, Norwich,

England. Mold cultures were maintained by weekly cultivation on Arabidopsis ecotype Ws-0, Weiningen, for P. parasitica pathovars Emwa, Wela and Noco, respectively.

Example 2: Investigation of mutants

Mr or T4 seeds were grown in soil for 2 weeks under 14 hours of light per day, misted with 0.33 mM INA (0.25 mg / ml made from 25% INA wettable powder; Ciba, Basel, Switzerland), and four inoculated days later by spraying with a P. parasitica conidis suspension containing 5-10 x 104 conidiophores per 20 ml of water. This fungus is normally virulent on the Arabidopsis Ws-O ecotype, unless resistance is first induced in these plants with isonicotinic acid (INA) or a similar compound. The plants were kept under moist conditions at 18 ° C for a week and then evaluated for spore formation by the fungus. Plants that showed fungal growth after INA treatment were selected as potential mutants.

After incubation in a high humidity environment, plants with visible disease symptoms were identified, usually 7 days after infection. These plants do not show resistance to the fungus despite the use of the resistance-inducing chemical compound and were therefore potential nim (non-inducible immunity) mutant plants. 75 possible nim mutants were identified from 360,000 plants.

These potentially mutant plants were isolated from the field and placed under low humidity conditions, after which the plant was allowed to form seeds. The plants obtained from this seed were similarly tested for susceptibility to the fungus Emwa, again after previous treatment with INA. The offspring that showed symptoms of infection were defined as m'm mutants. Thus, six m'm lines were identified. One line (niml-1) was isolated 5 from the T-DNA population and five (rtiml-2, niml-3, niml-4, niml-5, and niml-6) from the EMS population.

Example 3: Disease resistance of niml plants

Salicylic acid (SA) and benzo (1,2,3) thiadiazole-7-carbothioic acid-S-methyl ester 10 (BTH) are chemicals that, like INA, can induce a broad spectrum spectrum (SAR) disease resistance in wild type plants. Mutant plants were treated with SA, INA and BTH and then tested for resistance to Peronospora parasitica. P. parasitica isolate "Emwa" is a P.p. isolate compatible with the Ws ecotype. Compatible isolates are isolates capable of causing disease on a specific host. The P. parasitica isolate "Noco" is not compatible with Ws but compatible with on the Columbia ecotype. Incompatible pathogens are recognized by the potential host and cause a response in the host that prevents the onset of the disease.

Wild type seeds and seeds for each of the wW alleles (niml-1, -2, -3, -4, -20 5, -6) were sown on MetroMix 300 growth medium, covered with a clear plastic dome, and for three placed in the dark at 4 ° C for days. After 3 days of treatment at 4 ° C, the plants were transferred to a phytotron for 2 weeks. At about 2 weeks after post-planting, the germinated seedlings had developed 4 true leaves. The plants were then treated with H20, 5 mM 25 SA, 300 μΜ BTH or 300 μΜ INA. The chemicals were applied as a fine mist using a chromister to completely cover the seedlings. Water-treated control plants were repositioned in the phytotron used for growing while the chemically treated plants were kept in a separate but identical phytotron. 3 days after applying the chemicals, the water and chemically treated plants were inoculated with the compatible "Emwa" isolate. Grafting with "Noco" was performed only on water treated plants. After inoculation, the plants were covered with a clear plastic dome to avoid the high humidity required for successful infection with P.

.1 007779 74 parasitics and placed in a growth chamber with a daytime temperature of 19 ° C and a nighttime temperature of 17 ° C and a cycle of 8 hours of light and 16 hours of darkness.

To determine the relative strength of the different bl / s slates, each mutant was examined microscopically for growth of P. parasitica under normal growth conditions and after pretreatment with SA, INA or BTH at different times after inoculation. Under magnification, sporulation of the fungus could be observed at very early stages of disease onset. The percentage of plants / pot that showed sporulation at 5, 6, 7, 11 and 14 days after inoculation was determined and the density of the sporulation was also determined.

Table 1 shows, for each of the zW mutant plant lines, the percentage of plants showing some conidia on the surface on at least one leaf after infection with the Emwa variety of P. parasitica. P. parasitica was inoculated on the plants three days after treatment with water or chemicals. The table shows the number of days after infection that disease resistance was assessed.

Table 1

Percentage infection - Emwa / reference

Mutant Day 0 Day 5 Day 6 Day 7 Day 11 20 Ws WT 0 10 25 100 90 niml-1 0 75 95 100 100 niml-2 0 30 85 100 100 niml-3 0 30 90 100 100 niml-4 0 80 100 100 100 25 niml-5 0 0 5 100 100 niml-6 0 5 70 80 100

Percentage infection - Emwa / SA

Mutant Day 0 Day 5 Day 6 Day 7 Day 11 1 00 ^^ 9 75

Ws WT O 5 30 70 100 niml-1 0 5 95 100 100 niml-2 0 5 95 100 100 niml-3 0 10 90 100 100 5 niml-4 0 75 100 100 100 niml-5 0 0 20 75 100 niml- 6 0 80 100 100 100 1007779

Percentage infection - Emwa / INA

76

Mutant Day 0 Day 5 Day 6 Day 7 Day 11

Ws WT 0 0 0 0 0 .. 5 niml-1 0 5 80 100 100 niml-2 0 15 95 100 100 niml-3 0 10 60 100 100 niml-4 0 80 100 100 100 niml-5 0 0 0 5 5 10 niml-6 0 1 50 90 100

Percentage infection - Emwa / BTH

Mutant Day 0 Day 5 Day 6 Day 7 Day 11

Ws WT 0 0 0 0 0 15 niml-1 0 1 5 30 100 niml-2 0 0 25 90 100 niml-3 0 15 70 100 100 niml-4 0 80 100 100 100 niml-5 0 0 1 1 10 20 niml -6 0 1 90 100 100

As shown in Table 1, under normal growth, niml-1, niml-2, niml-3, niml-4 and niml-6 all showed approximately the same rate of fungal growth, which was slightly faster than the Ws-0 control. The exception were the niml-5 plants where fungal growth was delayed by several days compared to both the other mutants and the Ws-0 control, but eventually all niml-5 plants succumbed to the fungus.

After treatment with SA, the mutants could be divided into three groups: niml-4 and niml-6 showed relatively rapid fungal growth; niml-1, 30 niml-2, niml-3 plants showed a slightly slower rate of fungal growth; and fungal growth in niml-5 plants was even slower than in the untreated Ws-0 controls. After treatment with INA or BTH, the mutants also fell into three classes, of which niml-4 was most severely limited in its ability to inhibit fungal growth after chemical treatment; niml-J, niml-2, niml-3, and niml-6 were all moderately limited; and niml-5 was only slightly limited. In these experiments, Ws-0 gave no fungal growth after treatment with INA or BTH. Thus, with regard to inhibiting fungal growth after chemical treatment, the mutants fell into three classes, of which niml-4 was the most limited, niml-1, niml-2, niml-3, and niml-6 exhibited moderate fungal inhibition. , and niml-5 had only limited fungal resistance.

Table 2 shows the assessment of disease resistance by classification of the infection of the different mmi alleles as well as of the NahG plants at 7 and 11 days after inoculation with Peronospora parasitica. WsWt indicates the Ws wild-type parent line in which the m'm / alleles were found. The different niml alleles are indicated in the table and the NahG plant is also indicated.

A description of the NahG plant has been previously published (Delaney et al., Science 266, 1247-1250 (1994)). NahG Arabidopsis is also described in U.S. Patent Application Serial No. 08 / 454,876, incorporated herein by reference. NahG is a gene from Pseudomonas putida that encodes a salicylate hydroxylase that converts salicylic acid into catechol, and thus prevents the accumulation of salicylic acid, a necessary part of the signal transduction for SAR in plants. Therefore, NahG Arabidopsis plants do not exhibit a normal SAR, and exhibit much greater sensitivity to pathogens in general. However, the NahG plants still respond to the chemical inducers INA and BTH. NahG plants therefore serve as a general reference for sensitivity.

1007779

Infection rate - Emwa / water 78

Table 2

Mutant Day 7 Day 11

Ws WT 3 3 - 5 niml-1 4 4.5 niml-2 3 4 niml-3 4 4 niml-4 5 5 niml-5 1 3.5 10 niml-6 3 4.5

NahG 4 5

Degree of infection - Emwa / SA

Mutant Day 7 Day 11 15 Ws WT 3 4 niml-1 3 4.5 niml-2 3 4 niml-3 3 4 niml-4 4 5 20 niml-5 3 3 niml-6 4 4.5

NahG 4 5 1 007779

Degree of infection - Emwa / INA

79

Mutant Day 7 Day 11

Ws WT 0 0 niml-1 2.5 4 '5 niml-2 4 4 niml-3 3 3.5 niml-4 4 5 niml-5 1 2 niml-6 3 4.5 10 NahG 3 3

Degree of infection - Emwa / BTH

Mutant Day 7 Day 11

Ws WT 0 0 15 niml-1 2.5 4 niml-2 3.5 4 niml-3 3 3.5 niml-4 4 5 niml-5 1.5 2 20 niml-6 3 4

NahG 0 0

Table 2 shows that the niml-4 and niml-6 alleles showed the most serious Peronospora parasitica infections; this was most clearly observed at the early times. Furthermore, the niml-5 allele showed the greatest response to both INA and BTH, judging this to be the weakest niml allele. The NahG plants showed a very good response to both INA and BTH and were very similar to the niml-5-a \\ e \ en. However, at late times, day 11 in the Table, the induced disease resistance in the NahG plants began to decrease and there was a significant difference between INA and BTH, since the INA-induced resistance 1007779 80 decreased much faster and more severely. than the resistance induced by BTH in the NahG plants. As also shown in these experiments, INA and BTH induced very good resistance in Ws to Emwa and the niml-1, niml-2 and other niml alleles showed essentially no response to SA or INA in disease resistance.

The lack of response of the mm 7 plants to the SAR-inducing chemicals SA, INA and BTH implies that this mutation is downstream of the target point and of these chemicals in the signal transduction cascade leading to systemically acquired resistance.

Example 4: Northmanalysis of SAR gene expression

Since SA, INA and BTH did not induce SAR, or SAR gene expression, in any of the niml plants, it was important to investigate whether infection with a pathogen could induce SAR gene expression in these plants, such as in the wild type plants. Therefore, the formation of SAR gene mRNA was also used as a measure of characterization of the different mm7 alleles.

Wild type seeds and seeds for each of the m'm7 alleles (niml-1, -2, -3, -4, -5, -6) were sown on MetroMix 300 growth medium, covered with a clear plastic dome, and for Kept in the dark for three days at 4 ° C. After 3 days of treatment at 4 ° C, the plants were transferred to a 20 phytotron for 2 weeks. About two weeks after planting, the germinated seedlings had developed four true leaves. The plants were then treated with H20, 5mM SA, 300 μΜ BTH, or 300 μΜ INA. The chemicals were applied as a fine mist using a chromister until they completely covered the seedlings. The water treated reference plants were repositioned in the growth phthotron while the chemically treated plants were kept in a separate but identical phytotron. Three days after application of the chemicals, the water and chemically treated plants were inoculated with the compatible Emwa isolate. Grafting with noco was performed only on the water treated plants. After inoculation, the plants were covered with a clear plastic dome to maintain the high humidity required for successful infection with P. parasitica and placed in a growth chamber with a day temperature of 19 ° C and a night temperature of 17 ° C and a cycle of 8 hours of light and 16 hours of dark. RNA was extracted from the plants 3 days after treatment with water or chemical treatment, or 14 days after inoculation with the compatible P. parasitica Emwa isolate. The RNA was size fractionated by agarose gel electrophoresis and applied to GeneScreenPlus membranes (DuPont).

Figures 1-3 show the different RNA gel spots showing that SA, INA 5 and BTH SAR neither induce SAR gene expression in n / m7 plants. In Figure 1, replicated spots were hybridized to Arabidopsis gene stretches PR-1, PR-2 and PR-5 as described by Uknes et al. (1992). Unlike the wild-type plants, the chemicals did not induce RNA formation from any of these 3 SAR genes in niml-1 plants.

As shown in Figure 2, infection of the wild-type Ws-O plants with pathogen (Emwa) induced PR-1 gene expression within 4 days of infection. However, in niml-1 plants, PR-1 gene expression was not induced up to 6 days after infection and the level of expression was reduced relative to the wild type at this time. Therefore, after infection with pathogen, PR-1 gene expression in niml-1 plants was delayed and decreased relative to the wild type.

The RNA gel spot in Figure 3 shows that PR-1 mRNA is generated in large quantities after treatment of the wild-type plants with SA, INA or BTH or infection by P. parasitica. In the niml-1, niml-2 and niml-3 plants, the formation of PR-1 mRNA after chemical treatment was greatly reduced compared to the wild type. PR-1 mRNA formation was also reduced after infection with P. parasitica, but there was still some accumulation in these mutants. PR-1 mRNA formation was significantly reduced in the niml-4 and niml-6 plants than in the other alleles after chemical treatment (as evidenced by longer exposures) and significantly less PR-1 mRNA was formed after infection with 25 P parasitics, which supports the hypothesis that these are particularly strong mm7 alleles. PR-1 mRNA formation was enhanced in the niml-5 mutant, but was only moderately induced after chemical treatment or after infection with P. parasitica. Based on PR-1 mRNA formation and fungal infection, the mutants were classified into three classes: severely restricted alleles (niml-4 and 30 niml-6), moderately limited alleles (niml-1, niml-2, and niml -3) ·, and to some extent limited alleles (niml-5).

Example 5: Determination of SA-formin in m'm7 plants 1007779 82

Infection of the wild-type plants with pathogens causing a necrotic response leads to SA formation in the infected tissues. Endogenous SA is required for signal transduction in the SAR pathway, as degradation of the endogenous SA leads to a decrease in disease resistance. This characterizes the formation of SA as a marker in the SAR pathway (Gaffney et al., 1993, Science 261, 754-756). The phenim of niml plants indicates a disturbance in a component of the SAR pathway downstream of SA and upstream of SAR gene induction.

nim plants were tested for their ability to form SA after infection with a pathogen. Pseudomonas syringae tomato strain DC 3000, which carries the avrRpt2 gene 10, was injected into the leaves of 4 week old niml plants. The leaves were harvested 2 days later for analysis for SA as described by Delaney et al., 1995, PNAS 92, 6602-6606. This analysis showed that the w / m7 plants formed high levels of SA in infected leaves, as shown in Figure 4. Uninfected leaves also formed SA, but not in the same amounts as the infected leaves, which is similar to what is observed in wild-type Arabidopsis. This shows that the niml mutation is downstream of the SA marker in the signal transduction pathway. Furthermore, it has been shown that INA and BTH (not active in iw7 plants) stimulate a component in the SAR pathway downstream of SA (Vemooij et al. (1995); Friedrich et al.

20 (1996); and Lawton et al. (1996)). Furthermore, as described above, exogenously used SA niml plants did not protect against infection by Emwa.

Example 6: Genetic analysis

To determine the dominance of the various mutants displaying the niml phenotype 25, pollen from wild type plants was transferred to the stigmata of niml-1, -2, -3, -4, -5, -6. If the mutation were dominant, the w'mZ phenotype would be observed in the Fl plants thus obtained. If the mutation were recessive, the resulting Fl plants would exhibit a wild-type phenotype.

The data reported in Table 3 show that when niml-1, -2, -3, -4, and -6 were crossed with the wild type, the F1 plants thus obtained showed the wild type phenotype. Therefore, these mutations are recessive. In contrast, the offspring of niml-5X wild-type F1 all showed the m'mf phenotype, 1007779 83, indicating that this is a dominant mutation. After treatment with INA, no P. parasitica sporulation was observed on wild-type plants, while the F1 plants showed growth and some sporulation of P. parasitica. However, the rtiml phenotype in these F1 plants was less strong than observed when 5 niml-5 is homozygous.

To determine the alleles, pollen from kanamycin resistant niml-1 mutant plants was transferred to its stigma from nimJ-2, -3, -4, -5 and -6. The seeds obtained from this cross were plated on Murashige-Skoog B5 plates containing 25 µg / ml kanamycin to confirm the hybrid origin of the seed. Kanamycin resistant (Fl) plants were transferred to soil and examined for the nim 1 phenotype. Since the F1 progeny from the crossing of the niml-5 mutant with the Ws wild type showed an mm7 phenotype, an analysis of niml-5 X niml-1 F2 was also performed.

As can be seen from Table 3, all of the F1 plants thus obtained showed the nim 1 phenotype. Therefore, the mutation in niml-2, -3, -4, -5, -6 was not supplemented by the niml-1; these plants therefore fall within the same complementation group and are therefore allelic. F2 progeny from the niml-5 X niml-1 cross also showed the nim7 phenotype, confirming that niml-5 is a mm / -all.

20 1007779 84

Table 3. Genetic separation of the nim mutants

Phenotype

Mutant Generation Mother Father Wild- nimlb type8 niml-1 F1 wild-typec niml-1 24 0 5 F2 98 32 niml-2 FI niml-2 wild-type 3 0 niml-3 F1 niml-3 wild-type 3 0 niml- 4 F1 niml-4 wild type 3 0 niml-5 F1 niml-5 wild type 0 35 10 F1 wild type niml-5 0 18 niml-6 F1 niml-6 wild type 3 0 niml-2 F1 niml- 2 niml-1 0 15 niml-3 F1 niml-3 niml-1 0 10 niml-4 F1 niml-4 niml-1 0 15 15 niml-5 F1 niml-5 niml-1 0 14 F2 9 85 niml-6 F1 niml-6 niml-1 0 12 8 Number of plants with increased PR-1 mRNA formation and absence of P.

20 parasitics after treatment with INA.

b Number of plants without PR-1 mRNA formation and presence of P. parasitica after treatment with INA. c Wild type indicates the wild type Ws-0 strain.

25 B. Mapping the mm 7 mutation

The mapping of the mW mutation is described in great detail in U.S. Patent Application Serial No. 08 / 773,559, filed December 27, 1996, which is incorporated herein by reference in its entirety.

1007779 85

Example 7: Identification of markings in. and determination of the genetic map of the m> w7 locus

To determine a rough map for niml, 74 F2 nim plants from a cross between niml-1 (Ws-0) and Landsberg erecta (Ler) were identified based on their susceptibility to P. parasitica and the lack of formation of PR-1 mRNA after INA treatment. Using simple sequence length polymorphisms (SSLP) tags (Bell and Ecker 1994), niml-1 was determined to be about 2.8 centimorgans (cM) of ngal28 and 8.2 cM of ngalll on the lower arm of chromosome 1. It was further determined that niml-1 is between ngal 11 and 10 about 4 cM from the SSLP marker ATHGENEA. (Figure 5A).

To map the fine structure, 1138 nim plants from an F2 population obtained from a cross between niml-1 and Ler DP23 were identified for both their inability to form PR-1 mRNA and their ability to support fungal growth after treatment with INA. DNA was extracted from these plants and assessed for zygosity in both ATHGENEA and ngal 11. As shown in Figure 5A, 93 recombinant chromosomes were identified between ATHGENEA and niml-1, indicating a genetic distance of approximately 4.1 cM (93 of 2276 ), and 239 recombinant chromosomes were identified between ngal 11 and niml-1, indicating a genetic distance of about 10.5 cM (239 from 2276). Recombinants providing information in the ATHGENEA to ngal 11 interval were further analyzed using amplified fragment length polymorphism (AFLP) analysis (Vos et al., 1995).

AFLP mark between ATHGENEA and ngal 11 were identified and 25 were used to fabricate a low resolution map of the region (Figures 5A and 5B). AFLP marks W84.2 (1 cM from niml-1) and W85.1 (0.6 cM from niml-1) were used to isolate yeast artificial chromosome (YAC) clones from the CIC (for: Center d'Etude du Polymorphisme Humain, INRA and CNRS) library (Creusot et al., 1995). Two YAC clones, CIC12H07 and CIC12F04, were identified with W84.2 and two YAC clones CIC7E03 and CIC10G07 were identified with the W85.1 marker (Figure 5B). In order to bridge the space between the two sets of flanking YAC clones, bacterial artificial chromosome (BAC) and PI clones overlapping 1007779 86 were isolated and mapped with CIC12H07 and CIC12F04, and sequential displacement steps were performed to run the BAC / P1 continuous portion up to niml (Figure 5C; Liu et al., 1995; Chio et al., 1995). During the move, new AFLPs specific to BAC or PI 5 clones were developed and used to determine if the niml gene was crossed, niml was crossed when isolated BAC and PI clones containing both AFLP mark L84.6a and L84 .8. The AFLP marker L84.6a, as found on PI clones PI-18, PI-17 and PI-21, identified three recombinants and L84.8, as found on PI clones PI-20, PI-22, PI-23 and PI -24 and BAC clones, BAC-10 04, BAC-05 and BAC-06, identified one recombinant. Since these clones partially overlap to form a large contiguous portion (> 100 kb) and include AFLP markers flanking niml, it was deduced that the gene is within the contiguous portion. The BAC and PI clones that form the contiguous portion were used to generate further AFLP markers, indicating that niml is between L84.Y1 and L84.8, which is a region of about 0.09 cM.

C. Isolation of the niml gene. Example 8: Construction of a cosmid contiguous portion.

A cosmid library of the niml gene was constructed in the Agrobacterium-compatible T-DNA cosmid vector pCLD04541 using CsCl purified DNA from BAC-06, BAC-04 and PI-18. The DNAs from the three clones were mixed in equimolar amounts and partially digested with the restriction enzyme Sau3A. The 20-25 kb fragments were isolated using a sucrose gradient, collected and filled in with dATP and dGTP. Plasmid pCLD04541 was used as a T-DNA cosmid vector. This plasmid contains a broad host pRK290 based replicon, a tetracycline resistance gene for bacterial selection and the nptll gene for plant selection.

The vector was cut with Xhol and filled in with dCTP and dTTP. The prepared fragments were then inserted into the vector. The coupling mixture was packed and transduced into E. coli strain XL1-blue MR (Stratagene). The transformants thus obtained were evaluated by hybridization with the BAC04, 87 BAC06 and P1-18 clones and positive clones were isolated. Cosmid DNA was isolated from these clones and base strand DNA was prepared using the EC's ECORI / Msel and HindII / Msel. The AFLP fingerprint patterns thus obtained were analyzed to determine the order of the cosmid clones. A series 5 of 15 partially overlapping cosmids bridging the mm region was selected (Figure 5D). The cosmid DNAs were also digested with EcoRI, PstI, BssHII and SgrAI. This allowed estimating the sizes of the cosmid inserted portions and also allowed confirmation of the overlapping portions between the different cosmids, as determined from the AFLP 10 fingerprint.

Physical mapping showed that the physical distance between L84.Y 1 and L84.8 was> 90 kb, indicating a physical distance of approximately 1 megabase per cM. The DNA sequence of BAC04 was determined to facilitate the identification of the niml gene.

15

Example 9: Identification of a clone containing the niml-one.

Cosmids from clones spanning the niml region were transferred to Agrobacterium tumefaciens AGL-1 by conjugative transfer in a three-parent crossing with the auxiliary strain HB101 (pRK2013). These cosmids were then used to transform a canamicin-sensitive niml-1 Arabidopsis-Myn by vacuum infiltration (Bechtold et al., 1993; Mindrinos et al., 1994). Seeds from the infiltrated plants were harvested and the seed was allowed to germinate on GM agar plates containing 50 mg / ml kanamicin as a selection agent. Only young plants transformed with cosmid DNA were able to detoxify and survive the selection agent. Seedlings that survived the selection were transferred to soil about two weeks after plating and examined for the niml phenotype as described below. Transformed plants that no longer showed the niml phenotype indicated cosmid (s) containing an active niml gene.

30

Example 10: Supplementation of the niml phenotype

Soil-transferred plants were grown in a phytotron for about a week after transfer. Using a chromister, 1007779 88 300μπι ΙΝΑ was charged as a fine mist to completely cover the plants. After two days, the blades were harvested for RNA extraction and PR-1 expression analysis. The plants were then sprayed with Peronospora parasitica (isolate Emwa) and grown under high humidity conditions in a growth chamber with 5 cycles of 19 ° C day / 17 ° C night temperatures and 8 hours light / 16 hours dark. Eight to ten days after fungal infection, the plants were examined and rated positive or negative for fungal growth. WS and NIML plants were treated in the same manner to serve as references for each trial.

Total DNA was extracted from the collected tissue using a LiCl / phenol extraction buffer (Verwoerd et al., 1989). RNA samples were separated on a formaldehyde agarose gel and spotted onto GeneScreen Plus (DuPont) membranes. The spots were hybridized with a 32P-labeled PR-i cDNA test series. The stains thus obtained were exposed to a film to determine which transformants were able to induce PR-1-15 expression after treatment with INA. The results are summarized in Table 4, which shows filling of the niml phenotype by cosmid clones D5, E1 and D7.

10Ó7779 89

Table 4

Clone name Number of transformants Total number of plants with ΓΝΑ-induced PR-1 / total number of plants tested (%) A8 3 0/3 (0%) 'All 8 4/18 (22%) 5 C2 10 1/10 (10%) C7 33 1/32 (3%) D2 81 4/49 (8%) D5 6 5/6 (83%)

El 10 10/10 (100%) 10 D7 129 36/36 (100%) E8 9 0/9 (0%) F12 6 0/6 (%) E6 1 0/1 (0%) E7 34 0/4 (0%) 15 WS control (etc. NA 28/28 (100%) niml-1 phenotype control NA 0/34 (0%) NA - not indicated.

t 007 779 90

Example 11: Sequencing of the mmi gene region BAC04 DNA (25 µg, obtained from KeyGene) was the source of DNA used for the sequence analysis, since this BAC was the clone that completely replenishes the region of the nim] mutants included. BACO4 DNA was randomly fragmented in a nebulizer to generate fragments with an average length of about 2 kb. The ends of the broken fragments were repaired and the fragments purified. Purified DNA was coupled to EcoRV-cut pBRKanF4 (a derivative of pBRKanFI (Bhat 1993)). The kanamycin resistant colonies thus obtained were selected for isolation of the plasmid using the Wizard Plus 9600 Miniprep System (Promega). The plasmids were sequenced using dye terminator chemistry (Applied BioSystems, Foster City, CA) with basic arrays designed to sequence both strands of the plasmids (Ml3-21 forward and T7 reverse, Applied BioSystems). The data was collected on AB1377 DNA sequencers. The sequences were processed and assembled into contiguous sections using a Sequencher 3.0 (GeneCodes Corp., Ann Arbor, MI), the Staden genome assembly programs, phred, phrap and crossmatch (Phil Green, Washington University, St. Louis, MO) and consed (David Gorden, Washington University, St. Louis, MO). The DNA 20 sequences of the cosmids found to complement the niml-1 mutation were determined using assays designed by Oligo 5.0 Primer Analysis Software (National Biosciences, Inc., Plymouth, MN). Sequencing of Ws-0 DNA and the nimJ alleles and cDNAs was performed essentially as described above.

A region of about 9.9 kb, which is defined by the overlapping portion of cosmids E1 and D7, was determined to contain the niml region by complementation analysis. Test series flanking the site of insertion of the vector and specific to the cosmid backbone were designed using Oligo 5.0 Primer Analysis Software 30 (National Biosciences, Ine.). DNA was isolated from cosmids D7 and E1 using an adaptation of the ammonium acetate method (Traynor, P.L. 1990. BioTechniques 9 (6): 676). The DNA was sequenced directly using Dye Terminator chemistry. The sequence thus obtained made it possible to determine the end points of the additional region. The area defined by the overlapping portion of cosmids E1 and D7 is shown as SEQ ID NO: 1.

A truncated version of the BamH1-EcoRV fragment was also constructed, resulting in a construct that did not contain any part of the "Gene 3" region (Fig. 5D). The following approach was required due to the presence of HindII sites in the Bam-Spe region of the DNA. The BamHl-EcoRV construct was completely cut with Spel and then split into two separate reactions for two-fold cutting. One part was cut with BamHI, the other part with HindII. A 2816 bp BamHI-Spe fragment and a 1588 bp HindlII-Spe fragment were isolated from agarose gels (Qiaquick Gel extraction kit) and were linked to BamHI-HindlII-cut pSGCGO1. DH5a was transformed with the coupling mixture. The colonies thus obtained were examined for the correct insert by cutting with HindIII followed by preparation of DNA using Wizard Magic MiniPreps (Promega). A clone containing the correct construct was electroporated into Agrobacterium strain GV3101 for transformation of Arabidopsis plants.

Example 12: Sequence analysis and subcloning of the niml region.

The 9.9 kb region containing the niml gene was analyzed for the presence of open reading frames in all six reading frames using the Sequencher 3.0 and the GCG package. Four regions containing large ORFs were identified as potential genes (gene regions 1-4 in Figure 5D). These four regions were amplified by PCR from DNA of the wild-type parent plant and six different niml allelic variants niml-1, -2, -3, -4, -5 and -6. Test series for these amplifications were selected using oligo 5.0 (National Biosciences, Ine.) And synthesized by Integrated DNA Technologies, Ine. The PCR products were separated on 1.0% agarose gels and purified using the QIAquick Gel Extraction Kit. The sequences of the purified genomic PCR products were directly determined using the test sequences used for the original amplification and with further basic sequences that were sequenced from regions that could not be sequenced with the original 1007779 92 test series. A mean reading for these gene regions was about 3.5 readings / base.

The sequences were processed and pooled using Sequencher 3.0. Changes in the bases specific for the different 5 niml alleles were identified only in the region designated as "Gen region 2", as shown in Table 5, which reflects the differences in sequences between all six niml alleles.

1007779 93

Table 5

Gene region 1 2 (niml) 3 4 (bases 590-1090 (bases 1380-4100) (bases 5870- (bases 8140- 6840) 9210) .. 5 niml-I no changes t introduced on none none 2981: change of 7AA changes changes and early termination of the protein niml-2 no changes g and a at 2799: His in Tyr no no changes changes niml-3 no changes deletion of t at 3261: no no change of 10AA and changes changes early termination of the protein niml -4 no changes c in t at 2402: Arg in lys no no changes changes niml-3 no changes c in t at 2402: Arg in lys no no changes changes 10 niml-6 g in a at 734: asp in g in a at 2670: Gin in Stop I no I no yy lys changes changes WS no changes a in g at 1607: lie in Leu t in a at 5746 t in g at 8705 (compared a in c at 2344: intron a in t at 5751 g in t at 8729 with t in g at 2480: Gin in Pro t in a at 5754 g in g at 8 739

Columbia g in c at 2894: Ser in Trp c in t at 6728 g in t at 8784 ggc removed at 3449: a in t at 6815 c in a at 8789 loss of Ala t in c at 6816 c in t at 8812 c in t at 3490: Ala in Thr a in g at 8829 c in t at 3498: Ser in Asn t in g at 8856 a in t at 3873: non-coding a in c at 9004 g in a at 3992: non-coding a in t at 9011 g in a at 4026: non-coding a in g at 8461 g in a at 4061: non-coding 15 RNA No Yes No No observed 1007779 94

The positions listed in the table are SEQ ID NO: 1. All niml-1 to niml-6 alleles are of the WS strain. Columbia-0 is the wild type.

It is clear that the niml gene is within gene region 2, since in all six niml alleles there are changes in amino acids or changes in sequence within the open reading frame of gene region 2. At the same time, at least one of the m'm Alleles have no open reading frame changes within gene regions 1, 3 and 4. Thus, gene region 2 is the only gene region within the 9.9 kb region that can contain the niml gene.

The WS section of Table 5 shows the changes in the Arabsopsis Ws ecotype compared to the Arabidopsis Columbia ecotype. The sequences presented herein relate to the Arabidopsis Columbia ecotype, which contains the wild type gene in the experiments described herein. The changes are reported as amino acid changes within gene region 2 (the niml region) and are reported as base pair changes in the other regions.

The cosmid portion containing the niml gene was delimited by a BamHl-

EcoRV restriction fragment of about 5.3 kb. D7 cosmid DNA and plasmid DNA from pBlueScriptlI (pBSII) were digested with BamHI and with EcoRV (NEB).

The 5.3 fragment of D7 was isolated from agarose gels and purified using the QIAquick gel extraction kit (# 28796, Qiagen). The fragment was coupled to the Bam-EcoRV-cut pBSII overnight and the coupled mixture was transformed into the E. coli strain DH5a. Colonies containing the introduced portion were selected, DNA was isolated, and confirmed by HindIII cutting. The Bam-EcoRV fragment was then manipulated into a binary vector (pSGCGO1) for transformation into Arabidopsis.

Example 13: Northern analysis of the four gene regions

Identical Northern stain impressions were made from RNA samples isolated from water, SA, BTH and INA treated Ws and m'm / lines, as previously described by Delaney et al. (1995) . These spots were hybridized with PCR products generated from the four gene regions identified in the 9.9 kb mm ƒ gene region (SEQ ID NO: 1). Only the gene region containing the niml gene (gene region 2) showed observable hybridization 1007779 95 with RNA samples, indicating that only the m'm / region contains a detectable transcribed gene (Figure 5D and Table 5).

Example 14: Complementing with gene region 2 It was further demonstrated, by conducting further complementation experiments, that gene region 2 (Figure 5D) contains the functional niml gene. A BamHI / HindII genomic DNA fragment containing gene region 2 was isolated from cosmid D7 and cloned into the binary vector pSGCGO1, which contains the kanamycin resistance gene. The plasmid thus obtained was transformed into the Agrobacterium strain GV3101 and positive colonies were selected for kanamycin. PCR was used to confirm that the selected colony contained the plasmid. Kanamycin-sensitive niml-1 plants were infiltrated with these bacteria as described above. The seed thus obtained was harvested and planted on GM agar containing 50 µg / ml kanamycin. The plants that survived the selection were transferred to soil and examined for complementation. Transformed plants and reference WS and / / plants were sprayed with 300 µm INA. Two days later, the plants were harvested for RNA extraction and PR-1 expression analysis. The plants were then sprayed with Peronospora parasitica (isolate Emwa) and grown in the manner described above. Ten days after fungal infection, the plants were evaluated and rated positive or negative for fungal growth. All 15 transformed plants, as well as the Ws controls, were negative for fungal growth after treatment with INA, while the controls were positive for fungal growth. RNA was extracted from these transformants and controls and analyzed as described above. WS controls and all 15 transformants showed induction of the PR-1 gene after treatment with INA, while the controls did not show induction of PR-1 by INA.

Example 15: Isolation of a niml cDNA An Arabidopsis cDNA library made in the IYES expression vector (Elledge et al., 1991, PNAS 88, 1731-1735) was plated and the plaques were removed. Filters were hybridized with a 32 P-labeled PCR gene product obtained from gene region 2 (Figure 5D). Fourteen positives were identified from a study of approximately 150,000 1007779 96 plaques. Each plaque was purified and plasmid DNA recovered. cDNA inserted portions were excised from the vector using EcoRI and purified on agarose gel and sequenced. The sequence obtained from the longest cDNA 5 is shown in SEQ ID NO: 2 and in Figure 6. To confirm that the 5 'end of the cDNA was obtained, a Gibco BRL 5' RACE kit was used according to the manufacturer's instructions. The sequences of the RACE products thus obtained were determined to be found to contain the further bases shown in Figure 6. The transcribed region present in both 10 cDNA clones and observed in RACE is capitalized in Figure 6. Changes in the alleles are shown above the cDNA strand. Capital letters indicate the presence of the sequence in a cDNA clone or detected after RACE PCR.

The same RNA samples as obtained in the induction tests (Figure 3) were also tested with the niml gene using a full length cDNA clone as a test series. It can be seen from Figure 7 that INA induced the niml gene in the wild-type Ws alleles. However, the niml-1 mutation alleles showed a low basal level of expression of the niml gene and were not induced by INA. This was similar to what was observed in the niml-3 allele and the niml-6 allele. The niml-2 allele showed approximately normal levels in the untreated sample and showed similar induction to the wild-type sample, as did the niml-4 allele. The niml-5 allele appeared to show a higher basal level of expression of the niml gene and much stronger expression after induction with chemical inducers.

25 D. n / mV homologs

Example 16: BLAST study with the MW division

A multiple sequence equation was constructed using Clustal V (Higgins, Desmond G. and Paul M. Sharp (1989), Fast and sensitive multiple sequence allignments on a microcomputer, CABIOS 5: 151-153) as part of the DNA * ( 1228 South Park Street, Madison Wisconsin, 53715) Laser Gene Biocomputing Software Package for the Macintosh (1994). Certain 1007779 97 regions of the niml protein are amino acid sequence homologous to four different rice-derived cDNA protein products. The homologies were identified using the nim 1 sequences in a GenBank BLAST study. Comparisons of the areas of homology in niml and the rice cDNA products are shown in Figure 8 (see also SEQ ID NO: 3 and SEQ ID NO: 4-11). The m / m / protein fragments show 36-48% identical amino acid sequences with the four rice products.

Example 17: Isolation of homologous genes from other plants. Using niml cDNA as a test series, homologues of

Arabidopsis niml identified by probing genomic or cDNA libraries from different crops, such as, but not limited to, the crops listed in Example 22 below. Standard techniques for doing this include hybridization probing of plated DNA libraries (plaques or colonies; see, eg, Sambrook et al., Molecular Cloning, eds., Cold Spring Harbor Laboratory Press. (1989)) and PCR amplification using of oligonucleotide base arrays (see, e.g., Innis et al., PCR Protocols, a Guide to Methods and Applications, eds., Academic Press (1990)). The identified homologs were genetically engineered into the expression vectors described herein and transformed into the above crops. The transformants were evaluated for increased disease resistance using relevant pathogens for the crop plant examined.

nim 1 homologs were detected by DNA blot analysis in the cucumber, tomato, tobacco, corn, wheat and barley genomes. Genomic DNA was isolated from cucumber, tomato, tobacco, corn, wheat and barley, digested with enzymes BamHI, HindlII, Xbal or Sail, separated by electrophoresis on 0.8% agarose gels and transferred to nylon membrane by capillary spotting. After UV cross-linking to fix the DNA, the membrane was heated under moderate severe conditions [(1% BSA; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride) for 18-14 hour at 55 ° C] hybridized with 32P-

radiolabelled Arabidopsis thaliana niml cDNA. After hybridization, the spots were treated under moderately severe conditions [6XSSC for 15 minutes (X3) 3XSSC for 15 minutes (XI) at 55 ° C; 1XSSC is 0.15 M NaCl, 15 mM

1007779 98 sodium citrate (pH 7.0)] washed and exposed to X-ray film to reveal the bands corresponding to niml.

Furthermore, expressed sequence markers (EST) identified as resembling the niml gene, such as the rice-derived ESTs described in Example 16, can be used to isolate homologs. The rice-derived ESTs may be particularly useful for isolating niml homologs from other monocotyledonous plants.

Homologs can be obtained by PCR. In these methods, comparisons are made between known homologues (eg rice and Arabidopsis). Regions with a high degree of identity or identity in amino acids or DNA can then be used to create basic PCR arrays. Regions rich in M and W are the most useful, followed by regions rich in F, Y, C, H, Q, K and E as these amino acids are encoded by a limited number of codons. Once a suitable region has been identified, base strings for this region with different substitutions are made at the position of the third codon. These differences in third position substitution may be limited depending on the species targeted. For example, since corn is CG rich, base strings containing a C or a G in the third position are subjected, if possible.

A PCR reaction is performed under various standard conditions, starting from cDNA or genomic DNA. When a band is visible it is cloned and / or sequenced to determine if it is an m'm / homolog.

E. High-level expression of niml confers resistance to plant diseases

High level expression of the niml gene in transgenic plants to confer a CIM phenotype is also described in applicant's U.S. Patent Application Serial No. 08 / 773,554, filed December 27, 1996, which is incorporated herein by reference in its entirety.

Example 18: High-level expression of niml by an insert plate effect

To determine whether any of the transformants described in Example 10 / Table 4 above exhibited a high level of expression of niml due to an insert plate effect, primary transformants containing the D7, D5 or E1 cosmids (containing the niml gene) were contained self-crossed and the T2 seed was collected. Seeds from an E1 line, four D5 lines, and 95 D7 lines were sown on soil and grown in the manner described above. When the T2 plants had developed at least four true leaves, a single leaf from each plant was harvested separately. RNA was extracted from this tissue and analyzed for PR-1 and niml expression. The plants were then inoculated with P. parasitica (Emwa) and analyzed for fungal growth at 3-14 days, preferably 7-12 days after infection. Plants 10 exhibiting higher niml and PR-1 expression than normal and exhibiting resistance to fungus show that expression of niml at a high level confers a CIM phenotype.

Table 6 shows the results of testing different transformants for resistance to fungal infection. As can be seen from the table, some transformants showed less fungal growth than normal and some showed no visible fungal growth at all. RNA was prepared from collected samples and analyzed as described above (Delaney et al., 1995). Spots were hybridized with the Arabidopsis gene test series PR-1 (Uknes et al., 1992). Lines D7-74, D5-6 and E1-1 showed early induction of PR-1 gene expression, with 20 PR1 mRNA detectable as early as 24 or 48 hours after treatment with the fungus. These three lines also showed resistance to fungal infection.

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Table 6

Line P.parasi- Line P.parasi- Line P.parasitica growth tica growth tica growth D7-2 negative 52 + 90 + 3 + 53 + 91 + 5 9 + 54 +/- 92 + 11 + 56 + 93 + 12 + 57 + 94 + 13 + 58 + 95 + 14 + 59 + 96 + 10 17 + 60 + 97 + 18 + 61 + 98 +/- 19 + 62 + 100 +/- 20 + 63 + 101 +/- 21 + 64 + 102 +/- 15 22 + 66 + 103 + 23 + 67 + 104 + 24 + 68 + 106 + 25 + 69 + 107 + 28 + 70 + 108 + 20 29 + 71 + 114 + _ 31 + 72 + 115 + 32 + 73 + 118 + 33 + 74 negative 119 + 34 + 75 + 122 + 25 35 + 77 + 123 + 1007779 101

Table 6 (continued) 36 + 78 + 124 + 38 + 79 + 125 + • 5 39 + 80 +/- 126 + 42 + 81 + 128 + 43 + 82 + 129 + 46 + 83 + 130 + 47 + 84 + D5 -1 + 10 48 + 85 + 2 + 49 + 86 + 4 + 50 + 87 +/- 6 +/- 51 + 89 negative E1-1 negative. Plants were treated with P. parasitica isolate Emwa and assessed 10 days later.

+, normal fungus growth.

+/-, lower fungal growth than normal, negative, no visible fungal growth.

20

Example 19: 35S-guided expression of niml

Plants expressing the niml gene constitutively were also generated from transformation of Ws or Col wild type plants with the BamHI-HindlII Arabidopsis genomic fragment (Figure 6) cloned into pSCGGOl and transformed into the Agrobacterium strain GV3101 (pMP90, Koncz and Schell, 1986, Mol Gen Genet 204: 383-396). In another construct, the full-length niml cDNA was cloned into the EcoRI site of pCGN1761 ENX (Comai et al., 1990, Plant Mol. Biol. 15, 373-381). From the plasmid thus obtained, an Xbal fragment containing an amplified CaMV 35S promoter, the niml cDNA in the correct 1007779 102 orientation for transcription and a tml 3 'terminator was obtained. This fragment was cloned into the binary vector pCIB200 and transformed into GV3101.

WS or Col plants are infiltrated with any of these transformed strains as described above. The seed thus obtained was harvested and plated on 50 mg / ml GM agar. The young plants that survive are transferred to soil and examined.

Example 20: 35S guided expression of niml - alternative embodiment

A BamHI / HindlII Arabidopsis genomic fragment (Figure 6: bases 1249-10 5655) containing the niml promoter, gene, and upstream sequence cloned in pSGCGOl, is cut with the restriction endonuclease PstI and blunt ended with T4 polymerase. The fragment is further cut with the restriction endonuclease SpeI resulting in a 2162 bp fragment. The niml cDNA cloned in the EcoRI site of pCGN1761ENX (see above) is cut with the restriction endonuclease with Notl and blunt ended with T4 polymerase. This fragment is further cut with the restriction endonuclease Game. The 2162 bp genomic fragment obtained above is linked to the pCGN1761ENX vector containing 91 bp of the niml cDNA. The transformation cassette with the 35S promoter and tml terminator is released from pCGN1761ENX by partial cutting with the restriction enzyme Xbal and coupled in the Xbal site of dephosphorylated pCIB200. The plasmid thus obtained is transformed into GV3101 and WS or Col plants are infiltrated as described above. The seeds are plated and the young plants that survive are selected in the manner described above.

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Example 21: Evaluation of the CIM phenotype in the nim 1 transformants with high level expression

A leaf of each primary transformant is harvested, RNA is isolated (Verwoerd et al., 1989, Nuc. Acid Res., 2362) and examined for constitutive PR-1 expression and mm ƒ expression by RNA blot analysis (Uknes et al. 1992). Each transformant is evaluated for an increased disease resistance response indicative of constitutive SAR expression analysis (Uknes et al., 1992). Conidial suspensions of 5-10x104 spores / ml from two compatible P. parasitica isolates, Emwa and Noco (i.e., these fungal strains cause disease on wild-type Ws-0 and Col-0 plants, respectively) are prepared and transformants are sprayed with the appropriate isolate, depending on the ecotype of the transformant. Grafted plants are incubated under high humidity for 7 days. The plants are evaluated for disease on day 7 and a single leaf is harvested for RNA spot analysis using a test series, which provides a means of measuring fungal infection.

Transformants displaying a CIM phenotype are F1 generation and homozygous plants are identified. Transformants are subjected to a series of disease resistance tests. Fungus infection with Noco and Emwa is performed and leaves are stained with lactophenol blue to identify the presence of fungal hyphae, as described by Dietrich et al. (1994). Transformants are infected with the bacterial pathogen Pseudomonas syringae DC3000 to evaluate the spectrum of detectable resistance, as described by Uknes et al., (1993). Uninfected plants are evaluated for both free and glucose conjugated SA and the leaves are stained with lactophenol blue to evaluate for the presence of microscopic lesions. The resistant plants are sexually interbred with SAR mutants such as NahG and ndrl to determine the epistatic relationship between the resistant phenotype and the other mutants and assess how these dominant negative mutants of niml can affect SA dependent feedback.

Example 22: High level expression of niml in crop plants. These constructs, which confer a CIM phenotype on Col-0 or Ws-0 and others, are transformed into crop plants for evaluation. While the niml gene can be introduced into any plant cell that falls within these broad classes, it can be used in particular in crop plant cells such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, beans, peas, 30 chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, pumpkin, in particular cucurbita pepo, zucchini, cucumber, apple, pear , quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, pineapple, avocado, papaya, mango, banana, soy, tobacco, 1 007779 104 tomato, sorghum or sugar cane. Transformants are assessed for increased disease resistance. In a preferred embodiment of the invention, the expression of the niml gene is at a level at least twice higher than the expression level of the niml gene in the wild type plants, and preferably ten times above the expression level of the wild type .

F, Other general applications of plants with the m'm phenotype

Example 23: Use of mm mutants in disease research. Nim mutants are loaded with various pathogens, showing that they develop larger lesions faster than wild type plants. This phenotype is called UDS (general disease susceptibility) and is a result of the mutants unable to express SAR genes to provide a defense of the plant against pathogens. The UDS phenotype of the nim mutants makes them useful as reference plants for assessing disease symptoms in experimental lines in field pathogenesis trials where the natural resistance phenotype of the so-called wild type lines may diverge (i.e. against different pathogens and different pathotypes of the same pathogen). Therefore, under field conditions where natural infection by pathogens is used to assess the resistance of experimental lines, the inclusion of nim mutant lines of the appropriate crop plant species in the experiment may allow an assessment of the true level and spectrum of the load on the pathogen, without the variation inherent in the use of non-experimental lines.

25

Example 24: Assessment of the usefulness of transgenes for the purposes of disease resistance

Mm mutants are used as host plants for transforming transgenes to facilitate their assessment for use in disease resistance. For example, an Arabidopsis «m? Mutant line, characterized by its UDS phenotype, is used for subsequent transformation with potential genes for disease resistance, making it possible to assess the contribution of an individual gene to resistance from baseline of 1007779 105 the UDS mm mutant plants.

Example 25: mm mutants as an aid to investigate plant-pathogen interactions.

Mm mutants can be used to investigate plant pathogen interactions, and in particular to understand the processes used by the pathogen to enter the plant cells. This is because mm mutants do not give a systemic response to pathogen attack, and the uninhibited pathogen development is an ideal scenario for examining its biological interaction with the host.

Of further interest is the observation that a mm mutant used as a host may be sensitive to pathogens not normally associated with this particular host, but rather related to another host. For example, an Arabidopsis mm mutant such as niml-1, -2, -3, -4, -5 or -6 can be loaded with a number of pathogens that normally only infect the tobacco plant and prove to be susceptible to them. In this way, the mm mutation causing the UDS phenotype can lead to a change in the range of susceptibility to pathogens, which has considerable utility in the molecular, genetic and biochemical analysis of host-pathogen interaction.

20

Example 26: mm mutants for use in funeicide studies

Mm mutants are particularly useful in assessing new chemical compounds for fungicidal activity. Mm mutants selected in a specific host have significant utility in determining fungicides using this host and host pathogens. The advantage lies in the mutant UDS phenotype, which overcomes the problems that may arise because the host is sensitive to different pathogens and pathotypes to varying degrees, or even resistant to certain pathogens or pathotypes. For example, mm mutants in wheat can be effectively used to evaluate fungicides against a wide variety of pathogens and pathotypes in wheat, since the mutants do not give a resistance reaction to the introduced pathogen and do not exhibit differential resistance to different pathotypes, which would otherwise require the use of multiple wheat lines, each of which is sufficiently sensitive to a specific pathogen to be investigated. Wheat pathogens of particular interest include (but are not limited to) Erisyphe graminis (the causative agent of downy mildew), Rhizoctonia solani (the causative agent of "sharp eye blotch"), Pseudocercosporella herpotrichoides (the causative agent of "eye blotch) Puccinia spp. (the causative agents of leaf rust) and Septoria nodorum. Similarly, maize m'm mutants will be very sensitive to maize pathogens and thus can be used in determining fungicides with activity against maize diseases.

10 Mm mutants can further be used to examine a wide variety of pathogens and pathotypes in a heterologous host, i.e., in a host that is not normally within the host range of a specific pathogen and is particularly easy to handle ( such as Arabidopsis). Due to the presence of the UDS phenotype, the heterologous host 15 is sensitive to pathogens from other plant species, including crop plant species of economic importance. For example, the same Arabidopsis nim mutant can be infected with a wheat pathogen such as Erisyphe graminis (the downy mildew causative agent) or a maize pathogen such as Helminthosporium maydis and used to determine the efficacy of potential fungicides. Such an approach provides significant improvements in efficiency over the currently employed procedures of examining individual crop plant species and different cultivars of species with different pathogens and pathotypes that may be differentially virulent on the different crop plant cultivars. Furthermore, the use of Arabidopsis has advantages because of its small size, which makes it possible to do more experiments in limited available space.

Example 27: niml is a homolog of ΙκΒα

A multiple sequence comparison between the protein gene products of niml 30 and IkB was performed, finding that the w> n7 gene product is a homolog of ΙκΒα (Figure 9). Sequence homologies were investigated using BLAST (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The multiple sequence comparison was constructed using Clustal V (Higgins et al., CABIOS 5, 151-153 (1989)) as part of the Lasergene Biocomputing Software package from DNASTAR (Madison, WI). The sequences used in the equation in which niml (SEC ID no .: 3), mouse ΙκΒα (SEQ ID no .: 18, GenBank Accession #: 1022734), rat ΙκΒα (SEQ ID no .: 19) GenBank 5 Accession numbers 57674 and X63594; Tewari et al., Nucleic Acids Res. 20, 607 (1992)), and pigs ΙκΒα (SEQ ID NO: 20, GenBank accession number Z21968; de Martin et al., EMBO J. 12, 2773-2779 (1993); GenBank accession number 517193, de Martin et al. , Gene 152, 253-255 (1995)). The parameters used in the "Clustal" analysis were a "gap penalty" of 10 and a "gap length penalty" of 10 10. Distances of evolutionary differentiation were calculated using the PAM250 weight table (Dayhoff et al., " A model of evolutionary change in proteins. Matrices for detecting distant relationships. "In Atlas of Protein Sequence and Structure, volume 5, supplement 3, MO, Dayhoff, ed. (National Biomedical Research Foundation, Washington, DC), biz. 345- 358 (1978)). The degree of similarity in residues was calculated using an adjusted one

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) biz. 353-358 (1979). ; Gribskov and Burgess, Nucleic Acids Res. 14, 6745-6763 (1986)).

20 Homology studies indicate a degree of similarity between niml and ankyrin domains of different proteins, including: ankyrin, NF-κΒ and IkB. The best overall homology is with IkB and similar molecules (Figure 9). niml contains 2 serine residues at amino acid positions 55 and 59, the serine residue at position 59 being in a context (D / ExxxxxS) and in a position (N-terminal) that is 25 constitent with a role in a phosphorylation dependent, by ubiquitin transferred, inducible degradation. All IkBs have these N-terminal serine residues and they are required to inactivate IkB and the subsequent release of NF-κΒ. niml has ankyrin domains (amino acids 262-290 and 323-371). Ankyrin domains are believed to be involved in protein-protein interaction and are a required aspect of IkB and NF-κΒ molecules. The C ends of IkBs can be different. niml has some degree of homology with a QL-rich region (amino acids 491-499) that is found in the C-terminal portion of some IkBs.

1 007 779 108

Example 28: Generation of modified forms of niml - changing the serine residues 55 and 59 into alanine residues

The phosphorylation of serine residues is required in human ΙκΒα for the stimulus-activated degradation of ΙκΒα, activating NF-κΒ.

5 Mutagenesis of the serine residues (S32-S36) in human ΙκΒα to analin residues inhibits stimulus-induced phosphorylation, thereby blocking the proteosome-associated degradation of ΙκΒα (E. Britta-Mareen Traenckner et al., EMBO J. 14: 2876 2883 (1995); Brown et al., Science 267: 1485-1488 (1996); Broekman et al., Molecular and Cellular Biology 15: 2809-2818 (1995); Wang et al., Science 274: 784-10 787 (1996)).

This altered form of ΙκΒα acts as a dominant negative form in that it retains NF-κΒ in the cytoplasm, thereby blocking downstream signaling events. Based on sequence comparisons between niml and IkB, the serine residues 55 (S55) and 59 (S59) of niml are homologous to S32 and S36 in human ΙκΒα. To construct dominant-negative forms of niml, the serine residues at amino acid positions 55 and 59 are mutated to alanine residues. This can be performed by any method known to those skilled in the art, such as, for example, using the QuikChange Site Directed Mutagenesis Kit (# 200518: Stratagene).

Using a full length niml cDNA (SEQ ID NO: 21), including the 42 bp of the 5 'untranslated sequence (UTR) and 187 bp of the 3' UTR, the mutated construct are made according to the manufacturer's instructions using the following basic series (SEQ ID NO: 21, positions 192-226): 5'-CAA CAG CTT CGA AGC CGT CTT TGA CGC GCC GGA TG-3 '25 (SEQ ID no .: 32) and 5'-CAT CCG GCG CGT CAA AGA CGG CTT CGA AGC TGT TG-3 '(SEQ ID NO: 33), the underlined bases indicating the mutations. The strategy is as follows: the niml cDNA, cloned into vector pSE936 (Elledge et al., Proc. Nat. Acad. Sci. USA 88: 1731-1735 (1991)), is denatured and the base sequences are fused with the altered bases . DNA polymerase (Pfu) 30 complements the base strings by non-stranded vein placement leading to "nicked" spherical strands. DNA is cut with the restriction endonuclease Dpnl, which cuts only methylated sites (non-mutagenized template DNA). The remaining circular dsDNA is transformed into E.

1007779 109 coli strain XL 1-Blue. Plasmids from the colonies thus obtained are collected and sequenced to confirm the presence of the mutated bases and to confirm that no other mutations have occurred.

The mutated niml cDNA is cut with the restriction endonuclease EcoRI and cloned into pCGN1761 under the transcription control of the cauliflower mosaic virus 35S double promoter. The transformation cassette, including the 35S promoter, niml cDNA and tml terminator, is released from pCGN1761 by partial cutting with the restriction enzyme Xbal and coupled into the Xbal site of dephosphorylated pCIB200. SEQ ID NOS: 22 and 23 show the DNA coding sequence and the encoded amino acid sequence of this altered form of the niml gene, respectively.

The present invention also includes modified forms of alleles of niml, wherein the coding sequence of such allele hybridizes under the following conditions to the coding sequence shown in SEQ ID NO: 22: hybridization in 1% BSA; 520 mM NaPO4, Ph 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours, and washing in 6XSSC for 15 minutes (X3) 3XSSC for 15 minutes (XI) at 55 ° C. In these embodiments, the alleles of niml that hybridize under these conditions with SEQ ID NO: 22 are modified such that the encoded product contains alanine residues instead of serine residues at the amino acid positions corresponding to positions 55 and 59 of SEQ ID NO: 22 .

Example 29: Generation of altered forms of niml - N-terminal deletion Deletion of amino acids 1-36 (Broekman et al .; Sun et al.) Or 1-72 (Sun et al.) Of human ΙκΒα, containing K21, K22 , S32 and S36, leads to a dominant-negative ΙκΒα phenotype in transferred human cell cultures. An N-terminal deletion of approximately the first 125 amino acids of the encoded product of the niml cDNA removes eight lysine residues that may serve as potential ubiquitination sites and also removes potential phosphorylation sites at S55 and S59 (see Example 2). This altered gene construct can be produced in any manner known to those skilled in the art. For example, using the method of Ho et al., Gene 77: 51-59 (1989), a niml form can be generated from which the DNA encoding approximately the first 125 amino acids is 1007779 110. The following basic series provide a 1612-bp PCR product (SEQ ID NO: 21: 418 to 2011): 5'-gg aat tca-ATG GAT TCG GTT GTG ACT GTT TTG-3 '(SEQ ID NO: 34) and 5'-gga att cTA CAA ATC TGT ATA CCA TTG G-3 '(SEQ ID NO: 35) in which the synthetic start codon is underlined (ATG) and the EcoRI-5 coupling sequence is indicated in lower case. Amplification of these fragments uses a reaction mixture containing 0.1 to 100 ng of template DNA, 10 mM Tris pH 8.3 / 50 mM KCl / 2 mM MgCl 2 / 0.001% gelatin / 0.25 mM of each dNTP / 0. Contains 2 mM from each base series and 1 unit rTth DNA polymerase in a final volume of 50 ml and a Perkin Elmer Cetus 9600 PCR machine 10 is used. The conditions for PCR are as follows: 94 ° C 3 minutes: 35x (94 ° C 30 seconds: 52 ° C 1 minute: 72 ° C 2 minutes): 72 ° C 10 minutes. The PCR product is cloned directly into the pCR2.1 vector (Invitrogen). The PCR-introduced insert in the PCR vector is released by cutting with the restriction enzyme EcoRI and coupled into the EcoRI site of dephosphorylated pCGN176 under the transcriptional control of the 35S double promoter. The construct is sequenced to confirm the presence of the synthetic ATG start codon and to confirm that no further mutations had occurred during the PCR. The transformation cassette, including the 35S promoter, the modified niml cDNA and tml terminator, is released from pCGN1761ENX 20 by partial cutting with the Xbal restriction enzyme and coupled into the Xbal site of pCIB200. SEQ ID NOS: 24 and 25 show the DNA coding sequence and the encoded amino acid sequence of an altered form of the niml gene with an N-terminal amino acid deletion, respectively.

The present invention also encompasses altered 25 niml alleles, wherein the coding sequence of such an allele hybridizes to the coding sequence shown in SEQ ID NO: 24 under the following conditions: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours and washing in 6XSSC for 15 minutes (X3) 3XSSC for 15 minutes (XI) at 55 ° C. Under these conditions, the alleles of niml that hybridize under these conditions with SEQ ID NO: 24 have been modified such that the encoded product has an N-terminal deletion, removing lysine residues that may serve as potential ubiquitination sites, as well as the 1007779

Ill serine residues at the amino acid positions corresponding to positions 55 and 59 of the wild-type gene product.

Example 30: Generation of altered forms of niml - C-terminal deletion. It is believed that the removal of amino acids 261-317 from human ΙκΒα leads to increased intrinsic stability by blocking the constitutive phosphorylation of serine and threonine residues at the C-terminus. . A region rich in serine and threonine is located at amino acids 522-593 in the C-terminus of niml. The C-terminal coding region of the niml-gtn can be modified by removing the nucleotide sequences encoding amino acids 522-593. Using the method of Ho et al. (1989), the C-terminal coding region and the 3'UTR of the niml cDNA (SEQ ID NO: 21: 1606-2011) are removed by PCR, using a 1623 bp fragment is obtained, using the following base strings: 5'-15 cggttcGATCTCTTTAATTTGTGAATTT C-3 '(SEQ ID NO: 36) and 5'-ggttcTCAACAGTT CATAATCTGGTCG-3' (SEQ ID NO: 37) underlining a synthetic stop codon (TGA on the complementary strand) and the lowercase EcoRI link sequences are shown. The components of the PCR reaction are as described above and the parameters of the cycles are as follows: 94 ° C 3 minutes: 30x [(94 ° C 30 seconds: 52 ° C 1 minute: 72 ° C 2 minutes); 72 ° C, 10 minutes]. The PCR product is cloned directly into the pCR2.1 vector (in vitro). The PCR-introduced insert in the PCR vector is released by cutting with the restriction endonuclease EcoRI and coupled into the EcoRI site of dephosphorylated pCGN1761 containing the double 35S promoter. The construct is sequenced to confirm the presence of the synthetic in-frame stop codon and to confirm that no other mutations had occurred during PCR. The transformation cassette, including the promoter, modified niml cDNA and tml terminator, is released from pCG1761 by partial cutting with the restriction enzyme Xbal and coupled to 30 in the Xbal site of dephosphorylated pCIB200. SEQ ID NOS: 26 and 27 show the DNA coding sequence and the encoded amino acid sequence of an altered form of the niml gene with a C-terminal amino acid deletion, respectively.

The present invention also includes modified alleles of 1 007779 112 niml, wherein the coding sequence of such allele hybridizes to the coding sequence shown in SEQ ID NO: 26 under the following conditions: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours, and washing in 6XSSC for 15 minutes (X3) 3XSSC for 15 minutes (XI) at 55 ° C. In these embodiments, alleles of niml hybridizing under SEQ ID NO: 26 under the above conditions are modified such that the encoded product has a C-terminal deletion to remove serine and threonine residues.

10

Example 31: Generation of modified forms of niml - chimera with an N-terminal / C-terminal deletion

An N-terminal N-terminal and C-terminal deletion form is generated using a unique KpnI restriction site at position 819 (SEQ ID NO: 15 21). The N-terminal deletion shape (Example 3) is cut with the restriction

endonucleases EcoRI / Kpnl and the 415 bp fragment corresponding to the N-terminus is recovered by gel electrophoresis. Similarly, the C-terminal deletion form (Example 4) is cut with the restriction endonucleases EcoRI / Kpn1 and the 790 bp fragment corresponding to the modified C-terminus is recovered by gel electrophoresis. The fragments are taken at 15 ° C

coupled, cut with EcoRI to remove the EcoRI concatemers and cloned into the EcoRI site of dephosphorylated pCGN1761. The N / C terminal deletion form of niml is under the transcription regulation of the 35S double promoter. Similarly, a chimeric form of niml is generated consisting of the S35 / S59 mutagenized potential phosphorylation sites

(Example 2) fused to the C-terminal deletion (Example 4). This construct is generated in the manner described above. The sequences of the constructs are determined to ensure the reliability of the start and stop codons and to confirm that no mutations have occurred during cloning. The respective transformation cassettes, including the 35S promoter, / / w7 chimera and tml terminator, were released from pCGN1761 by partial cutting with restriction enzyme Xbal and coupled into the Xbal site of dephosphorylated pCIB200. SEQ ID NOS: 28 and 29 show the DNA, respectively

1007779 113 coding sequence and the encoded amino acid sequence of an altered form of the niml gene with both an N-terminal and a C-terminal amino acid deletion.

The present invention also encompasses modified 5 niml alleles, wherein the coding sequence of such allele hybridizes to the coding sequence shown in SEQ ID NO: 28 under the following conditions: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours, and washing in 6XSSC for 15 minutes (X3) 3XSSC for 15 minutes (XI) at 55 ° C. Under these conditions, alleles of niml that hybridize under SEQ ID NO: 28 under the above conditions are altered such that the encoded product has both an N-terminal deletion, removing lysine residues which may serve as potential ubiquitination sites, in addition to the serine residues at amino acid positions corresponding to positions 55 and 59 of the wild-type gene product; as a C-terminal deletion, removing serine and threonine residues.

Example 32: Generation of modified forms of niml - Ankrin domains niml shows homology with ankyrin motifs at approximately amino acids 103-362. Using the method of Ho et al. (1989), the DNA sequence encoding the possible ankyrin domains (SEQ ID NO: 2: 3093-3951) is amplified by PCR (conditions: 94 ° C 3 minutes: 35 x (94 ° C 30 seconds: 62 ° C 30 seconds: 72 ° C 2 minutes): 72 ° C 10 minutes) from the niml cDNA (SEQ ID NO: 21: 349-1128) using the following basic series: 5 "-25 ggttcaATGG ACT CC AAC AAC ACCGCCGC-3" (SEQ ID NO: 38) and 5 "ggttcTCAACCTTCCAAAGTTGCTTCTGATG-3" (SEQ ID NO: 39). The product thus obtained is cut with the restriction endonuclease EcoRI and then cleaved into the EcoRI site of dephosphorylated pCGN1761 under the transcription control of the double 35S promoter. The construct 30 is sequenced to confirm the presence of the synthetic start codon (ATG) and an in-frame stop codon (TGA) and to confirm that no other mutations have occurred during PCR. The transformation cassette, including the 35S promoter, ankyrin domains and tml terminator, is released from pCGN1761 1 007779 114 by partial cutting with the restriction enzyme Xbal and coupled into the Xbal site of dephosphorylated pCIB200. SEQ ID NOS: 30 and 31 show the DNA coding sequence and the encoded amino acid sequence of the ankyrin domain of niml, respectively.

The present invention also includes modified forms of alleles of niml, wherein the coding sequence of such an allele hybridizes to the coding sequence shown in SEQ ID NO: 30 under the following conditions: hybridization in 1% BSA; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours, 10 and washing in 6XSSC for 15 minutes (X3) 3XSSC for 15 minutes (XI) at 55 ° C. In these embodiments, alleles of niml that hybridize under SEQ ID NO: 30 under the above conditions are modified such that the encoded product consists essentially of the ankyrin domains of the wild type gene product.

15

Example 33: Construction of chimeric genes

To increase the likelihood of timely and appropriate expression of altered niml forms, a 4407 bp HindIII / BamHI fragment (SEQ ID NO: 2: bases 1249-5655) and / or a 5655 bp EcoRV / BamHI- fragment (SEQ ID NO: 2: bases 20 1-5655) containing the niml promoter and gene used to create the altered niml forms in Examples 28-32 above. Although the construction steps may be different, the underlying concepts are similar to the examples described above. Strong overexpression of the altered forms can potentially be fatal. Therefore, the altered forms of the niml gene described in Examples 28-32 may be brought under the control of promoters other than the endogenous niml promoter, including but not limited to the Rubi promoter or small subunit of Rubiscopromotor . Similarly, altered niml organisms can be expressed under the control of the pathogen-responsive promoter PR-1 (US Patent 5,614,395). Such expression allows enhanced expression of the altered forms only under load with a pathogen or under other SAR activating conditions. Furthermore, disease resistance may be detectable in transformants that altered forms under the control of 1007779 115 express the PR-1 promoter when treated with concentrations of SAR activating compounds (ie BTH or INA ) that do not normally activate SAR, and therefore activate a feedback cycle (Weymann et al., (1995) Plant Cell 7: 2013-2022).

5

Example 34: Transformation of modified forms of the nim] into Arabidovsis thaliana

The generated constructs (example 28-33) are introduced into Agrobacterium tumefaciens by electroporation into strain GV3101. These constructs are used to transform Arabidopsis ecotypes Col-0 and Ws-0 by vacuum infiltration (Mindrinos et al., Cell 78, 1089-1099 (1994)) or by standard root transformation. Seed from these plants is harvested and germinated on agar plates with kanamycin (or other suitable antibiotic) as a selection agent. Only young plants transformed with cosmid DNA can detoxify and survive the selection agent. Seedlings surviving the selection are transferred to soil and examined for a CIM (constitutive immunity) phenotype. Plants are judged for observable phenotype differences compared to the wild type plants.

Example 35: Assessment of the CIM phenotype in plants transformed with altered forms of niml

A leaf of each primary transformant is harvested, RNA is isolated (Verwoerd et al., 1989, Nuc Acid Res, 2362) and examined for constitutive PR-1 expression by RNA blot analysis (Uknes et al., 1992). Each transformant is evaluated for an increased disease resistance response indicative of constitutive SAR expression analysis (Uknes et al., 1992). Suspensions of conidia in 5-10x104 spores / ml of two compatible P. parasitica isolates, Emwa and Noco (i.e., these fungal strains cause disease on wild-type Ws-0 and Col-0 plants, respectively) are prepared and transformants are sprayed with the appropriate isolate depending on the ecotype of the transformant. Grafted plants are incubated under high humidity for 7 days. Plants are evaluated for disease on day 7 and a single leaf is harvested for RNA stain analysis using a test series, providing an agent 1007779 116 for measuring fungal infection.

Transformants displaying a CIM phenotype are led to the T1 generation and homozygous plants are identified. Transformants are subjected to a series of disease resistance tests as described below.

Infection with fungus 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 the bacterial pathogen Pseudomonas syringae DC3000 to assess the spectrum of observable resistance, as described in Uknes et 10 al. (1993). Uninfected plants are evaluated for both free and glucose conjugated SA and plants are stained with lactophenol blue to determine the presence of microscopic lesions. Resistant plants are sexually cross-bred with SAR mutants such as NahG (US Patent 5,614,395) and ndrl to determine the epistatic relationship of the resistant phenotype to other mutants and evaluate how these dominant-negative mutants of niml follow the SA-dependent feedback cycle. can influence.

Example 36: Isolation of niml homologs

Using the niml cDNA (SEQ ID NO: 21) as a test series 20, homologs of Arabidopsis niml are identified by probing genomic or cDNA libraries from various crop plants such as, but not limited to, the species listed in Example 11 above. Standard techniques for performing this include hybridization scanning of plated DNA libraries (i.e., plaques or colonies; see, e.g., Sambrook et al., Molecular Cloning, eds., Cold Spring Harbor Laboratory Press. (1989)) and PCR amplification using oligonucleotide base arrays (see, e.g., Innis et al., PCR Protocols, a Guide to Methods and Applications eds., Academic Press (1990)). Identified homologs are genetically engineered into the expression vectors described herein and transformed into the above crops. Transformants are assessed for increased disease resistance using relevant pathogens from the crop plant under investigation.

DNA stain analysis revealed nim 1 homologs in the genomes of 1007779 117 cucumber, tomato, tobacco, corn, wheat and barley. Genomic DNA was isolated from cucumber, tomato, tobacco, corn, wheat and barley, digested with restriction enzymes BamHI, HindlII, Xbal or Sail, separated electrophoretically on 0.8% agarose gels and transferred to nylon membrane by capillary spotting. After UV cross-linking to fix the DNA, the membrane was placed under moderately severe conditions [(1% BSA; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride) at 55 ° C for 18-24 hours] hybridized with 32 P radiolabelled Arabidopsis thaliana niml cDNA. After hybridization, the spots were washed under moderately severe conditions [6XSSC for 15 minutes (X3),

10 3XSSC for 15 minutes (XI) at 55 ° C; 1XSSC is 0.15M NaCl, 15 mM

sodium citrate (pH 7.0)] washed and exposed to X-ray film to reveal bands corresponding to niml.

Furthermore, identified expressed sequence markers (ESTs) that resemble the niml gene can be used to isolate homologs. For example, several expressed sequence markers (ESTs) from rice have been identified that resemble the niml gene. A multiple sequence comparison was constructed using Clustal V (Higgins, Desmond G. and Paul M. Sharp (1989), Fast and sensitive multiple sequence alignment on a microcomputer, CABIOS 5: 151-153) as part of the DNA * 20 ( 1228 South Park Street, Madison Wisconsin, 53715) Laser Gene Biocomputing

Software package for the Macintosh (1994). Certain regions of the niml protein are amino acid sequence homologous to 4 different rice-derived cDNA protein products. The homologies were identified using the m'wi sequences in a GenBank BLAST study.

Comparisons of the areas of homology between niml and the rice cDNA products are shown in Figure 8 (see also SEQ 1D No .: 3 and SEQ ID NO: 4-11). The w / w / protein fragments show amino acid sequences that are 36 to 48% identical to the 4 rice-derived products. These rice ESTs may be particularly useful for isolating nim 1 homologs from other monocotyledonous plants. Homologs can be obtained by PCR. In this method, comparisons are made between known homologues (e.g. rice and Arabidopsis). Regions with a high degree of identity or identity in amino acids and DNA are then used to make basic PCR-1007779 118 sequences. Regions rich in the amino acid residues M and W are most suitable, followed by regions rich in the amino acid residues F, Y, C, H, Q, K and E, because these amino acids are encoded by a limited number of codons. When a suitable region has been identified, ground arrays for this region 5 are made with various substitutions in place of the third codon. The diversity of substitution in the third position can be limited depending on the intended species. For example, since corn is GC-rich, base strings containing a G or a C in third place are used, if possible.

The PCR reaction is performed with cDNA or genomic DNA under a number of different standard conditions. When a band is observed, it is cloned and / or sequenced to determine if it is a niml homolog.

Example 37: Expression of altered forms of niml in crop plants. Those constructs that confer a CIM phenotype on Col-0 or Ws-0 are transformed into crop plants for evaluation. Alternatively, modified native mm / genes isolated from crops in the previous example can be reintroduced into the respective crops. While the niml gene can be introduced into any plant cell that falls within these broad classes, it is particularly useful in crop plant cells such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, beet, beans, peas, chicory , lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, pumpkin, in particular Cucurbita pepo, zucchini, cucumber, apple, pear, quince, melon, plum , cherry, peach, nectarine, apricot, strawberry, grape, raspberry, pineapple, avocado, papaya, mango, banana, soy, tobacco, tomato, sorghum or sugar cane. Transformants are assessed for increased disease resistance. According to a preferred embodiment of the invention, the expression of the altered form of the niml gene is at a level at least twice above the expression level of native niml gene in wild type plants, and preferably ten times above the expression level of the wild type.

1007779 119

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SEQUENCE LIST

(1) GENERAL INFORMATION: (i) APPLICANT:

(A) NAME: Novartis AG

(B) STREET: Schwarzwaldallee 215 (C) LOCATION: Basel (E) COUNTRY: Switzerland (F) POST CODE (ZIP): 4002 (G) PHONE: +41 61 69 11 11 (H) TELEFAX: + 41 61 696 79 76 (I) TELEX: 962 991 (v) COMPUTER READABLE FORM: (A) MEDIUM: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patent Release # 1.0, Version # 1.30 (ii) TITLE OF THE INVENTION: METHODS OF APPLYING THE NIM1 CONTENT TO GIVE PLANT RESISTANCE TO DISEASES.

(lil) NUMBER OF SEQUENCES: 39 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9919 base pairs (B) TYPE: nucleic acid (C) STRENGTH: single (D) TOPOLOGY: linear (ii TYPE OF MOLECULA: DNA (genomic)

(iii) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TGATCATGAA TTGCGTGTAG GGTTGTGTTT TAAAGATAGG GATGAGCTGA AGAAGGCGGT 60 1007779126 GGACTGGTGT TCCATTAGAG GGCAGCAAAA GTGTGTAGTA CAAGAGATTG AGAAGGACGA 120 GTATACG1TT AAATGCATCA GATGGAAATG CAA17GGTCG CGTCGGGCAG ATTGAATAGA 180 AGAACATGGA CHGHAAGA 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 CTCATCTAAT GGGCTACTTG TGGACTGCAA TATGAGCTTT TCCCTAATCC 480 TGAATTTGCA TCCTTCGGTG GCGCGTITTG GGCGTTTCCA CAGTCCATTG AAGGGTTTCA 540 ACACTGTAGA CCTCTGATCA TAGTGGATTC AAAAGACHG AACGGCAAGT ACCCTATGAA 600 ATTGATGATT TCCTCAGGAC TCGACGCTGA TGATTGCTTT TTCCCGCHG CCTTTCCGCT 660 TACCAAAGAA GTGTCCACTG ATAGTTGGCG TTGGTTTCTC ACTAATATCA GAGAGAAGGT 720 AACACAAAGG AAAGACGTTT GCCTCGTCTC CAGTCCTCAC CCGGACATAG π60Τ6ΠΑΤ 780 TAACGAACCC GGATCACTGT GGCAAGAACC TTGG GTCTAT CACAGGTTCT GTCTGGATTG 840 TTTTTGCTTA CAATTCCATG ATATTTTTGG AGACTACAAC CTGGTGAGCC TTGTGAAGCA 900 GGCTGGATCC ACAAGTCAGA AGGAAGAATT TGATTCCTAC ATAAAGGACA TCAAAAAGAA 960 GGACTCAGAA GCTCGG.AAAT GGTTAGCCCA ATTCCCTCAA AATCAGTGGG CTCTGGCTCA 1020 TGACCAGTGG TCGGAGATAT GGAGTCATGA CGATAGAAAC AGAAGATTTG AGGGCAATTT 1080 GTGAAAGCTT TCAGTCTCTT GGTCTATCAG TGACAGCGAA CGCACCTGCA CATGTGGGAA 1140 GTiTCAATCG AAGAAGTTTC CATGTATGCA CCCAGAAATG GTGCAAAGGA TTGTTAACTT 1200 GTGTCATTCA CAAATGTTGG ATGCAATGGA GCTGACTAGG AGAATGCACC HACACGCCC 1260 ACTCAGTGn CTCTTATCTC TAGACCTGAA ACTAACTTGC TGTGTAATTC GAGTTACAAA 1320 AGGTTAAAGG AAGAATTAGG AAGATACATA TAACATGAAT GHGCCAGAA GTTCAGGGAA 1380 CTTGAATAH CTmGGTTC TTGGTGGAAA ATATCCAACA GATGAACAAT TTGACATTAT 1440 1007779 127 TTCACACTTT GATTCTAGCA ACTCTGTAAC ACCATCATGG GTTATTGTTG ATGTACATAA 1500 ATATATATTA CAAATCTGTA TACCATTGGT TCAAATTGTT ACAACATTTG TTTGAAGCAC 1560 ACCTGCAGCA ATAATACACA GGATGCAAAA CGAAGAGCGA AACTATATGA CGCCAACGAT 1620 AGACATAAAC AGTTACAGTC ATCATGAAAA CAGATE TATA TGGTACAGCA AAAATTACAC 1680 TAAGAGGCAA GAGTCTCACC GACGACGATG AGAGAGTTTA CGGTTAGACC TCITTCCACC 1740 GGTTGATTTC GATGTGGAAG AAGTCGAATC TGTCAGGGAC GAATTTCCTA ATTCCAAATT 1800 GTCCTCACTA AAGGCOTCT HAGTGTCTC TTGTATTTCC ATGTACCTTT GCTTCTHTG 1860 TAGTCGTTTC TCAGCAGTGT CGTCHCTCC 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 AGGCTCGAGG CTAGTCACTA TGAACTCACA 2220 TGTTCCCTTC ATTTCGGCGA TCTCCATTGC AGCTTGTGCT TCCGHGGAA AAAGACGTTG 2280 AGCAAGTGCA ACTAAACAGT GGACGACACA AAGAATAGH ATCATTAGTT CACTCAGTTT 2340 CCTAATAGAG AGGACATAAA TTTAATTCAA ACATATAAGA AATAAGACTT GATAGATACC 2400 TCTATTTTCA AGATCGAGCA GCGTCATCH CAATTCATCG GCCGCCACTG CAAAAGAGGG 2460 AGGAACATCT CTAGGAATTT GTTCTCG1TT GTCTTCnGC TCTAGTATT T CTACACATAG 2520 TCGGCCTTTG AGAGAATGCT TGCATTGCTC CGGGATATTA TTACATTCAA CCGCCATAGT 2580 GGCTTGTTTT GCGATCATGA GTGCGGTTCT ACCTTCCAAA GTTGCTTCTG ATGCACTTGC 2640 ACCTTTTTCC AATAGAGATA GTATCAAHG TGGCTCCTTC CGCATCGCAG CAACATGAAG 2700 CACCGTATAT CCCCTCGGAT TCCTATGGTT GACATCGGCA AGATCAAGTT TTAAAAGATC 2760 TGTTGCGGTC TTCACAnGC AATATGCAAC AGCGAAATGA AGAGCACACG CATCATCTAG 2820 1007779 128 ATTGGTGTGA TCCTCTTTCA AAAGCAACTT GACTAACTCA ATATCATCCG AGTCAAGTGC 2880 CTTATGTACA HCGAGACAT GTTTCTTTAC TTTAGGTACC TCCAAACCAA GCTCTTTACG 2940 TCTATCAATT ATCTCTTTAA CAAGCTCTTC CGGCAATGAC TTTTCAAGAC TAACCATATC 3000 TACATTAGAC TTGACAATAA TCTCTTTACA TCTATCCAAT AGCTTCATAC AAGCTTTACC 3060 ACATATATTA GCAAGCTTGA GTATAACCAA TGTGTCCTCT ATAACAACH TGTCTACAAC 3120 GTCCAATAAG TGCCTCTGAA ATACAAATAC AAGTACTCAA GTAAGAACAT ATTCATGAAT 3180 GTGTAACCAT AGCTTAATGC AGATGGTGTT TTACCTGATA GAGAGTAATT AATTCAGGGA 3240 TCTTGAAGAT GAAAGCCAAA TAGAGAACCT CCAACATGAA ATCCACCGCC GGCCGGCAAG 3300 CCACGTGGCA GCAAHCTCG TCTGCGCATT CAGAAACTCC TTTAGGCG GC GGTCTCACTC 3360 TGCTGCTGTA AACATAAGCC AAAACAGTCA CAACCGAATC GAAACCGACT TCGTAATCCT 3420 TGGCAATCTC CTTAAGCTCG AGCTTCACGG CGGCGGTGTT GTTGGAGTCT TTCTCCTTCT 3480 TAGCGGCGGC TAAAGCGCTC HGAAGAAAG AGCHCTCGC TGACAAAACG CACCGGTGGA 3540 AAGAAACTTC CCGGCCGTCG GAGAGAACAA GCTTAGCGTC GCTGTAGAAA TCATCCGGCG 3600 AGTCAAAGAC GGATTCGAAG CTGHGGAGA GCAATTGCAG AGCAGATACA TCAGGTCCGG 3660 TGAGTACTTG TTCGGCGGCC AGATAAACAA TAGAGGAGTC GGTGTTATCG GTAGCGACGA 3720 AACTAGTGCT GCTGATTTCA TAAGAATCGG CGAATCCATC AATGGTGGTG TCCATCAACA 3780 GGTTCCGATG AAHGAAAH CACAAAHAA AGAGATCTCT GCTAATCAAC GAAGAGACCT 3840 TATCAACTGG ATTTGGTTAA AGATCGAAGA TAACCATTGA CGAGCAGAGC CAAGTCAAGT 3900 CAACGAGAGT GGTGGTGAGA TATGAAGAAG CATCCTCGTC CCACGGTTTA CATTTCACCA 3960 AAACCGGTAA ATTTCCAGGA AAGGAATCH TGTCAGAGAT CTTTTnAAA AAGATATAAC 4020 AGGAAGCTAA ACCGGTTCGG GTTATAAATG TTAGTATTTA TACCGGAGAC ATTTTGTGTT 4080 GCTAATTTTT GTATATGAGA AGTTCAATCC GGTTCGGTAA GCCCCTGAAC CAAACTAGAT 4140 TTGGAGATGA TATAAATATA TAAAATTTAT TTTTCATCCG GTTCGTTATT TTCATATAAA4200 1 007779 129 ΤΑΤΑΤΑΑΑΤΑ ΤΤΑΓΤΤΊ1 IA AATTTAAGAA TTAGATTTAC ATGTGAAAGT TACATTTCTG 4260 mATTTTCT TTGAAGTAAA ATGATAAAGG GAACGTATAT TAAGTTTCAT GCTTTATTCA 4320 CATAAGTTTT GTAATGTATA πΑΤΑΤΤΤπ CGTTTATTGA AAAAGTAATT TTCAGTGTTC 4380 AGCATGTTTA CACTATAAH AAATCAAGTC GAATATTTCC TGGAACTATT CTCCHGITC 4440 TATAGCAAAT GAAAACGCTC TTCACAACAA AATCATTATA GATATAGGAA TAAATTACAT 4500 TAAAAACATG AAAGTCATAA TGAATATATT ΤΓΠΤΑΑΤΤΑ GGATTTGAn TAAAAACAAT 4560 TATTGTATAC ATATAAAAGA CTTCTTTAGT TATTTGCCn 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 AHATGHGA ATAAACAGAG TACTTTATAT AGGAGTTATA ATAAGGTAAA 4920 TAAATTATTG CTTTCCGCGT TTTTTACTTT TGTATTTCTT AAATGATAAG TTAAATTAGG 4980 ATAAGATTTG TATGATTTTA AGTAAATTTA CA ATAACTCT CTATAACTCA ATAGCATCAC 5040 ATATTTAATT AATTTTACTA ATTATCTTTT GAACAATTTT ATGAAATAGT ΤΤΤΟΤΓΤΑΑ 5100 πΑΑΤΤΤΤπ AAAATGATAT ΑΠΑΤΑΑΑΑΤ TTAATTGAAT CAATCTGATA ΤΑΑΙΤΪ1111 5160 ATCTTCTACC ATCTATTATA G1TGATAAAT AHGTGATAA ACTTTAGATA AACACCCAAT 5220 TGCCAAATAT ΤΤΑΑΤΑΑΑπ HGTGTACCA TGCGTTTTTT HGGAGAATA TATATACGTG 5280 GACAGCATAC CGTACATATA TTGTATAAAA GCTTATAAAA CATAGATACG GGTTATATTG 5340 GTAAGCTATA AATATATGTA AACAATAGTA AGATATTACG TGnGTGTCT AAATATGTGT 5400 TGCTITAGAT ATTATGTATA TCTAATATAT TAAAATATCT TTTATTAACT AATATATTAT 5460 TTAAGAGAGA AAAHGGGAC ACTATTTTCT ATACAGTAAC TGTTTTCAAC TATAAACAGG 5520 AACCCTTGAT ATAATAAAAT AACTAGCCAA AAAATCAGAT TAAATATTCA TAAAACAATG 5580 1007779 130 ITTGGTATTA TTACATAAAC CTAAGAAACA AAATTCAATA TTCCTTTTTA CCHATAAAA 5640 AACAAHAAA CATCACTAGA TATATTTATG CCCCACAATG AGCGAGCCAA TTGAGACTTG 5700 AGACTTGAGA TCCTTGTCAA CTACG1TTGC AITTGTCGGC (ΧΑΙΙΙΓΠΊ Taii Π I 11 I 5760 TTAAAGTGTC GGCCCGTTGC TTCÏÏCCGÏÏ CAGATCAACC CTCTCGTAAT C AGAACAAAA 5820 CGGAAAACAA ACGAAAGAAC AATCAGATCC CTCITTTIIT GCATAAACTA AATTCAACTT 5880 CTCTGCGTTT ATGTTGTAGA GGCAACCACG ATCACTACTA CGAAACAATA CAACGTCGH 5940 GCTTGGAGTC CACGTAATCA AATCTACTCC AATGCTTTTA ATATCTTTCA CTTTAACCCA 6000 CGACTTTTCA AAACTGCTCT HAAAACCCA TAACTCGTGA ACATCTTCTT GATC1TTGTT 6060 TGTCCACTGA CGAATAGCAC CTAGCTTCCC TTCGTATCTG ACTAATCCTG AGAAAACATC 6120 AGAGTTCGGA GTATGGAAGA AGGACCAAGT TTCGGHTTG 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 TTTGTATTrT ATACCGGAAA GGGTTCAATC CGATTACCGC 6420 TAAACCCAAT GACATATCCC AACCCTTCAC TTCTGGCTTT GGTATGACCT GATACTGTTT 6480 AGTGGTTGGT TTGAAGACTA TGTATCCACG TGATGGTTTT GTATACTTAA CACAAAGCAA 6540 TATCCCATGA CTTGCATCAC AAGCTTCGAT CITTATCATT CCGGGTGGCA GAAAGTCGAT 6600 GGAGACTCCA TTGTTTTGTA AATCACTCCT CTCATGGACA AAACTGGTTC GAAGTTCGTG 6660 TCCTTTTACT ATGTAGTGH GTATGAAGTA TCCCGAAATA CGAHGGTTC TAAGGAGATT 6720 AAGAHGACA 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 1 007 779 131 TCTTTGTTTA AGGAAAGTAT CAAAA1TTGC CTTmCTTC GCTAGTCCTA AAACAAACAA 7020 ATTAACCAAA AGATAAAATC TTTCATGATT AATGTTACTT GTGATACCTT AAGCCAAAAC 7080 TTTATCTTTA GACTTTTAAC CAAATCTACA GTAATTTAAT TGCTAGACTT AGGAAACAAC 7140 ill II11 ΠΙ ACCCAACAAT CTTTGGATrT TAATTGTTTT TTTTTCTACT AATAGATTAA 7200 CAACTCAHA TATAATAATG TTTCTATCAT AATTGACAAT TCTTTCTTrT TAATAAACAT 7260 CCAGCTTGTA TAATAATCCA CAAGTCAATT TCACCATTTT GGCCAATTTA TTTTCTTATA 7320 AAAAHAGCA CAAAAAAGAT TATCATTGTT TAGCAGATTT AATTTCTAAT TAACTTACGT 7380 AATTTCCATT TTCCATAGAT TTATCTTTCT TTTTATTTCC TTAGTTATCT TAGTACTTTC 7440 TTAGFICCT TAGTAATTH AAATTTTAAG ATAATATAT GAAATTAAAA GAAGAAAAA A 7500 AACTCTAGH ATACTTTTGT TAAATGTTTC ATCACACTAA CTAATAATTT TTTTTAGTTA 7560 AATTACAATA TATAAACACT GAAGAAAGTT TTTGGCCCAC ACTTTTTTGG 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 CClimGAT TATTTTATGC CCTACGTTGA GCCTTGTTGA 7920 CTAGTTTGCA TTTGTCGGTC CATTTCTTCT TCCGTCCAGA TCAACCCTCT CGTAATCAGA 7980 ACAAAAGGGG AAACAAACGT AAGAGGCAAA ATCCTTGTTT GTATGAACTA AGTTTAACH 8040 CTCTGTGTTT AAGTTGTAGA GGCAAACATG ATCCCAACTA GAAAGCATTA CGACGTCGTT 8100 GCTTGGTATC CACGTAATAT GCTCTACTCC AATGCTTTCA ATATCTTTCA CmTTCCCA 8160 CGACTTTTCA AAACTGCTCT TTAAAACCCA TAATCTGTGA ACATCTTCTT GATTGTTGTT 8220 TATCCAGTGA CGAATAACAC CTAGCTTCCC TTCGTAGCTG ACTAACTCTG GGAATAAACC 8280 AACGTTTGGA GTATGTAAGA AAGACCAAGT TTCGGTTTTG GGACATAACC GGATCACATT 8340 1 0 0 7 ^ 132 GTGGTTCCAT GATCTCCAAT GCAAGAACCC TGAAGOTGT ACCGGGTTTG AAAGAATTAG 8400 ACCGTCTGTT CTCGGTAGAC GCAAATTTTT TAATCTCTTC CACATAAACG AATCGGAATC 8460 AAAAACTTCG CACGCAAAAG TTCTGAGATT CCGAGTCATA CCAGGCGATT TCGAAAGCCT 8520 AAATATTTTA TACCGGAAAG GCTGCAATCC GGTTACCGTT AGACCTAATG ACTTATCACA 8580 ACTCCTCACT TrTGGGTTTG GTATGATCTG ATACTGUTT GTTGTTGGTT TGCAGACTAT 8640 GTATTCCGGT ATTGGTCTTG TATCATTATA ACAAAGCAAT ATCCCATGAC GTGCATCACA 8700 AGCITTGATC TTTACCTCTC CTTGTGGCAG AAAATCGATG GAGACTCCTT TGTTATCCAA 8760 ATCTCTCCTC TCATGGAAAA AACTGGTATC AAGTTTGTAT CCTCTTTCGT AGCGTTCTAG 8820 GAAGTATCCA ΘΑΟΑΤΑπθΤ TGGHCGATG GAGATTTAGG TTGACAAACC AAGACTCGTA 8880 GCTTCTCTTG TTGCACTCTT TATTGATGAG CCTCAAim CCGATTTCGG ACCCCCGAAG 8940 ATAAGAAAGA ACCTCTTGGA TCGTGTCCTG ATTTATCACC GGAGAACTCA TGATCUATT 9000 GGAAAAAAGA AAGAAAGAGA TGAGCACGAT CAGTGAATGA GATATATAGA AATCAGGATT 9060 GGTAGAGAAC CGACGATGAT GAATATACAA GTGTTTATAA GTATCACAAA nGCCTnTT 9120 CTTCGCTAGT CCCAAAACAA GCAAAHAAC 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 ATCCATITTC TTTAAAAATT AGCAAGTTTG TTCTTATATC 9480 ATCATTCAGC AGATTTCTTA ATTAAACTTA GTGATTTCCA TTTTGCACCT ATATGTTTCT 9540 CTTTCTTAGT TTAGTACTTT AAATTTTCAT ΑΤΑΤΑΤΑΑπ TATTAAAATT AAAAGTAAAA 9600 ACTCCAGTTT AACTTATGTT AAATGTTTCA TCACACTAAA AGAGCAHAA GTAATAAATA 9660

TlTTAGCrn ATGAAAAAAA ATATCAAATC ACTGAAGACA TTÏGTTGGCC TATACTCTAT 9720 1007779 133 TTTTTATTTG GCCAATTAGT AATAGACTAA TAGTAACTCA TATGATATCT CTCTAATTCT 9780 GGCGAAACGA ATATTCTGAT TCTAAAGATA GTAAAAATGA ATTTTGATGA AGGGAATACT 9840 ATTTCACACA CCTAGAAAGA GTAAGGTAGA AACCTTTTIT TTTTTGGTCA GATTCTTGTA 9900 TCAAGAAGTT CTCATCGAT 9919 (2) INFOR MATE FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: ( A) LENGTH: 5655 base pairs (B) TYPE: nucleic acid (C) STRENGTH: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA: DNA (genomic)

(iii) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(ix) SPECIAL ASPECT: (A) NAME / SIGN: exon (B) PLACE: 2787..3347 (D) FURTHER INFORMATION: / product = "1st exon of ΝΙΜΓ (ix) SPECIAL ASPECT: (A) NAME / SIGN: exon (B) LOCATION: 3427..4162 (D) FURTHER INFORMATION: / product = "2nd exon of ΝΙΜΓ '(ix) SPECIAL ASPECT: (A) NAME / SIGN: exon (B) LOCATION: 4271..4474 (D ) FURTHER INFORMATION: / product = "3rd exon of NIM1" (ix) SPECIAL ASPECT: (A) NAME / SIGN: exon (B) LOCATION: 4586..4866 (D) FURTHER INFORMATION: / product = "3rd exon of NIM1 "(ix) SPECIAL ASPECT:

(A) NAME / SIGN: CDS

(B) PLACE: merge (2787..3347, 3427..4162. 4271..4474.

1 O 07 134 7 7 q 4586..4866) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: .. TGTGATGCAA GTCATGGGAT ATTGCTTTGT GTTAAGTATA CAAAACCATC ACGTGGATAC 60 ATAGTCTTCA AACCAACCAC TAAACAGTAT CAGGTCATAC CAAAGCCAGA AGTGAAGGGT 120 TGGGATATGT CATTGGG1TT AGCGGTAATC GGA1TGAACC CTTTCCGGTA TAAAATACAA 180 AGGCTTTCGC AGTCTCGGCG TATGTGTATG TCTCGGGGTA TCTACCATTT GAATCACAGA 240 ACmTATGT GCGAAGTTTT CGATTCTGAT TCGTTTACCT GGAAGAGAH AGAAAATTTG 300 CGTCTACCAA AAACAGACAG ATTAATTTTT TCCAACCCGA TACAAGTTTC GGGGTTCTTG 360 CATTGGATAT CACGGAACAA CAATGTGATC CGGiTTTGTC TCAAAACCGA AACTTGGTCC 420 TTCTTCCATA CTCCGAACTC TGATGTTTTC TCAGGATTAG TCAGATACGA AGGGAAGCTA 480 GGTGCTATTC GTCAGTGGAC AAACAAAGAT CAAGAAGATG TTCACGAG17 ATGGGTTTTA 540 AAGAGCAGn TTGAAAAGTC GTGGGTTAAA GTGAAAGATA TTAAAAGCAT TGGAGTAGAT 600 TTGATTACGT GGACTCCAAG CAACGACGTT GTATTGTTTC GTAGTAGTGA TCGTGGTTGC 660 CTCTACAACA TAAACGCAGA GAAGTTGAAT TTAGTTTATG CAAAAAAAGA GGGATCTGAT 720 TGTTCTTTCG mGTTTTCC GTTTTGTTCT GATTACGAGA GGGTTGGCTCT GAACGGAAGA 780 CGGAGAGAGA AAAAAAAT AAAAAAAATG GGCCGACAAA TGCAAACGTA 840 GTTGACAAGG ATCTCAAGTC TCAAGTCTCA ATTGGCTCGC TCAHGTGGG GCATAAATAT 900 ATCTAGTGAT GTTTAATTGT TTTTTATAAG GTAAAAAGGA ATATTGAATT TTGTTTCTTA 960 GG1TTATGTA ATAATACCAA ACATTGTTTT ATGAATATTT AATCTGA1TT TTTGGCTAGT 1020 ΤΑΓΠΤΑΤΤΑ TATCAAGGGT TCCTGTTTAT AGTTGAAAAC AGTTACTGTA TAGAAAATAG 1080 TGTCCCAATT TTCTCTCTTA AATAATATAT TAGTTAATAA AAGATATTTT AATATAHAG 1140 ATATACATAA TATCTAAAGC AACACATATT TAGACACAAC ACGTAATATC TTACTATTGT 1200 HACATATAT TTATAGCTTA CCAATATAAC CCGTATCTAT GTTTTATAAG CTTTTATACA 1260 1007779 135 ATATATGTAC GGTATGCTGT CCACGTATAT ATATTCTCCA AAAAAAACGC ATGGTACACA 1320 AAATTTATTA AATATTTGGC AAHGGGTGT TTATCTAAAG TTTATCACAA TATTTATCAA 1380 CTATAATAGA TGGTAGAAGA TAAAAAAA1T ATATCAGATT GATTCAAHA ΑΑΤΤΓΤΑΤΑΑ 1440 ΤΑΤΑΤΟΑΠΤ TAAAAAATTA AHAAAAGAA AACTATTTCA TAAAATTGTT CAAAAGATAA 1500 HAGTAAAAT TAAHAAATA TGTGATGCTA HGAGHATA GAGAGTTATT GTAAAITTAC 1560 TTAAAATCAT ACAAATCTTA TCCTAATTTA ΑΟπΑΤΟΑΠ TAAGAAATAC AAAAGTAAAA 1620 AACGCGGAAA GCAATAATTT ATTTACCTTA TTATAACTCC TATATAAAGT ACTCTGTTTA 1680 TTCAACATAA TCTTACGTTG TTGTATTCAT AGGCATCTTT AACCTATCTT TTCATTTTCT 1740 GATCTCGATC GTTTTCGATC CAACAAAATG AGTCTACCGG TGAGGAACCA AGAGGTGATT 1800 ATGCAGATTC CTTCTTCTTC TCAG1TTCCA GCAACATCGA GTCCGGAAAA CACCAATCAA 1860 GTGAAGGATG AGCCAAATTT GTTTAGACGT GTTATGAATT TGCTTTTACG TCGTAGTTAT 1920 TGAAAAAGCT GATTTATCGC ATGATTCAGA ACGAGAAGTT GAAGGCAAAT AACTAAAGAA 1980 GTCTTTTATA TGTATACAAT AATTGTTTTT AAATCAAATC CTAATTAAAA AAATATATTC 2040 ATTATGACTT TCATGTTTTT AATGTAA1TT ATTCCTATAT CTATAATGAT TTTGTTGTGA 2100 AGAGCGTTTT CATTTGCTAT AGAACAAGGA GAATAGTTCC AGGAAATATT CGACTTGATT 2160 TAAHATAGT GTAAACATGC TGAACACTGA AAATTACTTT TTCAATAAAC GAAAAATATA 2220 ATATACATTA CAAAACTTAT GTGAATAAAG CATGAAACTT AATATACGTT CCC1TTATCA 2280 TTTTACTTCA AAGAAAATAA ACAGAAATGT AACTTTCACA TGTAAATCTA ATTCnAAAT 2340 TTAAAAAATA ATATTTATAT ATTTATATGA AAATAACGAA CCGGATGAAA AATAAATTTT 2400 ΑΤΑΤΑΠΤΑΤ ATCATCTCCA AATCTAGTTT GGTTCAGGGG CTTACCGAAC CGGATTGAAC 2460 TTCTCATATA CAAAAATTAG CAACACAAAA TGTCTCCGGT ATAAATACTA ACAHTATAA 2520 CCCGAACCGG TTTAGCTTCC TGTTATATCT TTTTAAAAAA GATCTCTGAC AAAGATTCCT 2580 TTCCTGGAAA TTTACCGGTT TTGGTGAAAT GTAAACCGTG GGACGAGGAT GCTTCTTCAT 2640 1 007 779 136 ATCTCACCAC CACTCTCGTT GACTTGACTT GGCTCTGCTC GTCAATGGTT ATCTTCGATC 2700 TTTAACCAAA TCCAGTTGAT AAGGTCTCTT CGTTGATTAG CAGAGATCTC mAATTTGT 2760 GAATTTCAAT TCATCGGAAC CTGTTG ATG GAC ACC ACC ATT GAT GGA TTC GCC 2813

With Asp Thr Thr lie Asp Gly Phe Ala 1 5 GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC GTC GCT ACC GAT AAC ACC 2861

Asp Ser Tyr Glu lie Ser Ser Thr Ser Phe 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 He Val Tyr Leu Ala Ala Glu Gin Val Leu Thr Gly Pro 30 35 40 GAT GTA TCT GCT CTG CAA TTG CTC TCC AAC AGC TTC GAA TCC GTC TTT 2957

Asp Val Ser Ala Leu Gin Leu Leu Ser Asn Ser Phe Glu Ser Val Phe 45 50 55 GAC TCG CCG GAT GAT TTC TAC AGC GAC GCT AAG CTT GTT CTC TCC GAC 3005

Asp Ser Pro Asp Asp Phe Tyr Ser Asp Ala Lys Leu Val Leu Ser Asp 60 65 70 GGC CGG GAA GTT Tπ πC CAC CGG TGC GTT TTG TCA GCG AGA AGC TCT 3053

Gly Arg Glu Val Ser Phe His Arg Cys Val Leu Ser Ala Arg Ser Ser 75 80 85 TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAG GAG AAA GAC TCC AAC 3101

Phe Phe Lys Ser Ala Leu Ala Ala Ala Lys Lys Glu Lys Asp Ser Asn 90 95 100 105 AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG ATT GCC AAG GAT TAC 3149

Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu He Ala Lys Asp Tyr 110 115 120 GAA GTC GGT TTC GAT TCG GTT GTG ACT GTT TTG GCT TAT GTT TAC AGC 3197

Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser 125 130 135 AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT GAA TGC GCA GAC GAG 3245

Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu 140 145 150 AAT TGC TGC CAC GTG GCT TGC CGG 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 1 007779 137 155 160 165 GTT CTC TAT TTG GCT TTC ATC TTC AAG ATC CCT GAA TTA ATT ACT CTC 3341

Val Leu Tyr Leu Ala Phe lie Phe Lys lie Pro Glu Leu He Thr Leu 170 175 180 185 TAT CAG GTAAAACACC ATCTGCATTA AGCTATGGTT ACACATTCAT GAATATGTTC 3397

Tyr Gin TTACTTGAGT ACTTGTAHT GTATTTCAG AGG CAC TTA JIG GAC GTT GTA GAC 3450

Arg His Leu Leu Asp Val Val Asp 190 195 AAA GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA 3498

Lys Val Val He Glu Asp Thr Leu Val He Leu Lys Leu Ala Asn He 200 205 210 TGT GGT AAA GCT TGT ATG AAG CTA TTG GAT AGA TGT AAA GAG ATT ATT 3546

Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu lie He 215 220 225 GTC AAG TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA 3594

Val Lys Ser Asn Val Asp With Val Ser Leu Glu Lys Ser Leu Pro Glu 230 235 240 GAG CTT GTT AAA GAG ATA ATT GAT AGA CGT AAA GAG CTT GGT TTG GAG 3642

Glu Leu Val Lys Glu He He 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 AAG TTG CTT TTG AAA GAG GAT CAC ACC 3738

Ser Asp Asp He Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr 280 285 290 AAT CTA GAT GAT GCG TGT 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 AAG ACC GCA ACA GAT CTT TTA AAA CTT GAT CTT GCC GAT GTC AAC 3834

Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn 310 315 320 1 007779 138 CAT A6G AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG 3882

His Arg Asn Pro Arg Gly Tyr 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 GCA AGT GCA 3930

Lys Glu Pro Gin Leu lie Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala 340 345 350 355 TCA GAA GCA 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 lie Ala Lys Gin 360 365 370 GCC ACT ATG GCG GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT 4026

Ala Thr Met Ala Val Glu Cys Asn Asn lie Pro Glu Gin Cys Lys His 375 380 385 TCT CTC AAA GGC CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA 4074

Ser Leu Lys Gly Arg Leu Cys Val Glu He Leu Glu Gin Glu Asp Lys 390 395 400 CGA GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TTT GCA GTG GCG GCC 4122

Arg Glu Gin He Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala 405 410 415 GAT GAA TTG AAG ATG ACG CTG CTC GAT CTT GAA AAT AGA G 4162

Asp Glu Leu Lys With Thr Leu Leu Asp Leu Glu Asn Arg 420 425 430 GTATCTATCA AGTCTTATTT CTTATATGTT TGAATTAAT TTATGTCCTC TCTaTTAGGA 4222 AACTGAGTGA ACATATATA ACTATTCTTT GTGTCGTCCA CTGTTTAG π GCA CTCA

Val Ala Leu 435 GCT CAA CGT CTT ITT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC 4326

Ala Gin Arg Leu Phe Pro Thr Glu Ala Gin Ala Ala Met Glu He Ala 440 445 450 GAA ATG AAG GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC 4374

Glu Met Lys Gly Thr Cys Glu Phe He Val Thr Ser Leu Glu Pro Asp 455 460 465 CGT CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAG ATA GCA CCT 4422

Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys He Ala Pro 470 475 480 1007779 139 TTC AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA 4470

Phe Arg lie Leu Glu Glu His Gin 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 TG GAA CTC GGG AAA CGA TTC TTC CCG CGC TGT TCG GCA GTG CTC 4629

Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu 505 510 515 GAC CAG ATT ATG AAC TGT GAG GAC TTG ACT CAA CTG GCT TGC GGA GAA 4677

Asp Gin lie Met Asn Cys Glu Asp Leu Thr Gin Leu Ala Cys Gly Glu 520 525 530 GAC GAC ACT GCT GAG AAA CGA CTA CAA AAG AAG CAA AGG TAC ATG GAA 4725

Asp Asp Thr Ala Glu Lys Arg Leu Gin Lys Lys Gin Arg Tyr Met Glu 535 540 545 ATA CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GAA TTA 4773 lie Gin Glu Thr Leu Lys Ala Phe Ser Glu Asp Asn Leu Glu Leu 550 555 560 GGA AAT TCG TCC CTG ACA GAT TCG ACT TCT TCC ACA TCG AAA TCA ACC 4821

Gly Asn Ser Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser Lys Ser Thr 565 570 575 GGT GGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT CGT CGG TGA 4866

Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg Arg * 580 585 590 GACTCTTGCC TCTTAGTGTA ATTTTTGCTG TACCATATAA TTCTGTTTTC ATGATGACTG 4926 TAACTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT 4986 ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG 5046 ATTTGTAATA TATATTTATG TACATCAACA ATAACCCATG ATGGTGTTAC AGAGTTGCTA 5106 GAATCAAAGT GTGAAATAAT GTCAAATTGT TCATCTGTTG GATATTTTCC ACCAAGAACC 5166 AAAAGAATAT TCAAGTTCCC TGAACTTCTG GCAACATTCA TGTTATATGT ATCTTCCTAA 5226 1007 '' '' 140 TTCTTCCTTT AACCTTTTGT AACTCGMTT ACACAGCMG 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 TTCCTTCTG A 5646 CTTGTGGAT 5655 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE FEATURES: (A) LENGTH: 594 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION : SEQ ID N0: 3:

With Asp Thr Thr IIe Asp Gly Phe Al a Asp Ser Tyr Glu IIe Ser Ser 15 10 15

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

Al a Ala Glu Gin Val Leu Thr Gly Pro Asp Val Ser Al a Leu Gin Leu 35 40 45

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

Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His 65 70 75 80

Arq Cys Val Leu Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Al a 85 90 95 1007779 141

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

Glu Leu Lys Glu lie Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val 115 120 125

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

Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys 145 150 155 160

Arg Pro Ala Val Asp Phe With Leu Glu Val Leu Tyr Leu Ala Phe lie 165 170 175

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

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

Ala Asn He Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys 210 215 220

Glu He He Val Lys Ser Asn Val Asp With Val Ser Leu Glu Lys Ser 225 230 235 240

Leu Pro Glu Glu Leu Val Lys Glu He lie Asp Arg Arg Lys Glu Leu 245 250 255

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

Ala Leu Asp Ser Asp Asp He Glu Leu Val 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 Asp Leu Ala 305 310 315 320

Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala 325 330 335

Ala Met Arg Lys Glu Pro Gin Leu He Leu Ser Leu Leu Glu Lys Gly 1007779 142 340 345 350

Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met He 355 360 365

Ala Lys Gin Ala Thr With Ala Val Glu Cys Asn Asn lie Pro Glu Gin 370 375 380

Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu He Leu Glu Gin 385 390 395 400

Glu Asp Lys Arg Glu Gin He Pro Arg Asp Val Pro Pro Ser Phe Ala 405 410 415

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

Val Ala Leu Ala Gin Arg Leu Phe Pro Thr Glu Ala Gin Ala Ala With 435 440 445

Glu He Ala Glu With Lys Gly Thr Cys Glu Phe He Val Thr Ser Leu 450 455 460

Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys 465 470 475 480

He Ala Pro Phe Arg He Leu Glu Glu His Gin Ser Arg Leu Lys Ala 485 490 495

Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser 500 505 510

Ala Val Leu Asp Gin He Met Asn Cys Glu Asp Leu Thr Gin Leu Ala 515 520 525

Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gin Lys Lys Gin Arg 530 535 540

Tyr Met Glu He Gin Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn 545 550 555 560

Leu Glu Leu Gly Asn Ser Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser 565 570 575

Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg 580 585 590 1007779

Arg * 143 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 amino acids (B) TYPE: amino acid (C) STRENGTH: not relevant (D) TOPOLOGY: not relevant (ii) TYPE MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4: I1e Arg Arg With Arg Arg Ala Leu Asp Ala Ala Asp I1e Glu Leu Val 15 10 15

Lys Leu Met Val With Gly Glu Gly Leu Asp Leu Asp Asp Al a Leu Al a 20 25 30

Val His Tyr Ala Val Gin His Cys Asn 35 40 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 amino acids (B) TYPE: amino acid (C) STRING: not relevant (D) TOPOLOGY: not relevant (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Pro Thr Gly Lys Thr Ala Leu His Leu Al a Ala Glu Met Val Ser Pro 15 10 15 1007779 144

Asp With Vól Ser Val Leu Leu Asp His His Ala Asp Xaa Asn Phe Arg 20 25 30

Thr Xaa Asp Gly Val Thr 35 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE FEATURES: (A) LENGTH: 41 amino acids (B) TYPE: amino acid (C) STRENGTH: not relevant (D) TOPOLOGY: not relevant (li) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: lie Arg Arg With Arg Arg Ala Leu Asp Ala Ala Asp lie Glu Leu Val 15 10 15

Lys Leu With Val With Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala 20 25 30

Val His Tyr Ala Val Gin His Cys Asn 35 40 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 amino acids (B) TYPE: amino acid (C) STRICT: not relevant (D) TOPOLOGY: not relevant (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Arg Arg Pro Asp Ser Lys Thr Ala Leu His Leu Ala Ala Glu Met Val 1007779 145 15 10 15

Ser Pro Asp With Val Ser Val Leu Leu Asp Gin 20 25 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 amino acids (B) TYPE: amino acid (C) STRICT: not relevant ( D) TOPOLOGY: not relevant (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: lie Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp lie Glu Leu Val 15 10 15

Lys Leu With Val With Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala 20 25 30

Val His Tyr Ala Val Gin His Cys Asn 35 40 (2) INFORMATION FOR SEQ ID N0: 9: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 amino acids (B) TYPE: amino acid (C) STRING: not relevant (D) TOPOLOGY: not relevant (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Arg Arg Pro Asp Ser Lys Thr Ala Leu His Leu Ala Ala Glu Met Val 15 10 15 1007779 146

Ser Pro Asp Met Val Ser Val Leu Leu Asp Gin 20 25 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 amino acids (B) TYPE: amino acid (C) STRICT: not relevant ( D) TOPOLOGY: not relevant (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: lie Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp lie Glu Leu Val 15 10 15

Lys Leu With Val With Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala 20 25 30

Val His Tyr Ala Val Gin His Cys Asn 35 40 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) STRING: not relevant (D) TOPOLOGY: not relevant (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Pro Thr Gly Lys Thr Ala Leu His Leu Ala Ala Glu Met Val Ser Pro 15 10 15

Asp With Val 1007779 147 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE FEATURES: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRENGTH: single (D) TOPOLOGY: linear (ii ) TYPE OF MOLECULE: Other Nucleic Acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: ATTATE COAT CATGCCGATC GG 22 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE FEATURES: (A ) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRENGTH: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: AATTCCGATC GGCATGCTTT A 21 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRING: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid 1007779 148 (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 14: ATATTATAAC CATGGCGATC GG 22 (2) INFORMATION FOR SEQ ID NO: 15: (i) SE QUENTITY CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRING: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION : SEQ ID NO: 15: AATTCCGATC GCCATGGTTT A 21 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRING: single (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 16: CCAGCTGGAA TTCCG 15 (2) INFORMATION FOR SEQ ID NO: 17: 1 007779 149 ( i) SEQUENCE CHARACTERISTICS; (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRENGTH: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CGGAATTCCA GCTGGCATG 19 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 314 amino acids (B) TYPE: amino acid (C) STRICT: not relevant (D ) TOPOLOGY: not relevant (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

With Phe Gin Pro Ala Gly His Gly Gin Asp Trp Ala With Glu Gly Pro 15 10 15

Arg Asp Gly Leu Lys Lys Glu Arg Leu Val Asp Asp Arg His Asp Ser 20 25 30

Gly Leu Asp Ser With Lys Asp Glu Glu Tyr Glu Gin With Val Lys Glu 35 40 45

Leu Arg Glu He Arg Leu Gin Pro Gin Glu Ala Pro Leu Ala Ala Glu 50 55 60

Pro Trp Lys Gin Gin Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu 65 70 75 80 1 007 779 150

Ala lie lie His Glu Glu Lys Pro Leu Thr Met Glu Val lie Gly Gin 85 90 95

Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gin Asn Asn Leu Gin Gin 100 105 110

Thr Pro Leu His Leu Ala Val He Thr Asn Gin Pro Gly He 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 Gin Gly Cys Leu Ala Ser Val 145 150 155 160

Ala Val Leu Thr Gin Thr Cys Thr Pro Gin His Leu His Ser Val Leu 165 170 175

Gin Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser Thr 180 185 190

His Gly Tyr Leu Ala He Val Glu His Leu Val Thr Leu Gly Ala Asp 195 200 205

Val Asn Ala Gin Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala 210 215 220

Val Asp Leu Gin Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly 225 230 235 240

Ala Asp Val Asn Arg Val Thr Tyr Gin Gly Tyr Ser Pro Tyr Gin Leu 245 250 255

Thr Trp Gly Arg Pro Ser Thr Arg He Gin Gin Gin Leu Gly Gin Leu 260 265 270

Thr Leu Glu Asn Leu Gin With Leu Pro Glu Ser Glu Asp Glu Glu Ser 275 280 285

Tyr Asp Thr Glu Ser Glu Phe Thr Glu Asp Glu Leu Pro Tyr Asp Asp 290 295 300

Cys Val Phe Gly Gly Gin Arg Leu Thr Leu 305 310 1007779 151 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 314 amino acids (B) TYPE: amino acid (C) STRICT: not relevant (D) TOPOLOGY: not relevant (i1) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

With Phe Gin Pro Ala Gly His Gly Gin Asp Trp Ala With Glu Gly Pro 15 10 15

Arg Asp Gly Leu Lys Lys Glu Arg Leu Val Asp Asp Arg His Asp Ser 20 25 30

Gly Leu Asp Ser With Lys Asp Glu Asp Tyr Glu Gin With Val Lys Glu 35 40 45

Leu Arg Glu IIe Arg Leu Gin Pro Gin Glu Al a Pro Leu Al a Ala Glu 50 55 60

Pro Trp Lys Gin Gin Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu 65 70 75 80

Ala I1e I1e His Glu Glu Lys Thr Leu Thr Met Glu Val Ile Gly Gin 85 90 95

Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gin Asn Asn Leu Gin Gin 100 105 110

Thr Pro Leu His Leu Ala Val lie Thr Asn Gin Pro Gly Ile 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 Gin Gly Cys Leu Ala Ser Val 145 150 155 160

Ala Val Leu Thr Gin Thr Cys Thr Pro Gin His Leu His Ser Val Leu 1007779 165 170 175 152

Gin Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser lie 180 185 190

His Gly Tyr Leu Gly lie Val Glu His Leu Val Thr Leu Gly Ala Asp 195 200 205

Val Asn Ala Gin Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala 210 215 220

Val Asp Leu Gin Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly 225 230 235 240

Ala Asp Val Asn Arg Val Thr Tyr Gin Gly Tyr Ser Pro Tyr Gin Leu 245 250 255

Thr Trp Gly Arg Pro Ser Thr Arg lie Gin Gin Gin Leu Gly Gin Leu 260 265 270

Thr Leu Glu Asn Leu Gin Thr Leu Pro Glu Ser Glu Asp Glu Glu Ser 275 280 285

Tyr Asp Thr Glu Ser Glu Phe Thr Glu Asp Glu Leu Pro Tyr Asp Asp 290 295 300

Cys Val Phe Gly Gly Gin Arg Leu Thr Leu 305 310 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 314 amino acids (B) TYPE: amino acid (C) STRING: not relevant (D ) TOPOLOGY: not relevant (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

With Phe Gin Pro Ala Glu Pro Gly Gin Glu Trp Ala With Glu Gly Pro 1 0 07779 153 15 10 15

Arg Asp Ala Leu Lys Lys Glu Arg Leu Leu Asp Asp Arg Arg His Asp Ser 20 25 30

Gly Leu Asp Ser With Lys Asp Glu Glu Tyr Glu Gin With Val Lys Glu 35 40 45

Leu Arg Glu lie Arg Leu Glu Pro Gin Glu Ala Pro Arg Gly Ala Glu 50 55 60

Pro Trp Lys Gin Gin Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu 65 70 75 80

Ala lie He His Glu Glu Lys Ala Leu Thr Met Glu Val Val Arg Gin 85 90 95

Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gin Asn Asn Leu Gin Gin 100 105 110

Thr Pro Leu His Leu Ala Val He Thr Asn Gin Pro Glu He 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 Gin Gly Cys Leu Ala Ser Val 145 150 155 160

Gly Val Leu Thr Gin Pro Arg Gly Thr Gin His Leu His Ser He Leu 165 170 175

Gin Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser He 180 185 190

His Gly Tyr Leu Gly He Val Glu Leu Leu Val Ser Leu Gly Ala Asp 195 200 205

Val Asn Ala Gin Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala 210 215 220

Val Asp Leu Gin Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly 225 230 235 240

Ala Asp Val Asn Arg Val Thr Tyr Gin Gly Tyr Ser Pro Tyr Gin Leu 245 250 255 1007779 154

Thr Trp Gly Arg Pro Ser Thr Arg He Gin Gin Gin Leu Gly Gin Leu 260 265 270

Thr Leu Glu Asn Leu Gin With Leu Pro Glu Ser Glu Asp Glu Glu Ser 275 280 285

Tyr Asp Thr Glu Ser Glu Phe Thr Glu Asp Glu Leu Pro Tyr Asp Asp 290 295 300

Cys Val Leu Gly Gly Gin Arg Leu Thr Leu 305 310 (2) INFORMATION FOR SEQ ID N0: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2011 base pairs (B) TYPE: nucleic acid (C) STRING: single (D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: cDNA

(vi) ORIGINAL SOURCE: (A) ORGANISM: Arabidopsis thaliana (ix) SPECIAL ASPECT: (A) NAME / SIGN: various aspects (B) LOCATION: 1..2011 (D) FURTHER INFORMATION: / note = "NIM1 cDNA sequence "(ix) SPECIAL ASPECT:

(A) NAME / SIGN: CDS

(B) LOCATION: 43..1824 (D) FURTHER INFORMATION: / product = "NIM1 protein" (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 21: GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC 54

With Asp Thr Thr 1 ATT GAT GGA TTC GCC GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC GTC 102

He Asp Gly Phe Ala Asp Ser Tyr Glu lie Ser Ser Thr Ser Phe Val 5 10 15 20 1 007 779 155 GCT ACC GAT AAC ACC GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA 150

Ala Thr Asp Asn Thr Asp Ser Ser He Val Tyr Leu Ala Ala Glu Gin 25 30 35 GTA CTC ACC GGA CCT GAT GTA TCT GCT CTG CAA TTG CTC TCC AAC AGC 198

Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gin Leu Leu Ser Asn Ser 40 45 50 TTC GAA TCC GTC ITT GAC TCG CCG GAT GAT TTC TAC AGC GAC GCT AAG 246

Phe Glu Ser Val Phe Asp Ser Pro Asp Asp Phe Tyr Ser Asp Ala Lys 55 60 65 CTT GTT CTC TCC GAC GGC CGG GAA GTT TCT TTC CAC CGG TGC GTT TTG 294

Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His Arg Cys Val Leu 70 75 80 TCA GCG AGA AGC TCT TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAG 342

Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala Ala Ala Lys Lys 85 90 95 100 GAG AAA GAC TCC AAC AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG 390

Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu 105 110 115 ATT GCC AAG GAT TAC GAA GTC GGT TTC GAT TCG GTT GTG ACT GTT TTG 438

He Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu 120 125 130 GCT TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT 486

Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Lys Gly Val Ser 135 140 145 GAA TGC GCA GAC GAG ATAT TGC TGC CAC GTG GCT TGC CGG CCG GCG 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 TTG GCT TTC ATC TTC AAG ATC CCT 582

Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe He Phe Lys lie Pro 165 170 175 180 GAA TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA 630

Glu Leu He Thr Leu Tyr Gin Arg His Leu Leu Asp Val Val Asp Lys 185 190 195 GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT 678

Val Val He Glu Asp Thr Leu Val He Leu Lys Leu Ala Asn He Cys 1 007 779 156 200 205 210 GGT AM GCT TGT ATG MG CTA TTG GAT AGA TGT AM GAG ATT ATT GTC 726

Gly Lys Ala Cys With Lys Leu Leu Asp Arg Cys Lys Glu lie lie Val 215 220 225 MG TCT MT GTA GAT ATG GTT AGT CTT GM MG TCA TTG CCG GM GAG 774

Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu 230 235 240 CTT GTT AM GAG ATA ATT GAT AGA CGT AM GAG CTT GGT TTG GAG GTA 822

Leu Val Lys Glu He lie Asp Arg Arg Lys Glu Leu Gly Leu Glu Val 245 250 255 260 CCT AM GTA MG AM CAT GTC TCG MT GTA CAT MG 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 GTC MG TTG CTT TTG AM GAG GAT CAC ACC MT 918

Asp Asp lie Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn 280 285 290 CTA GAT GAT GCG TGT GCT CH CAT TTC GCT GTT GCA TAT TGC MT GTG 966

Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val 295 300 305 MG ACC GCA ACA GAT CTT TTA AM CTT GAT CTT GCC GAT GTC MC CAT 1014

Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His 310 315 320 AGG MT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG MG 1062

Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys 325 330 335 340 GAG CCA CM TTG ATA CTA TCT CTA TTG GM AM GGT GCA AGT GCA TCA 1110

Glu Pro Gin Leu He Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser 345 350 355 GM GCA ACT TTG GM GGT AGA ACC GCA CTC ATG ATC GCA AM CM GCC 1158

Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met He Ala Lys Gin Ala 360 365 370 ACT ATG GCG GTT GM TGT MT MT ATC CCG GAG CM TGC MG CAT TCT 1206

Thr Met Ala Val Glu Cys Asn Asn He Pro Glu Gin Cys Lys His Ser 375 380 385 1007 779 157 CTC AAA G6C CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA CGA 1254

Leu Lys Gly Arg Leu Cys Val Glu He Leu Glu Gin Glu Asp Lys Arg 390 395 400 GAA CAA ΑΠ CCT AGA GAT GTT CCT CCC TCT TTT GCA GTG GCG GCC GAT 1302

Glu Gin lie Pro Arg Asp Val Pro Pro Ser Phe Ala Val 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 With Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala 425 430 435 CAA CGT CTT TTT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC GAA 1398

Gin Arg Leu Phe Pro Thr Glu Ala Gin Ala Ala Met Glu lie Ala Glu 440 445 450 ATG AAG GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC CGT 1446

With Lys Gly Thr Cys Glu Phe He Val Thr Ser Leu Glu Pro Asp Arg 455 460 465 CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAG ATA GCA CCT TTC 1494

Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys He Ala Pro Phe 470 475 480 AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA ACC 1542

Arg He Leu Glu Glu His Gin Ser Arg Leu Lys Ala Leu Ser Lys Thr 485 490 495 500 GTG GAA CTC GGG AAA CGA TTC TTC CCG CGC TGT TCG GCA GTG CTC GAC 1590

Val Glu Leu Gly Lys Arg Phe Phe Pro Arg 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

Gin He Met Asn Cys Glu Asp Leu Thr Gin Leu Ala Cys Gly Glu Asp 520 525 530 GAC ACT GCT GAG AAA CGA CTA CAA AAG AAG CAA AGG TAC ATG GAA ATA 1686

Asp Thr Ala Glu Lys Arg Leu Gin Lys Lys Gin Arg Tyr Met Glu He 535 540 545 CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTC GAA TTA GGA 1734

Gin Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn Leu Glu Leu Gly 550 555 560 AAT TTC TCC CTG ACA GAT TCG ACT TCT TCC ACA TCG AAA TCA ACC GGT 1782

Asn Leu Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser Lys Ser Thr Gly 1 0 07 779 158 565 570 575 580 GGA MG AGG TCT MC CGT AM 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 TACCATATM TTCTGTTTTC ATGATGACTG 1884 TMCTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT 1944 ATTATTGCTG CAGGTGTGCT TCAMCAMT GTTGTMCM TTTGMCCM TGGTATACAG 2004 ATTTGTA 2011 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2011 base pairs (B) TYPE: nucleic acid (C) STRENGTH: single (D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: cDNA

(ix) SPECIAL ASPECT:

(A) NMM / SIGN: CDS

(B) PLMTS: 43..1824 (D) FURTHER INFORMATION: / product = "modified form of ΝΙΜΓ / note =" Serine residues at amino acid positions 55 and 59 in the wild-type NIM1 gene product have been changed to alanine residues. "( ix) SPECIAL ASPECT: (A) NMM / SIGN: various_aspects (B) PLMTS: 205..217 (D) FURTHER INFORMATION: / note = "nucleotides 205 and 217 changed from T's to G's compared to the wild-type sequence." (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: GATCTCTTTA ATTTGTGMT TTCMTTCAT CGGMCCTGT TG ATG GAC ACC ACC 54

Met Asp Thr Thr 1 1 007779 159 Απ GAT GGA TTC GCC GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC GTC 102 lie Asp Gly Phe Al a Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val 5 10 15 20 GCT ACC GAT AAC ACC GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA 150

Al a Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu Al a Ala Glu Gin 25 30 35 GTA CTC ACC GGA CCT GAT GTA TCT GCT CTG CAA TTG CTC TCC AAC AGC 198

Val Leu Thr Gly Pro Asp Val Ser Al a Leu Gin Leu Leu Ser Asn Ser 40 45 50 TTC GAA GCC GTC TTT GAC GCG CCG GAT GAT TTC TAC AGC GAC GCT AAG 246

Phe Glu Ala Val Phe Asp Al a Pro Asp Asp Phe Tyr Ser Asp Ala Lys 55 60 65 CTT GTT CTC TCC GAC GGC CGG GAA GTT TCT TTC CAC CGG TGC GTT TTG 294

Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His Arg Cys Val Leu 70 75 80 TCA GCG AGA AGC TCT TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAG 342

Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Al a Ala Ala Lys Lys 85 90 95 100 GAG AAA GAC TCC AAC AAC ACC GCC GCC GTG AAG CTC GAG CU AAG GAG 390

Glu Lys Asp Ser Asn Asn Thr Al a Ala Val Lys Leu Glu Leu Lys Glu 105 110 115 ΑΠ GCC AAG GAT TAC GAA GTC GGT TIC GAT TCG GH GTG ACT GH TTG 438

Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu 120 125 130 GCT TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA GH TCT 486

Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Lys Gly Val Ser 135 140 145 GAA TGC GCA GAC GAG ATAT TGC TGC CAC GTG GCT TGC CGG CCG GCG GTG 534

Glu Cys Al a Asp Glu Asn Cys Cys His Val Al a Cys Arg Pro Ala Val 150 155 160 GAT HC ATG TTG GAG GH CTC TAT TTG GCT TTC ATC TTC AAG ATC CCT 582

Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe lie Phe Lys lie Pro 165 170 175 180 GAA ACT Απ ACT CTC TAT CAG AGG CAC πΑ TTG GAC GTT GTA GAC AAA 630

Glu Leu Ile Thr Leu Tyr Gin Arg His Leu Leu Asp Val Val Asp Lys 1 00? 77q 185 190 195 160 Gπ GIT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT 678

Val Val lie Glu Asp Thr Leu Val I1e Leu Lys Leu Ala Asn IIe Cys 200 205 210 GGT AAA GCT TGT ATG AAG CTA TTG GAT AGA TGT AAA GAG ATT ATT GTC 726

Gly Lys Al a Cys Met Lys Leu Leu Asp Arg Cys Lys Glu 11th 11th Val 215 220 225 AAG 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 AGA CGT AAA GAG CTT GGT TTG GAG GTA 822

Leu Val Lys Glu lie I1e Asp Arg Arg Lys Glu Leu Gly Leu Glu Val 245 250 255 260 CCT AAA GTA AAG AAA CAT GTC TCG 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 HOLE GAT ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG GAT CAC ACC AAT 918

Asp Asp IIe Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn 280 285 290 CTA GAT GAT GCG TGT GCT CTT CAT TTC GCT GTT GCA 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 Al a 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 Tyr Thr Val Leu His Val Al a 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 Gin Leu IIe Leu Ser Leu Leu Glu Lys Gly Ala 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 Al a Leu Met IIe Ala Lys Gin Al a 360 365 370 1007779 161 ACT ATG GCG GTT GAA TGT AAT ATAT CCG GAG CAA TGC AAG CAT TCT 1206

Thr Met Ala Val Glu Cys Asn Asn lie Pro Glu Gin 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 lie Leu Glu Gin Glu Asp Lys Arg 390 395 400 GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TTT GCA GTG GCG GCC GAT 1302

Glu Gin He Pro Arg Asp Val Pro Pro Ser Phe Ala Val 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 With Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala 425 430 435 CAA CGT CTT ΉΤ CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC GAA 1398

Gin Arg Leu Phe Pro Thr Glu Ala Gin Ala Ala Met Glu He Ala Glu 440 445 450 ATG AAG GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC CGT 1446

With Lys Gly Thr Cys Glu Phe He Val Thr Ser Leu Glu Pro Asp Arg 455 460 465 CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAG ATA GCA CCT TTC 1494

Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys He Ala Pro Phe 470 475 480 AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA ACC 1542

Arg lie Leu Glu Glu His Gin Ser Arg Leu Lys Ala Leu Ser Lys Thr 485 490 495 500 GTG GAA CTC GGG AAA CGA TTC TTC CCG CGC TGT TCG GCA GTG CTC GAC 1590

Val Glu Leu Gly Lys Arg Phe Phe Pro Arg 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

Gin lie Met Asn Cys Glu Asp Leu Thr Gin Leu Ala Cys Gly Glu Asp 520 525 530 GAC ACT GCT GAG AAA CGA CTA CAA AAG AAG CAA AGG TAC ATG GAA ATA 1686

Asp Thr Ala Glu Lys Arg Leu Gin Lys Lys Gin Arg Tyr Met Glu He 535 540 545 CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GAA TTA GGA 1734

Gin Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn Leu Glu Leu Gly 1007779 162 550 555 560 AAT TTG TCC CTG ACA GAT TCG ACT TCT TCC ACA TCG AAA TCA ACC GGT 1782

Asn Leu Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser Lys Ser Thr Gly ·. 565 570 575 580 GGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT CGT CGG TGA 1824

Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg Arg * 585 590 GACTCTTGCC TCTTAGTGTA ATTTTTGCTG TACCATATAA TTCTGTTTTC ATGATGACTG 1884 TAACTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT 1944 ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG 2004 ATTTGTA 2011 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 594 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

With Asp Thr Thr He Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser 15 10 15

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

Al a Ala Glu Gin Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gin Leu 35 40 45

Leu Ser Asn Ser Phe Glu Ala Val Phe Asp Al a Pro 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 Arg Ser Ser Phe Phe Lys Ser Ala Leu Al a 1 0 07 7 79 163 85 90 95

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

Glu Leu Lys Glu lie Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val 115 120 125

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

Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys 145 150 155 160

Arg Pro Ala Val Asp Phe With Leu Glu Val Leu Tyr Leu Ala Phe lie 165 170 175

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

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

Ala Asn He Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys 210 215 220

Glu He He Val Lys Ser Asn Val Asp With Val Ser Leu Glu Lys Ser 225 230 235 240

Leu Pro Glu Glu Leu Val Lys Glu He He Asp Arg Arg Lys Glu Leu 245 250 255

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

Ala Leu Asp Ser Asp Asp He Glu Leu Val 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 Asp Leu Ala 305 310 315 320

Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala 325 330 335 1007779 164

Ala Met Arg Lys Glu Pro Gin Leu lie Leu Ser Leu Leu Glu Lys Gly 340 345 350

Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met He 355 360 365

Ala Lys Gin Ala Thr With Ala Val Glu Cys Asn Asn lie Pro Glu Gin 370 375 380

Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu He Leu Glu Gin 385 390 395 400

Glu Asp Lys Arg Glu Gin He Pro Arg Asp Val Pro Pro Ser Phe Ala 405 410 415

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

Val Ala Leu Ala Gin Arg Leu Phe Pro Thr Glu Ala Gin Ala Ala With 435 440 445

Glu He Ala Glu With Lys Gly Thr Cys Glu Phe He Val Thr Ser Leu 450 455 460

Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys 465 470 475 480

He Ala Pro Phe Arg He Leu Glu Glu His Gin Ser Arg Leu Lys Ala 485 490 495

Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser 500 505 510

Ala Val Leu Asp Gin He Met Asn Cys Glu Asp Leu Thr Gin Leu Ala 515 520 525

Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gin Lys Lys Gin Arg 530 535 540

Tyr Met Glu He Gin Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn 545 550 555 560

Leu Glu Leu Gly Asn Leu Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser 565 570 575

Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg 1007779 165 580 585 590

Arg * (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1597 base pairs (B) TYPE: nucleic acid (C) STRING: single (D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: cDNA

(ix) SPECIAL ASPECT:

(A) NAME / SIGN: CDS

(B) PLACE: 1..1410 (D) FURTHER INFORMATION: / product = "Modified form of NIM1" / note = "N-terminal deletion compared to the wild-type NIM1 sequence." (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 24: ATG GAT TCG GTT GTG ACT GTT TTG GCT TAT GTT TAC AGC AGC AGA GTG 48

With Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val 15 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 Ala Asp Glu Asn Cys Cys 20 25 30 CAC GTG GCT TGC CGG CCG GCG 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 ATT ACT CTC TAT CAG AGG 192

Leu Ala Phe I1e Phe Lys IIe Pro Glu Leu IIe Thr Leu Tyr Gin Arg 50 55 60 CAC TTA TTG GAC GTT GTA GAC AAA GTT GTT ATA GAG GAC ACA TTG GTT 240

His Leu Leu Asp Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val 65 70 75 80

1 nri777Q

166 ATA CTC AAG CTT GCT AAT ATA TGT GGT AAA GCT TGT ATG AAG CTA TTG 288 11 e Leu Lys Leu Al a Asn Ile Cys Gly Lys Al a Cys Met Lys Leu Leu 85 90 95 GAT AGA TGT AAA GAG ATT ATT GTC AAG TCT AAT GTA GAT ATG GTT AGT 336

Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser 100 105 110 CTT GAA AAG 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 Ile Asp Arg 115 120 125 CGT AAA GAG CTT GGT TTG GAG GTA CCT AAA GTA AAG AAA CAT GTC TCG 432

Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser 130 135 140 AAT GTA CAT AAG GCA CTT GAC TCG GAT GAT ATT GAG TTA GTC AAG TTG 480

Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu 145 150 155 160 CTT TTG AAA GAG GAT CAC ACC AAT CTA GAT GAT GCG TGT GCT CTT CAT 528

Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His 165 170 175 TTC GCT GTT GCA TAT TGC AAT GTG 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 GTC AAC CAT AGG AAT CCG AGG GGA TAT ACG GTG 624

Leu Asp Leu Al a Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val 195 200 205 CTT CAT GTT GCT GCG ATG CGG AAG GAG CCA CAA TTG ATA CTA TCT CTA 672

Leu His Val Ala Ala With Arg Lys Glu Pro Gin Leu Ile Leu Ser Leu 210 215 220 TTG GAA AAA GGT GCA AGT GCA TCA GAA GCA ACT HG GAA GGT AGA ACC 720

Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr 225 230 235 240 GCA CTC ATG ATC GCA AAA CAA GCC ACT ATG GCG GTT GAA TGT AAT AAT 768

Al a Leu Met Ile Ala Lys Gin Al a Thr Met Ala Val Glu Cys Asn Asn 245 250 255 ATC CCG GAG CAA TGC AAG CAT TCT CTC AAA GGC CGA CTA TGT GTA GAA 816 lie Pro Glu Gin Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu 1007779 167 260 265 270 ATA CTA GAG CAA GAA GAC AAA CGA GAA CAA ATT CCT AGA GAT GTT CCT 864 lie Leu Glu Gin Glu Asp Lys Arg Glu Gin IIe Pro Arg Asp Val Pro 275 280 285 CCC TCT TTT GCA GTG GCG GCC GAT GAA TTG AAG ATG ACG CTG CTC GAT 912

Pro Ser Phe Ala Val Al a Ala Asp Glu Leu Lys Met Thr Leu Leu Asp 290 295 300 CTT GAA AAT AGA GTT GCA CTT GCT CAA CGT CTT TTT CCA ACG GAA GCA 960

Leu Glu Asn Arg Val Ala Leu Ala Gin Arg Leu Phe Pro Thr Glu Ala 305 310 315 320 CAA GCT GCA ATG GAG ATC GCC GAA ATG AAG GGA ACA TGT GAG TTC ATA 1008

Gin Ala Ala With Glu He Ala Glu With Lys Gly Thr Cys Glu Phe lie 325 330 335 GTG ACT AGC CTC GAG CCT GAC CGT CTC ACT GGT ACG AAG 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 AAG ATA GCA CCT TTC AGA ATC CTA GAA GAG CAT CAA AGT 1104

Pro Gly Val Lys lie Ala Pro Phe Arg He Leu Glu Glu His Gin Ser 355 360 365 AGA CTA AAA GCG CTT TCT AAA ACC GTG GAA CTC GGG AAA CGA TTC TTC 1152

Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe 370 375 380 CCG CGC TGT TCG GCA GTG CTC GAC CAG ATT ATG AAC TGT GAG GAC TTG 1200

Pro Arg Cys Ser Ala Val Leu Asp Gin He 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 Gin Leu Ala Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gin 405 410 415 AAG AAG CAA AGG TAC ATG GAA ATA CAA GAG ACA CTA AAG AAG GCC TTT 1296

Lys Lys Gin Arg Tyr With Glu He Gin Glu Thr Leu Lys Lys Ala Phe 420 425 430 AGT GAG GAC AAT TTG GAA 17A GGA AAT TTG TCC CTG ACA GAT TCG ACT 1344

Ser Glu Asp Asn Leu Glu Leu Gly Asn Leu Ser Leu Thr Asp Ser Thr 435 440 445 10077? N 168 TCT TCC ACA TCG 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 CGG TGA 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 ATTTGTA 1597 (2) INFORMATION FOR SEQ ID N0: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 470 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

With Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val 15 10 15

Arq Pro Pro Pro Lys Gly Val Ser Glu Cys 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 lie Phe Lys lie Pro Glu Leu He Thr Leu Tyr Gin Arg 50 55 60

His Leu Leu Asp Val Val Asp Lys Val Val He Glu Asp Thr Leu Val 65 70 75 80

He Leu Lys Leu Ala Asn He Cys Gly Lys Ala Cys With Lys Leu Leu 85 90 95

Asp Arg Cys Lys Glu He He Val Lys Ser Asn Val Asp Met Val Ser 100 105 110 1 007779 169

Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu lie lie Asp Arg 115 120 125

Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser 130 135 140

Asn Val His Lys Ala Leu Asp Ser Asp Asp lie Glu Leu Val Lys Leu 145 150 155 160

Leu Leu Lys Glu Asp His Thr 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 With Arg Lys Glu Pro Gin Leu He Leu Ser Leu 210 215 220

Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr 225 230 235 240

Ala Leu With He Ala Lys Gin Ala Thr With Ala Val Glu Cys Asn Asn 245 250 255

He Pro Glu Gin Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu 260 265 270

He Leu Glu Gin Glu Asp Lys Arg Glu Gin He Pro Arg Asp Val Pro 275 280 285

Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys With Thr Leu Leu Asp 290 295 300

Leu Glu Asn Arg Val Ala Leu Ala Gin Arg Leu Phe Pro Thr Glu Ala 305 310 315 320

Gin Ala Ala With Glu He Ala Glu With Lys Gly Thr Cys Glu Phe He 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 He Ala Pro Phe Arg He Leu Glu Glu His Gin Ser 1 007779 170 355 360 365

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

Pro Arg Cys Ser Ala Val Leu Asp Gin lie Met Asn Cys Glu Asp Leu 385 390 395 400

Thr Gin Leu Ala Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gin 405 410 415

Lys Lys Gin Arg Tyr With Glu lie Gin Glu Thr Leu Lys Lys Ala Phe 420 425 430

Ser Glu Asp Asn Leu Glu Leu Gly Asn Leu Ser Leu Thr Asp Ser Thr 435 440 445

Ser Ser Thr Ser Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu 450 455 460

Ser His Arg Arg Arg * 465 470 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE FEATURES: (A) LENGTH: 1608 base pairs (B) TYPE: nucleic acid (C) STRING: single (D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: cDNA

(ix) SPECIAL ASPECT:

(A) NAME / SIGN: CDS

(B) PLACE: 43..1608 (D) FURTHER INFORMATION: / product = "Modified form of NIM1" / note = "C-terminal deletion compared to wild-type NIM1." (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC 54

Met Asp Thr Thr 1 1007779 171 ATπ GAT GGA TTC GCC GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC GTC 102 I1e Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val 5 10 15 20 GCT ACC GAT AAC ACC GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA 150

Al a Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu Al a Ala Glu Gin 25 30 35 GTA CTC ACC GGA CCT GAT GTA TCT GCT CTG CAA TTG CTC TCC AAC AGC 198

Val Leu Thr Gly Pro Asp Val Ser Al a Leu Gin Leu Leu Ser Asn Ser 40 45 50 TTC GAA TCC GTC TTT GAC TCG CCG GAT GAT TTC TAC AGC GAC GCT AAG 246

Phe Glu Ser Val Phe Asp Ser Pro Asp Asp Phe Tyr Ser Asp Ala Lys 55 60 65 CTT G "T CTC TCC GAC GGC CGG GAA GTT TCT TTC CAC CGG TGC GTT TTG 294

Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His Arg Cys Val Leu 70 75 80 TCA GCG AGA AGC TCT TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAG 342

Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Al a Ala Ala Lys Lys 85 90 95 100 GAG AAA GAC TCC AAC AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG 390

Glu Lys Asp Ser Asn Asn Thr Al a Ala Val Lys Leu Glu Leu Lys Glu 105 110 115 ΑΠ GCC AAG GAT TAC GAA GTC GGT TTC GAT TCG GTT GTG ACT GH TTG 438

Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu 120 125 130 GCT TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA GH TCT 486

Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Lys Gly Val Ser 135 140 145 GAA TGC GCA GAC GAG ATAT TGC TGC CAC GTG GCT TGC CGG CCG GCG GTG 534

Glu Cys Al a Asp Glu Asn Cys Cys His Val Al a Cys Arg Pro Ala Val 150 155 160 GAT HC ATG HG GAG GTT CTC TAT TTG GCT TTG ATC TTC AAG ATC CCT 582

Asp Phe Met Leu Glu Val Leu Tyr Leu Al a Phe lie Phe Lys lie Pro 165 170 175 180 GAA ΠΑ Απ ACT CTC TAT CAG AGG CAC πΑ TTG GAC GTT GTA GAC AAA 630

Glu Leu Ile Thr Leu Tyr Gin Arg His Leu Leu Asp Val Val Asp Lys 1007779 185 190 195 172 GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT 678

Val Val lie Glu Asp Thr Leu Val lie Leu Lys Leu Ala Asn He Cys 200 205 210 GGT AAA GCT TGT ATG AAG CTA TTG GAT AGA TGT AAA GAG ATT ATT GTC 726

Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu He He Val 215 220 225 AAG 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 AGA CGT AAA GAG CTJ GGT TTG GAG GTA 822

Leu Val Lys Glu He He Asp Arg Arg Lys Glu Leu Gly Leu Glu Val 245 250 255 260 CCT AAA GTA AAG AAA CAT GTC TCG AAT GTA CAT AAG GCA CTJ GAC TCG 870

Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser 265 270 275 HOLE GAT ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG GAT CAC ACC AAT 918

Asp Asp He Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn 280 285 290 CTA GAT GAT GCG TGT GCT CTT CAT TTC GCT GTT GCA 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 ACG GTG CTT CAT GTT GCT GCG ATG CGG AAG 1062

Arg Asn Pro Arg Gly Tyr 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 Gin Leu He Leu Ser Leu Leu Glu Lys Gly Ala 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 With He Ala Lys Gin Ala 360 365 370 1 007779 173

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

Thr Met Ala Val Glu Cys Asn Asn lie Pro Glu Gin 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 lie Leu Glu Gin Glu Asp Lys Arq 390 395 400 GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT ITT GCA GTG GCG GCC GAT 1302

Glu Gin He Pro Arg Asp Val Pro Pro Ser Phe Ala Val 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 With Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala 425 430 435 CAA CGT CTT TTT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC GAA 1398

Gin Arg Leu Phe Pro Thr Glu Ala Gin Ala Ala Met Glu He Ala Glu 440 445 450 ATG AAG GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC CGT 1446

With Lys Gly Thr Cys Glu Phe He Val Thr Ser Leu Glu Pro Asp Arg 455 460 465 CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAG ATA GCA CCT TTC 1494

Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys He Ala Pro Phe 470 475 480 AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA ACC 1542

Arg He Leu Glu Glu His Gin Ser Arg Leu Lys Ala Leu Ser Lys Thr 485 490 495 500 GTG GAA CTC GGG AAA CGA TTC TTC CCG CGC TGT TCG GCA GTG CTC GAC 1590

Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu Asp 505 510 515 CAG ATT ATG AAC TGT TGA 1608

Gin He Met Asn Cys * 520 (2) INFORMATION FOR SEQ ID N0: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 522 amino acids (B) TYPE: amino acid 1 007779 174 (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE : protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

With Asp Thr Thrlie Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser 15 10 15

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

Al a Ala Glu Gin Val Leu Thr Gly Pro Asp Val Ser Al a Leu Gin Leu 35 40 45

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

Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His 65 70 75 80

Arg Cys Val Leu Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala 85 90 95

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

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

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

Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys 145 150 155 160

Arg Pro Ala Val Asp Phe With Leu Glu Val Leu Tyr Leu Ala Phe Ile 165 170 175

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

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

Ala Asn lie Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys 1007779 210 215 220 175

Glu He lie Val Lys Ser Asn Val Asp With Val Ser Leu Glu Lys Ser 225 230 235 240

Leu Pro Glu Glu Leu Val Lys Glu lie lie Asp Arg Arg Lys Glu Leu 245 250 255

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

Ala Leu Asp Ser Asp Asp He Glu Leu Val 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 Asp Leu Ala 305 310 315 320

Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala 325 330 335

Ala Met Arg Lys Glu Pro Gin Leu He Leu Ser Leu Leu Glu Lys Gly 340 345 350

Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met He 355 360 365

Ala Lys Gin Ala Thr With Ala Val Glu Cys Asn Asn He Pro Glu Gin 370 375 380

Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu He Leu Glu Gin 385 390 395 400

Glu Asp Lys Arg Glu Gin He Pro Arg Asp Val Pro Pro Ser Phe Ala 405 410 415

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

Val Ala Leu Ala Gin Arg Leu Phe Pro Thr Glu Ala Gin Ala Ala With 435 440 445

Glu He Ala Glu With Lys Gly Thr Cys Glu Phe He Val Thr Ser Leu 450 455 460 1 007779 176

Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys 465 470 475 480 lie Ala Pro Phe Arg lie Leu Glu Glu His Gin Ser Arg Leu Lys Ala 485 490 495

Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser 500 505 510

Ala Val Leu Asp Gin He Met Asn Cys * 515 520 (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE FEATURES: (A) LENGTH: 1194 base pairs (B) TYPE: nucleic acid (C) STRING: single (D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: cDNA

(ix) SPECIAL ASPECT:

(A) NAME / SIGN: CDS

(B) PLACE: 1..1194 (D) FURTHER INFORMATION: / product = "Modified form of NIM1" / note = "N-terminal / C-terminal chimera." (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: ATG GAT TCG GTT GTG ACT GTT TTG GCT TAT GTT TAC AGO AGC AGA GTG 48

With Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val 15 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 Ala Asp Glu Asn Cys Cys 20 25 30 CAC GTG GCT TGC CGG CCG GCG 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 ATT ACT CTC TAT CAG AGG 192

Leu Ala Phe He Phe Lys He Pro Glu Leu He Thr Leu Tyr Gin Arg 1007779 177 50 55 60 CAC TTA TTG GAC GTT GTA GAC AAA GTT GTT ATA GAG GAC ACA TTG GTT 240

His Leu Leu Asp Val Val Asp Lys Val Val lie Glu Asp Thr Leu Val 65 70 75 80 ATA CTC AAG C1T GCT AAT ATA TGT GGT AAA GCT TGT ATG AAG CTA TTG 288

He Leu Lys Leu Ala Asn lie Cys Gly Lys Ala Cys Met Lys Leu Leu 85 90 95 GAT AGA TGT AAA GAG ATT ATT GTC AAG TCT AAT GTA GAT ATG GTT AGT 336

Asp Arg Cys Lys Glu He He Val Lys Ser Asn Val Asp Met Val Ser 100 105 no CTT GAA AAG 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 lie lie Asp Arg 115 120 125 CGT AAA GAG CTT GGT TTG GAG GTA CCT AAA GTA AAG AAA CAT GTC TCG 432

Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser 130 135 140 AAT GTA CAT AAG GCA CTT GAC TCG GAT GAT ATT GAG TTA GTC AAG TTG 480

Asn Val His Lys Ala Leu Asp Ser Asp Asp He Glu Leu Val Lys Leu 145 150 155 160 CTT TTG AAA GAG GAT CAC ACC AAT CTA GAT GAT GCG TGT GCT CTT CAT 528

Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His 165 170 175 TTC GCT GTT GCA TAT TGC AAT GTG 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 GTC 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 GCG ATG CGG AAG GAG CCA CAA TTG ATA CTA TCT CTA 672

Leu His Val Ala Ala With Arg Lys Glu Pro Gin Leu He Leu Ser Leu 210 215 220 TTG GAA AAA GGT GCA AGT GCA TCA GAA GCA ACT TTG GAA GGT AGA ACC 720

Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr 225 230 235 240 1007779 178 GCA CTC ATG ATC GCA AAA CAA GCC ACT ATG GCG GTT GAA TGT AAT AAT 768

Ala Leu Met lie Ala Lys Gin Ala Thr Met Ala Val Glu Cys Asn Asn 245 250 255 ATC CCG GAG CAA TGC AAG CAT TCT CTC AAA GGC CGA CTA TGT GTA GAA 816 lie Pro Glu Gin Cys Lys 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

He Leu Glu Gin Glu Asp Lys Arg Glu Gin He Pro Arg Asp Val Pro 275 280 285 CCC TCT ITT GCA GTG GCG GCC GAT GAA TTG AAG ATG ACG CTG CTC GAT 912

Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp 290 295 300 CTT GAA AAT AGA GTT GCA CTT GCT CAA CGT CTT ITT CCA ACG GAA GCA 960

Leu Glu Asn Arg Val Ala Leu Ala Gin Arg Leu Phe Pro Thr Glu Ala 305 310 315 320 CAA GCT GCA ATG GAG ATC GCC GAA ATG AAG GGA ACA TGT GAG TTC ATA 1008

Gin Ala Ala Met Glu lie Ala Glu Met Lys Gly Thr Cys Glu Phe He 325 330 335 GTG ACT AGC CTC GAG CCT GAC CGT CTC ACT GGT ACG AAG 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 AAG ATA GCA CCT TTC AGA ATC CTA GAA GAG CAT CAA AGT 1104

Pro Gly Val Lys He Ala Pro Phe Arg He Leu Glu Glu His Gin Ser 355 360 365 AGA CTA AAA GCG CTT TCT AAA ACC GTG GAA CTC GGG AAA CGA TTC TTC 1152

Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe 370 375 380 CCG CGC TGT TCG GCA GTG CTC GAC CAG ATT ATG AAC TGT TGA 1194

Pro Arg Cys Ser Ala Val Leu Asp Gin He Met Asn Cys * 385 390 395 (2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE FEATURES: (A) LENGTH: 398 amino acids (B) TYPE: amino acid 1007779 179 (D ) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

With Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val 15 10 15

Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Al a Asp Glu Asn Cys Cys 20 25 30

His Val Al a Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr 35 40 45

Leu Ala Phe I1e Phe Lys lie Pro Glu Leu I1e Thr Leu Tyr Gin Arg 50 55 60

His Leu Leu Asp Val Val Asp Lys Val Val I1e Glu Asp Thr Leu Val 65 70 75 80 II e Leu Lys Leu Al a Asn IIe Cys Gly Lys Al a Cys Met Lys Leu Leu 85 90 95

Asp Arg Cys Lys Glu I1e IIe Val Lys Ser Asn Val Asp Met Val Ser 100 105 110

Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu 11th 11th Asp Arg 115 120 125

Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser 130 135 140

Asn Val His Lys Ala Leu Asp Ser Asp Asp 11 e Glu Leu Val Lys Leu 145 150 155 160

Leu Leu Lys Glu Asp His Thr 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 Al a Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val 195 200 205

Leu His Val Al a Ala Met Arg Lys Glu Pro Gin Leu IIe Leu Ser Leu 1 007779 180 210 215 220

Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr 225 230 'Ala Leu Met He Ala Lys Gin Ala Thr Met Ala Val Glu Cys Asn Asn 245 lie Pro Glu Gin Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu 260

Iln, Λ11 p-. r-. p-ι. I vs Arg Glu Gin He Pro Arg Asp Val Pro lie Leu Glu Gin Glu Asp Ly3 285 275

Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys With Thr Leu Leu Asp 290 295 300

Leu Glu Asn Arg Val Ala LeU Ala Gin Arg Leu Phe Pro Thr Glu Ala 305 310 13

Gin Ala Ala Met Glu lie Ala Glu Met Lys Gly Thr Cys Glu Phe lie 325

Val Thr Ser Leu Glu Pro ASP Arg Leu Thr Gly Thr Lys Arg Thr Ser 340

Pro Gly Val Lys lie Ala Pro ^ Ar9 Ile Leu Glu G '“His G, n Ser 355

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

Pro Arg Cys Ser Ala Val Leu Asp Gin Ile Met Asn Cys * 385 390 395 (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE FEATURES: (A) LENGTH: 786 base pairs (B) TYPE: nucleic acid (C) STRENGTH : single (D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: cDNA

1007779 181 (ix) SPECIAL ASPECT:

(A) NAME / SIGN: CDS

(B) LOCATION: 1..786 (D) FURTHER INFORMATION: / product = "Changed form of NIM1" / note = "Ankyrin areas of NIMl." (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 30: ATG GAC TCC AAC AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG ATT 48

With Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu lie 15 10 15 GCC AAG GAT TAC GAA GTC GGT TTC GAT TCG GTT GTG ACT GTT TTG GCT 96

Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Ala 20 25 30 TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT GAA 144

Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Lys Gly Val Ser Glu 35 40 45 TGC GCA GAC GAG ATAT TGC TGC CAC GTG GCT TGC CGG CCG GCG GTG GAT 192

Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp 50 55 60 TTC ATG TTG GAG GTT CTC TAT TTG GCT TTC ATC TTC AAG ATC CCT GAA 240

Phe Met Leu Glu Val Leu Tyr Leu Ala Phe He Phe Lys lie Pro Glu 65 70 75 80 TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA GTT 288

Leu He Thr Leu Tyr Gin Arg His Leu Leu Asp Val Val Asp Lys Val 85 90 95 GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT GGT 336

Val He Glu Asp Thr Leu Val He Leu Lys Leu Ala Asn He Cys Gly 100 105 110 AAA GCT TGT ATG AAG CTA TTG GAT AGA TGT AAA GAG ATT ATT GTC AAG 384

Lys Ala Cys With Lys Leu Leu Asp Arg Cys Lys Glu He He Val Lys 115 120 125 TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG 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 AGA CGT AAA GAG CTT GGT TTG GAG GTA CCT 480 1 007 779 182

Val Lys Glu IIe lie Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro 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 CTT TTG AAA GAG GAT CAC ACC AAT CTA 576

Asp IIe Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Leu 180 185 190 GAT GAT GCG TGT GCT CTT CAT TTC GCT GTT GCA 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 GCC GAT GTC AAC CAT AGG 672

Thr Ala Thr Asp Leu L eu Lys Leu Asp Leu Ala Asp Val Asn His Arg 210 215 220 AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG AAG GAG 720

Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys Glu 225 230 235 240 CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA GAA 768

Pro Gin Leu I1e Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu 245 250 255 GCA ACT TTG GAA GGT TGA 786

Ala Thr Leu Glu Gly * 260 (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE FEATURES: (A) LENGTH: 262 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein ( (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

With Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu He 15 10 15 1 007 779 183

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

Tyr Val Tyr Ser Ser Arg Val Arg Pro 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 Glu Val Leu Tyr Leu Ala Phe He Phe Lys lie Pro Glu 65 70 75 80

Leu lie Thr Leu Tyr Gin Arg His Leu Leu Asp Val Val Asp Lys Val 85 90 95

Val He Glu Asp Thr Leu Val He Leu Lys Leu Ala Asn He Cys Gly 100 105 110

Lys Ala Cys With Lys Leu Leu Asp Arg Cys Lys Glu He He Val Lys 115 120 125

Ser Asn Val Asp With Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu 130 135 140

Val Lys Glu He He Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro 145 150 155 160

Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser Asp 165 170 175

Asp lie Glu Leu Val Lys Leu Leu Leu Lys 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 Tyr Thr Val Leu His Val Ala Ala Met Arg Lys Glu 225 230 235 240

Pro Gin Leu He Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu 245 250 255

Ala Thr Leu Glu Gly * 1007779 260 184 (2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRING: single (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: CAACAGCTTC GAAGCCGTCT TTGACGCGCC GGATG 35 (2) INFORMATION FOR SEQ ID NO: 33: (i ) SEQUENCE FEATURES: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRENGTH: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid CA) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION : SEQ ID NO: 33: CATCCGGCGC GTCAAAGACG GCTTCGAAGC TGTTG 35 (2) INFORMATION FOR SEQ ID N0: 34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRING: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid 1007779 185 (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: HOLE TATE GGATTCGGTT GTGACT GTiT TG 32 (2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE FEATURES: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRENGTH: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: GGAATTCTAC AAATCTGTAT ACCATTGG 28 (2) INFORMATION FOR SEQ ID N0: 36: (1) SEQUENCE FEATURES: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRINE: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: CGGAATTCGA TCTCTTTAAT TTGTGAATTT C 31 (2) INFORMATION FOR SEQ ID NO: 37: 1007779 186 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRING: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: GGAATTCTCA ACAGTTCATA ATCTGGTCG 29 (2) INFORMATION FOR SEQ ID NO: 38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRENGTH: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = " oligonucleotide "(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: HOLE TATE GGACTCCAAC AACACCGCCG C 31 (2) INFORMATION FOR SEQ ID NO: 39: (i) SEQUENCE FEATURES: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRING: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" 1007779 187 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 39: GGAATTCTCA ACCTTCCAAA GTTGCTTCTG AT77 33 - 64

Claims (36)

DNA molecule, characterized in that it encodes an altered form of a NIM1 protein. DNA molecule according to claim 1, characterized in that it acts as a dominant negative control of the SAR signal transduction pathway. DNA molecule according to claim 1, characterized in that the modified form of the NIM1 protein has alanine residues instead of serine residues at the 10 amino acid positions corresponding to positions 55 and 59 of SEQ ID NO: 3. DNA molecule according to claim 3, characterized in that the altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO: 23. DNA molecule according to claim 4, characterized in that the DNA molecule comprises the nucleotide sequence shown in SEQ ID NO: 22 as well as whole DNA. DNA molecule according to claim 1, characterized in that the altered form of the NIM1 protein is a truncated version of the MM7 gene product. 7. DNA molecule according to claim 1, characterized in that the altered form of the NIM1 protein has an N-terminal truncation of amino acids corresponding approximately to amino acid positions 1-125 of SEQ ID NO: 3. DNA molecule according to claim 7, characterized in that the modified form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO: 25. DNA molecule according to claim 8, characterized in that the DNA molecule comprises the nucleotide sequence shown in SEQ ID NO: 24. DNA molecule according to claim 1, characterized in that the modified form of the NIM1 protein has a C-terminal truncation of amino acids corresponding approximately to amino acid positions 522-593 of SEQ ID NO: 3. DNA molecule according to claim 10, characterized in that the altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO: 27. DNA molecule according to claim 11, characterized in that the DNA molecule comprises the nucleotide sequence shown in SEQ ID NO: 26. DNA molecule according to claim 1, characterized in that the altered form of the NIM1 protein has an N-terminal truncation of amino acids corresponding approximately to amino acid positions 1-125 of SEQ ID NO: 2 and a C- 5 has amino acid terminal truncation which corresponds approximately to amino acid positions 522-593 of SEQ ID NO: 3. DNA molecule according to claim 13, characterized in that the altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO: 29. DNA molecule according to claim 14, characterized in that the DNA molecule comprises the nucleotide sequence shown in SEQ ID NO: 28. DNA molecule according to claim 1, characterized in that the modified form of the NIM1 protein consists essentially of ankyrin motifs which correspond approximately to amino acid positions 103-362 of SEQ ID NO: 3. DNA molecule according to claim 16, characterized in that the altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO: 31. DNA molecule according to claim 17, characterized in that the DNA molecule comprises the nucleotide sequence shown in SEQ ID NO: 30. DNA molecule according to claim 1, characterized in that the DNA molecule hybridizes under the following conditions to a nucleotide sequence selected from the group consisting of 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 NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55 ° C for 18-24 hours, and washing in 6XSSC for 15 minutes (X3) 3XSSC for 15 minutes (XI) at 55 ° C. 20. Chimeric gene, characterized in that it comprises a plant-active promoter operably linked to a DNA molecule according to any one of claims 1-19. A recombinant vector, characterized in that it comprises a chimeric gene according to claim 20, wherein the vector can be stably transformed into a host cell. - - < A method of activating SAR in a plant, characterized in that the plant is transformed with a recombinant vector according to claim 21, wherein an altered form of the NIM1 protein is expressed in the transformed plant and SAR in this plant activates. A method of conferring a broad spectrum of disease resistance to a plant, characterized in that the plant is transformed with a recombinant vector according to claim 21, wherein an altered form of the NIM1 protein is expressed in the transformed plant and confers disease resistance with a broad spectrum of action to this plant. A method of conferring a CIM phenotype to a plant, characterized in that the plant is transformed with a recombinant vector according to claim 21, wherein an altered form of the NIM1 protein is expressed in the transformed plant and imparts a CIM phenotype to the plant. Host cell, characterized in that it has been stably transformed with a vector according to claim 21. Host cell according to claim 25, characterized in that this cell is a plant cell. Plant, plant cells or their progeny, characterized in that they comprise a chimeric gene according to claim 20 and exhibit disease resistance with a broad spectrum of action. 28. Plant, plant cells or their progeny, characterized in that a NIM1 protein involved in the signal transduction cascade leading to systemically acquired resistance in plants is expressed in the transformed plant at higher levels than in a wild -type plant. Plant, plant cells or the progeny thereof according to claim 27 or 28, characterized in that the plant is selected from the group comprising gymnosperms, monocotyledonous plants and dicotyledonous plants. Plant, plant cells or the progeny thereof according to claim 27 or 28, characterized in that the plant is a crop plant. 31. Plant, plant cells or their progeny according to claim 27 or 28, characterized in that the plant is selected from the series comprising rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, beans , peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, brinjal, pepper, celery, carrot, pumpkin, in particular cucurbita pepo, zucchini, cucumber, apple, pear, quince , melon, plum, cherry, peach, nectarine, apricot, 5 strawberry, grape, raspberry, pineapple, avocado, papaya, mango, banana, soy, tobacco, tomato, sorghum or sugar cane. 32. A method of conferring a CIM phenotype on a plant cell, a plant or its progeny, characterized in that the plant is transformed with a recombinant vector comprising a chimeric gene, which gene comprises a plant-active promoter operably linked to a DNA molecule encoding a NIM1 protein involved in the signal transduction cascade leading to systemically acquired resistance in plants, wherein the vector can be stably transformed into a host, and NIM1 protein in the transformed plant is expressed at higher levels than in a wild type plant. 33. A method of activating systemically acquired resistance in a plant cell, a plant or its progeny, characterized in that the plant is transformed with a recombinant vector comprising a chimeric gene, which gene comprises a plant-active promoter which acts in an effective manner is linked to a DNA molecule encoding an NIM1 protein involved in the signal transduction cascade leading to systemically acquired resistance in plants, where the vector can be stably transformed into the host, and the NIM1 protein in the transformed plant is expressed at higher levels than in a wild type plant. A method of conferring broad spectrum disease resistance to a plant cell, a plant or its progeny, characterized in that the plant is transformed with a recombinant vector comprising a chimeric gene, which gene comprises a plant-active promoter operably linked to a DNA molecule encoding a NIM1 protein involved in the signal transduction cascade leading to systemically acquired resistance in plants, wherein the vector can be stably transformed into a host, and wherein the NIM1 protein in the transformed plant is expressed at higher levels than in a HjQT7 sn wild type plant. Use of a transgenic plant or its progeny in an agricultural method, wherein the plant comprises a chimeric gene according to claim 20. 36. A commercially suitable container comprising transgenic plant seed comprising at least an altered form of an NIM1 protein or comprising an NIM1 protein expressed at higher levels in this transformed plant than in a wild type plant, in an amount sufficient to act as a dominant-negative control of the SAR-10 signal transduction pathway; together with a suitable carrier and along with labeling instructions for its use for conferring disease resistance with a wide spectrum of action to plants. *** J U l '' "