AU727179B2 - Methods of using the NIM1 gene to confer disease resistance in plants - Google Patents

Description

WO 98/26082 PCT/EP97/07012 METHODS OF USING THE NIM1 GENE TO CONFER DISEASE RESISTANCE IN PLANTS The present invention generally relates to broad-spectrum disease resistance in plants, 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 create transgenic plants having broad-spectrum disease resistance. The present invention relates further to high-level expression of the cloned NIM1 gene in transgenic plants that have broad-spectrum disease resistance.

Plants are constantly challenged by a wide variety of pathogenic organisms including viruses, bacteria, fungi, and nematodes. Crop plants are particularly vulnerable because they are usually grown as genetically-uniform monocultures; when disease strikes, losses can be severe. However, most plants have their own innate mechanisms of defense against pathogenic organisms. Natural variation for resistance to plant pathogens has been identified by plant breeders and pathologists and bred into many crop plants. These natural disease resistance genes often provide high levels of resistance to or immunity against pathogens.

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

5,614,395, incorporated by reference herein in its entirety). SAR is a particularly important aspect of plant-pathogen responses because it is a pathogen-inducible, systemic resistance against a broad spectrum of infectious agents, including viruses, bacteria, and fungi. When the SAR signal transduction pathway is blocked, plants become more susceptible to pathogens that normally cause disease, and they also become susceptible to some infectious agents that would not normally cause disease (Gaffney et al., Science 261, 754- 756 (1993), incorporated by reference herein in its entirety; Delaney et al., Science 266, 30 1247-1250 (1994), incorporated by reference herein in its entirety; Delaney et al., Proc.

Natl. Acad. Sci. USA 92, 6602-6606 (1995), incorporated by reference herein in its entirety; Delaney, Plant Phys.. 113, 5-12 (1997), incorporated by reference herein in its entirety; Bi et al., Plant J. 8, 235-245 (1995), incorporated by reference herein in its entirety; Mauch-Mani and Slusarenko, Plant Cell8, 203-212 (1996), incorporated by reference herein in its WO 98/26082 PCrT/I'po7/LnM1i -2entirety). These observations indicate that the SAR signal transduction pathway is critical for maintaining plant health.

Conceptually, the SAR response can be divided into two phases. In the initiation phase, a pathogen infection is recognized, and a signal is released that travels through the phloem to distant tissues. This systemic signal is perceived by target cells, which react by expression of both SAR genes and disease resistance. The maintenance phase of SAR refers to the period of time, from weeks up to the entire life of the plant, during which the plant is in a quasi steady state, and disease resistance is maintained (Ryals et al., 1996).

Salicylic acid (SA) accumulation appears to be required for SAR signal transduction.

Plants that cannot accumulate SA due to treatment with specific inhibitors, epigenetic repression of phenylalanine ammonia-lyase, or transgenic expression of salicylate hydroxylase, which specifically degrades SA, also cannot induce either SAR gene expression or disease resistance (Gaffney et al., 1993; Delaney et al., 1994; Mauch-Mani and Slusarenko 1996; Maher et al., Proc. Natl. Acad. Sci. USA 91, 7802-7806 (1994), incorporated by reference herein in its entirety; Pallas et al., Plant J. 10, 281-293 (1996), incorporated by reference herein). Although it has been suggested that SA might serve as the systemic signal, this is currently controversial and, to date, all that is known for certain is that if SA cannot accumulate, then SAR signal transduction is blocked (Pallas et al., 1996; Shulaev et al., 1995Plant Cell7, 1691-1701 (1995), incorporated by reference herein in its entirety; Vernooij et al., Plant Cell6, 959-965 (1994), incorporated by reference herein in its entirety).

Recently, Arabidopsis has emerged as a model system to study SAR (Uknes et al., Plant Cell4, 645-656 (1992), incorporated by reference herein in its entirety; Uknes et al., Mol. Plant-Microbe Interact. 6, 692-698 (1993), incorporated by reference herein in its entirety; Cameron et al., Plant J. 5, 715-725 (1994), incorporated by reference herein in its entirety; Mauch-Mani and Slusarenko, Mol. Plant-Microbe Interact. 7, 378-383 (1994), incorporated by reference herein in its entirety; Dempsey and Klessig, Bulletin de L'lnstitut Pasteur93, 167-186 (1995), incorporated by reference herein in its entirety). It has been demonstrated that SAR can be activated in Arabidopsis by both pathogens and chemicals, such as SA, 2 ,6-dichloroisonicotinic acid (INA) and benzo(1, 2 3 )thiadiazole-7-carbothioic acid S-methyl ester (BTH) (Uknes et al., 1992; Vernooij et al., Mol. Plant-Microbe Interact. 8, 228-234 (1995), incorporated by reference herein in its entirety; Lawton et al., Plant J. 71-82 (1996), incorporated by reference herein in its entirety). Following treatment with either INA or BTH or pathogen infection, at least three pathogenesis-related (PR) protein genes, namely, PR-1, PR-2, and PR-5 are coordinately induced concomitant with the onset WO 98/26082 PCT/EP97/07012 -3of resistance (Uknes et al., 1992, 1993). In tobacco, the best characterized species, treatment with a pathogen or an immunization compound induces the expression of at least nine sets of genes (Ward et al., Plant Cell3, 1085-1094 (1991), incorporated by reference herein in its entirety). Transgenic disease-resistant plants have been created by transforming plants with various SAR genes Patent No. 5,614,395).

A number of Arabidopsis mutants have been isolated that have modified SAR signal transduction (Delaney, 1997). The first of these mutants are the so-called Isd (lesions simulating disease) mutants and acd2 (accelerated cell death) (Dietrich et al., Cell 77, 551- 563 (1994), incorporated by reference herein in its entirety; Greenberg et al., Cell77, 551- 563 (1994), incorporated by reference herein in its entirety). These mutants all have some degree of spontaneous necrotic lesion formation on their leaves, elevated levels of SA, mRNA accumulation for the SAR genes, and significantly enhanced disease resistance. At least seven different Isd mutants have been isolated and characterized (Dietrich et al., 1994; Weymann et al., Plant Cell7, 2013-2022 (1995), incorporated by reference herein in its entirety). Another interesting class of mutants are cim (constitutive immunity) mutants (Lawton et al., 1993 The molecular biology of systemic aquired resistance" in Mechanisms of Defence Responses in Plants, B. Fritig and M. Legrand, eds (Dordrecht, The Netherlands: Kluwer Academic Publishers), pp. 422-432 (1993), incorporated by reference herein in its entirety). See also, International PCT Application WO 94/16077, both of which are incorporated by reference entirety herein in their entireties. Like Isd mutants and acd2, cim mutants have elevated SA and SAR gene expression and resistance, but in contrast to Isd or acd2, do not display detectable lesions on their leaves. cprl (constitutive expresser of PR genes) may be a type of cim mutant; however, because the presence of microscopic lesions on the leaves of cprl has not been ruled out, cprl might be a type of Isd mutant (Bowling et al., Plant Cell6, 1845-1857 (1994), incorporated by reference herein in its entirety).

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

nprl (nonexpresser of PR genes) is a mutant that cannot induce expression of the SAR signaling pathway following INA treatment (Cao et al., Plant Cell6, 1583-1592 (1994), incorporated by reference herein in its entirety). eds (enhanced disease susceptibility) mutants have been isolated based on their ability to support bacterial infection following WO 98/26082 PCT/EPo7/n7l i 4 inoculation of a low bacterial concentration (Glazebrook et al., Genetics 143, 973-982 (1996), incorporated by reference herein in its entirety; Parker et al., Plant Cell 8, 2033- 2046 (1996), incorporated by reference herein in its entirety). Certain eds mutants are phenotypically very similar to nprl, and, recently, eds5 and eds53 have been shown to be allelic to nprl (Glazebrook et al., 1996). niml (noninducible immunity) is a mutant that supports P. parasitica causal agent of downy mildew disease) growth following INA treatment (Delaney et al., 1995; International PCT Application WO 94/16077). Although niml can accumulate SA following pathogen infection, it cannot induce SAR gene expression or disease resistance, suggesting that the mutation blocks the pathway downstream of SA. niml is also impaired in its ability to respond to INA or BTH, suggesting that the block exists downstream of the action of these chemicals (Delaney et al., 1995; Lawton et al., 1996).

Recently, two allelic Arabidopsis genes have been isolated and characterized, mutants of which are responsible for the niml and nprl phenotypes, respectively (Ryals et al., Plant Cell9, 425-439 (1997), incorporated by reference herein in its entirety; Cao etal., Cell88, 57-63 (1997), incorporated by reference herein in its entirety). The wild-type NIM1 gene product is involved in the signal transduction cascade leading to both SAR and genefor-gene disease resistance in Arabidopsis (Ryals et al., 1997). Ryals et al., 1997 also report the isolation of five additional alleles of niml that show a range of phenotypes from weakly impaired in chemically induced PR-1 gene expression and fungal resistance to very strongly blocked. Transformation of the wild-type NPR1 gene into nprl mutants not only complemented the mutations, restoring the responsiveness of SAR induction with respect to PR-gene expression and disease resistance, but also rendered the transgenic plants more resistant to infection by P. syringae in the absence of SAR induction (Cao et 1997).

NF-xB/IKB Signal Transduction Pathways NF-KB/IKB signaling pathways have been implicated in disease resistance responses in a range of organisms from Drosophila to mammals. In mammals,

NF-KB/IKB

signal transduction can be induced by a number of different stimuli including exposure of cells to lipopolysaccharide, tumor necrosis factor, interleukin 1 or virus infection (Baeuerle and Baltimore, Cell87, 13-20 (1996); Baldwin, Annu. Rev. Immunol. 14, 649-681 (1996)). The activated pathway leads to the synthesis of a number of factors involved in inflammation and immune responses, such as IL-2, IL-6, IL-8 and granulocyte/macrophagecolony stimulating factor (deMartin et al., Gene 152, 253-255 (1995)). In transgenic mouse studies, the knock out of NF-KB/IKB signal transduction leads to a defective immune WO 98/26082 PCT/EP97/07012 response including enhanced susceptibility to bacterial and viral pathogens (Beg and Baltimore, Science 274, 782-784 (1996); Van Antwerp et al., Science 274, 787-789 (1996); Wang et al., Science 274, 784-787 (1996); Baeuerle and Baltimore (1996)). In Arabidopsis, SAR is functionally analogous to inflammation in that normal resistance processes are potentiated following SAR activation leading to enhanced disease resistance (Bi et al., 1995; Cao et al., 1994; Delaney et al., 1995; Delaney et al., 1994; Gaffney et al., 1993; Mauch-Mani and Slusarenko 1996; Delaney, 1997). Furthermore, inactivation of the pathway leads to enhanced susceptibility to bacterial, viral and fungal pathogens.

Interestingly, SA has been reported to block NF-KB activation in mammalian cells (Kopp and Ghosh, Science 265, 956-959 (1994)), while SA activates signal transduction in Arabidopsis. Bacterial infection of Drosophila activates a signal transduction cascade leading to the synthesis of a number of antifungal proteins such as cercropin B, defensin, diptericin and drosomycin (Ip et al., Ce1/75, 753-763 (1993); Lemaitre et al., Ce1186, 973- 983 (1996)). This induction is dependent on the gene product of dorsal and dif, two NF-KB homologs, and is repressed by cactus, an IKB homolog, in the fly. Mutants that have decreased synthesis of the antifungal and antibacterial proteins have dramatically lowered resistance to infection.

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

The following definitions will assist in the understanding of the present invention.

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

Plant tissue: a group of plant cells organized into a structural and functional unit.

Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced WO 98/26082 PCT/EP97/07012 -6by this definition is not intended to be exclusive of any other type of plant tissue.

Protoplast: a plant cell without a cell wall.

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

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

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

Recombinant DNA technology: Technology which produces recombinant DNA in vitro and transfers the recombinant DNA into cells where it can be expressed or propagated (See, Concise Dictionary of Biomedicine and Molecular Biology, Ed. Juo, CRC Press, Boca Raton (1996)), for example, transfer of DNA into a protoplast(s) or cell(s) in various forms, including, for example, naked DNA in circular, linear or supercoiled forms,

DNA

contained in nucleosomes or chromosomes or nuclei or parts thereof, DNA complexed or associated with other molecules, DNA enclosed in liposomes, spheroplasts, cells or protoplasts or DNA transferred from organisms other than the host organism (ex.

Agrobacterium tumefiaciens). These and other various methods of introducing the recombinant DNA into cells are known in the art and can be used to produce the transgenic cells or transgenic plants of the present invention.

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

Also included as recombinant DNA technology is the insertion of a peroxidase coding sequence lacking selected expression signals into a monocot and assaying the transgenic monocot plant for increased expression of peroxidase due to endogenous control sequences in the monocot. This would result in an increase in copy number of peroxidase coding sequences within the plant.

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

Chimeric ene: A DNA molecule containing at least two heterologous parts, e.g., parts derived from pre-existing DNA sequences which are not associated in their pre-existing states, these sequences having been preferably generated using recombinant P\OPERMR\SPECM663 1.9 spc.do.3Il1lA)O -7- DNA technology.

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

Coding sequence: a DNA molecule which, when transcribed and translated, results in the formation of a polypeptide or protein.

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

The present invention describes the identification, isolation, and characterization of the NIM1 gene, which encodes a protein involved in the signal transduction cascade responsive to biological and chemical inducers that leads to systemic acquired resistance in plants.

Hence, according to one embodiment of the present invention there is provided a 15 recombinant DNA molecule that encodes an altered from of a NIM1 protein acting as a dominant-negative regulator of the SAR signal transduction pathway wherein said 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; 520mM NaPO 4 pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55 0 C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (Xl) at 55 0

C.

Within the scope of the present invention a DNA molecule is described that S encodes the NIM1 protein hybridizing under the following conditions to clone BAC-04, ATCC Deposit No. 97543: hybridization in 1%BSA; 520mM NaPO 4 pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55 0 C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55 0 C. In an especially preferred embodiment, the NIM1 gene is comprises within clone BAC-04, ATCC Deposit No. 97543.

-7A- Further described is a DNA molecule that encodes the NIM1 protein hybridizes under the following conditions to cosmid D7, ATCC Deposit No. 97736: hybridization in 1%BSA; 520mM NaPO 4 pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 550C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (Xl) at 550C. In an especially preferred embodiment, the NIM1 gene is comprised within cosmid D7, ATCC Deposit No. 97736.

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

Further described is an encoded NIM1 protein comprising the amino acid sequence set forth in SEQ ID NO:3. Further described is the NIM1 gene coding sequence hybridizing under the following conditions to the coding sequence set forth in SEQ ID NO:2: hybridization in 1%BSA; 520mM NaPO4, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 550C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C. In an especially preferred embodiment, the NIM1 gene coding sequence comprises the coding sequence set forth in SEQ ID NO:2.

The present invention also describes a chimeric gene comprising a promoter active in plants operatively linked to a NIM1 gene coding sequence, a recombinant vector comprising such a chimeric gene, wherein the vector is capable of being 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 following agronomically important crops: rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.

P:OPER\MKR\SPEC%631-9A s.doc-31J/07 -7B- In an especially preferred embodiment of the present invention there is provided a plant, plant cells and the descendants thereof, wherein NIM1 protein involved in the signal transduction cascade leading to systemic acquired resistance in plants is expressed in said transformed plant at higher levels than in a wild type plant.

In another embodiment the present invention is directed to a method of conferring a CIM phenotype to a plant cell, a plant and the descendants thereof, comprising transforming the plant with the recombinant vector comprising the chimeric gene comprising a promoter active in plants operatively linked to the DNA molecule that encodes a NIM1 protein involved in the signal transduction cascade leading to systemic acquired resistance in plants, wherein said vector is capable of being stably transformed into a host wherein said NIM1 protein is expressed in said transformed plant at higher levels than in a wild type plant.

In a further embodiment the present invention is directed to a method of activating systemic acquired resistance in a plant cell, a plant and the descendants thereof, 15 comprising transforming the plant with the recombinant vector comprising the chimeric gene comprising a promoter active in plants operatively linked to the DNA molecule that encodes a NIM1 protein involved in the signal transduction cascade leading to systemic acquired resistance in plants, wherein said vector is capable of being stably transformed into a host, wherein said NIM1 protein is expressed in said transformed plant at higher .20 levels than in a wild type plant.

In a further embodiment the present invention isdirected to a method of conferring broad spectrum disease resistance to a plant cell, a plant and the descendants thereof, S"comprising transforming the plant with the recombinant vector comprising the chimeric gene comprising a promoter active in plants operatively linked to the DNA molecule that encodes a NIM1 protein involved in the signal transduction cascade leading to systemic acquired resistance in plants, wherein said vector is capable of being stably transformed into a host, wherein said NIM1 protein is expressed in said transformed plant at higher levels than in a wild type plant.

P:OPER\MKR\SPECI36631-98 pe.doc-31M7O -8- In another embodiment the present invention is directed to a commercial bag comprising seed of a transgenic plant comprising at least one altered form of a NIM1 protein or a NIM1 protein that is expressed in said transformed plant at higher levels than in a wild type plant together with a suitable carrier in an amount sufficient to act as a dominant-negative regulator of the SAR signal transduction pathway, together with lable instructions for the use thereof for conferring broad spectrum disease resistance to plants.

Another aspect of the present invention exploits both the recognition that the SAR pathway in plants shows functional parallels to the NF-KB/kB regulation scheme in mammals and flies, as well as the discovery that the NIM1 gene product is a structural homologue of the mammalian signal transduction factor IKB subclass a. Mutations of IKB have been described that act as super-repressors or dominant-negatives of the NF-KB/IKB regulation scheme. The present invention encompasses altered forms of wild-type NIM1 gene (SEQ NO: 2) that act as dominant-negative regulators of the SAR signal transduction oooo o* ooo o« WO 98/26082 PCT/EP97/07012 -9pathway. These altered forms of NIM1 confer the opposite phenotype in plants transformed therewith as the niml mutant; plants plants transformed with altered forms of NIM1 exhibit constitutive SAR gene expression and a CIM phenotype.

Also comprised by the present invention are DNA molecules that hybridize to a DNA molecule according to the invention as defined hereinbefore, but preferably to an oligonucleotide probe obtainable from said DNA molecule comprising a contiguous portion of the coding sequence for the said altered forms of NIM1 at least 10 nucleotides in length, under moderately stringent conditions.

Factors that affect the stability of hybrids determine the stringency of the hybridization.

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

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

In one embodiment of the present invention, the NIM1 gene is altered so that the encoded product has alanines instead of serines in the amino acid positions corresponding to positions 55 and 59 of the 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, which results in changes of these serine residues to alanine residues, is presented in SEQ ID NO:22. An exemplary dominant-negative form of the NIM1 protein with alanines instead of serines at amino acid positions 55 and 59 is shown in SEQ ID NO:23. The present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under moderate stringent conditions to the coding sequence set forth in SEQ ID NO:22, especially preferred are the following conditions: hybridization in 1%BSA; 520mM NaPO4, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for min. (Xl) at 55 C. In these embodiments, alleles of NIM1 hybridizing to SEQ ID NO:22 under the above conditions are altered so that the encoded product has alanines instead of serines in the amino acid positions that correspond to positions 55 and 59 of SEQ ID NO:22.

In another embodiment of the present invention, the NIM1 gene is altered so that the encoded product has an N-terminal truncation, which removes lysine residues that may serve as potential ubiquitination sites in addition to the serines at amino acid positions corresponding to positions 55 and 59 of the wild-type protein. An example of a preferred WO 98/26082 PCT/EP97/07012 embodiment of this altered form of the NIM1 gene, which encodes a gene product having an N-terminal deletion, is presented 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 also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under moderate stringent conditions to the coding sequence set forth in SEQ ID NO:24; especially preferred are the following conditions: hybridization in 1%BSA; 520mM NaPO4, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55°C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55 C. In these embodiments, alleles of NIM1 hybridizing to SEQ ID NO:24 under the above conditions are altered so that the encoded product has an Nterminal deletion that removes lysine residues that may serve as potential ubiquitination sites in addition to the serines at amino acid positions corresponding to positions 55 and 59 of the wild-type gene product.

In still another embodiment of the present invention, the NIM1 gene is altered so that the encoded product has a C-terminal truncation, which is believed to result in enhanced intrinsic stability by blocking the constitutive phosporylation of serine and threonine residues in the C-terminus of the wild-type gene product. An example of a preferred embodiment of this altered form of the NIM1 gene, which encodes a gene product having a C-terminal deletion, is presented 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 encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under moderate stringent conditions to the coding sequence set forth in SEQ ID NO:26; especially preferred are the following conditions: hybridization in 1%BSA; 520mM NaPO 4 pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55 C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 550C.

In these embodiments, alleles of NIM1 hybridizing to SEQ ID NO:26 under the above conditions are altered so that the encoded product has a C-terminal deletion that removes serine and threonine residues.

In yet another embodiment of the present invention, the NIM1 gene is altered so that the encoded product has both an N-terminal deletion and a C-terminal truncation, which provides the benefits of both the above-described embodiments of the invention.

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

WO 98/26082 PCT/EP97/07012 -11 An example of a preferred embodiment of this altered form of the NIM1 gene, which encodes a gene product having both an N-terminal and a C-terminal deletion, is presented in SEQ ID NO:28. An exemplary dominant-negative form of the NIM1 protein with a Cterminal deletion is shown in SEQ ID NO:29. The present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under the moderate stringent conditions to the coding sequence set forth in SEQ ID NO:28; especially preferred are the following conditions: hybridization in 1%BSA; 520mM NaPO 4 pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55 0 C for 18- 24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55 0 C. In these embodiments, alleles of NIM1 hybridizing to SEQ ID NO:28 under the above conditions are altered so that the encoded product has both an N-terminal deletion, which removes lysine residues that may serve as potential ubiquitination sites in addition to the serines at amino acid positions corresponding to positions 55 and 59 of the wild-type gene product, as well as a C-terminal deletion, which removes serine and threonine residues.

In even another embodiment of the present invention, the NIM1 gene is altered so that the encoded product consists essentially of only the ankyrin domains of the wild-type gene product. Preferred is an isolated DNA molecule, wherein said altered form of the NIM1 protein consists essentially of ankyrin motifs corresponding approximately to amino acid positions 103-362 of SEQ ID NO:3. An example of a preferred embodiment of this altered form of the NIM1 gene, which encodes the ankyrin domains, is presented in SEQ ID An exemplary dominant-negative form of the NIM1 protein consists essentially of only the ankyrin domains is shown in SEQ ID NO:31. The present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under the moderate stringent conditions to the coding sequence set forth in SEQ ID especially preferred are the following conditions: hybridization in 1%BSA; 520mM NaPO 4 pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 550C for 18- 24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55 0 C. In these embodiments, alleles of NIM1 hybridizing to SEQ ID NO:30 under the above conditions are altered so that the encoded product consists essentially of the ankyrin domains of the wildtype gene product.

Thus, the present invention concerns DNA molecules encoding altered forms of the NIM1 gene, such as those described above and all DNA molecules hybridizing therewith using moderate stringent conditions.

The present invention also encompasses a chimeric gene comprising a promoter active in plants operatively linked to one of the above-described altered forms of the NIM1 gene, a recombinant vector comprising such a chimeric gene, wherein the vector is capable WO 98/26082 PCT/EP97/07012 -12of 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 and the descendants thereof from, for example, one of the following agronomically important crops: rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.

The present invention is also directed to a method of conferring a CIM phenotype to a plant by transforming the plant with a recombinant vector comprising a chimeric gene that itself comprises a promoter active in plants operatively linked to one of the above-described altered forms of the NIM1 gene, wherein the encoded dominant-negative form of the NIM1 protein is expressed in the transformed plant and confers a CIM phenotype to the plant.

Further, the present invention is directed to a method of activating systemic acquired resistance in a plant by transforming the plant with a recombinant vector comprising a chimeric gene that itself comprises a promoter active in plants operatively linked to one of the above-described altered forms of the NIM1 gene, wherein the encoded dominantnegative form of the NIM1 protein is expressed in the transformed plant and activates systemic acquired resistance in the plant.

In addition, the present invention is directed to a method of conferring broad spectrum disease resistance to a plant by transforming the plant with a recombinant vector comprising a chimeric gene that itself comprises a promoter active in plants operatively linked to one of the above-described altered forms of the NIM1 gene, wherein the encoded dominant-negative form of the NIM1 protein is expressed in the transformed plant and confers broad spectrum disease resistance to the plant.

In yet another aspect, the present invention is directed to a method of screening for a NIM1 gene involved in the signal transduction cascade leading to systemic acquired resistance in a plant, comprising probing a genomic or cDNA library from said plant with a NIM1 coding sequence that hybridizes under the following set of conditions to the coding sequence set forth in SEQ ID NO:2: hybridization in 1%BSA; 520mM NaPO 4 pH7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 550C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (Xl) at 550C.

Further subjects encompassed by the invention are: An isolated DNA molecule according to the invention wherein said altered form of the NIM1 protein has alanines instead of serines in amino acid positions corresponding to positions WO 98/26082 PCT/EP97/07012 -13and 59 of SEQ ID NO:3, wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID NO:22: hybridization in 1%BSA; 520mM NaPO4, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55°C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 550C.

An isolated DNA molecule according to the invention wherein said altered form of the NIM1 protein has an N-terminal truncation of amino acids corresponding approximately to amino acid positions 1-125 of SEQ ID NO:3, wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID NO:24: hybridization in 1%BSA; 520mM NaPO 4 pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55°C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for min. (X1) at 550C.

An isolated DNA molecule according to the invention wherein said altered form of the NIM1 protein has a C-terminal truncation of amino acids corresponding approximately to amino acid positions 522-593 of SEQ ID NO:3, wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID NO:26: hybridization in 1%BSA; 520mM NaPO4, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55 C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for min. (X1) at 550C.

An isolated DNA molecule according to the invention, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO:28, wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID NO:28: hybridization in 1%BSA; 520mM NaPO4, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55°C for 18-24h, and wash in 6XSSC for min. (X3) 3XSSC for 15 min. (X1) at 550C.

An isolated DNA molecule according to the invention wherein said altered form of the NIM1 protein consists essentially of ankyrin motifs corresponding approximately to amino acid positions 103-362 of SEQ ID NO:3, wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID NO:30: hybridization in 1%BSA; 520mM NaPO4, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55 C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for min. at WO 98/26082 PCT/EP9/0712 WO 9826082PCTEP97O701 2 -14- An altered form of a NIM1 gene according to the invention, which has been constructed by mutagenization.

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

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

Use of an isolated DNA molecule according to the invention to confer a CIM phenotype to a plant cell, a plant and the descendants thereof.

Use of resistant plants and the descendants thereof according to the invention to incorporate the disease resistant trait into plant lines through breeding.

Use of variants of the NIM1 gene to confer disease resistance and activate SAR gene expression in plants transformed therewith.

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

A method of producing transgenic descendants of a transgenic parent plant comprising an isolated DNA molecule encoding an altered form of a NIM1 protein according to the invention comprising transforming said parent plant with a recombinant vector molecule according to the invention and transferring the trait to the descendants of said transgenic parent plant involving 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 preparing a nucleotide probe capable of specifically hybridizing to an altered form of a NIM1 gene or mRNA, wherein said probe comprises a contiguous portion of the coding sequence for an altered form of a NIM1 of at least 10 nucleotides length; probing for other altered forms of a NIM1 coding sequence in populations of cloned genomic DNA fragments or cDNA fragments from a chosen organism using the nucleotide probe prepared according to step and isolating and multiplying a DNA molecule comprising a DNA portion containing a DNA portion encoding an altered form of a NIM1 protein.

WO 98/26082 PCT/EP97/07012 A method of isolating a DNA molecule comprising a DNA portion containing an altered form of a NIM1 sequence comprising preparing a nucleotide probe capable of specifically hybridizing to an altered form of a NIM1 gene or mRNA, wherein said probe comprises a contiguous portion of the coding sequence for an altered form of a NIM1 protein from a plant of at least 10 nucleotides length; probing for other altered forms of NIM1 sequences in populations of cloned genomic DNA fragments or cDNA fragments from a chosen organism using the nucleotide probe prepared according to step and isolating a DNA molecule comprising a DNA portion containing an altered form of a NIM1 gene.

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

A method of producing transgenic plants that express higher-than-wild-type levels of the NIM1 gene, or functional variants and mutants thereof, wherein the expression of the NIM1 gene is at a level which is at least two-fold above the expression level of the NIM1 gene in wild-type plants.

A method of producing transgenic plants that express higher-than-wild-type levels of the NIM1 gene, or functional variants and mutants thereof, wherein the expression of the NIM1 gene is at a level which is at least ten-fold above 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 are defective in their normal response to pathogen infection in that they do not express genes associated with SAR. These mutants are referred to as nim mutants (for non-inducible immunity) and are "universal disease susceptible" (UDS) by virtue of their being susceptible to many strains and pathotypes of pathogens of the host plant and also to pathogens that do not normally infect the host plant, but that normally infect other hosts.

Such mutants can be selected by treating seeds or other biological material with mutagenic agents and then selecting descendant plants for the UDS phenotype by treating WO 98/26082 PCT/EP97/07012 -16descendant plants with known chemical inducers INA) of SAR and then infecting the plants with a known pathogen. Non-inducible mutants develop severe disease symptoms under these circumstances, whereas wild type plants are induced by the chemical compound to systemic acquired resistance. nim mutants can be equally selected from mutant populations generated by chemical and irradiation mutagenesis, as well as from populations generated by T-DNA insertion and transposon-induced mutagenesis.

Techniques of generating mutant plant lines are well known in the art.

nim mutants provide useful indicators of the evaluation of disease pressure in field pathogenesis tests where the natural resistance phenotype of so-called wild type nonmutant) plants may vary and therefore not provide a reliable standard of susceptibility.

Furthermore, nim plants have additional utility for the testing of candidate disease resistance transgenes. Using a nim stock line as a recipient for transgenes, the contribution of the transgene to disease resistance is directly assessable over a base level of susceptibility. Furthermore, the nim plants are useful as a tool in the understanding of plant-pathogen interactions. nim host plants do not mount a systemic response to pathogen attack, and the unabated development of the pathogen is an ideal system in which to study its biological interaction with the host.

As nim host plants may also be susceptible to pathogens outside of the host-range they normally fall, these plants also have significant utility in the molecular, genetic, and biological study of host-pathogen interactions. Furthermore, the UDS phenotype of nim plants also renders them of utility for fungicide screening. nim mutants selected in a particular host have considerable utility for the screening of fungicides using that host and pathogens of the host. The advantage lies in the UDS phenotype of the mutant, which circumvents the problems encountered by hosts being differentially susceptible to different pathogens and pathotypes, or even resistant to some pathogens or pathotypes.

nim mutants have further utility for the screening of fungicides against a range of pathogens and pathotypes using a heterologous host, i.e. a host that may not normally be within the host species range of a particular pathogen. Thus, the susceptibility of nim mutants of Arabidopsis to pathogens of other species crop plant species) facilitates efficacious fungicide screening procedures for compounds against important pathogens of crop plants.

The Arabidopsis thaliana nim Mutant An Arabidopsis thaliana mutant called nimi (noninducible immunity) that supports P.

parasitica causal agent of downy mildew disease) growth following INA treatment is WO 98/26082 PCT/EP97/07012 -17described in Delaney et al., 1995. Although niml can accumulate SA following pathogen infection, neither SAR gene expression nor disease resistance can be induced, suggesting that the mutation blocks the pathway downstream of SA. niml is also impaired in its ability to respond to INA or BTH, suggesting that the block exists downstream of the action of these chemicals (Delaney et al., 1995; Lawton et al., 1996). This first Arabidopsis niml mutant (herein designated 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 fungal growth after INA treatment were selected as putative mutants. Five additional mutants (herein designated niml-2, niml-3, niml-4, niml-5, and niml-6) were isolated from 280,000 M 2 plants from an ethyl methanesulfonate (EMS)-mutagenized Ws-0 population.

To determine whether the mutants were dominant or recessive, Ws-0 plants were used as pollen donors to cross to each of these mutants. The F, plants were then scored for their ability to support fungal growth following INA treatment. As shown in Table 3 of the Examples, all niml-1, nim1-2, niml-3, niml-4, and niml-6 F, plants were phenotypically wild type, indicating a recessive mutation in each line. nim1-5 showed the nim phenotype in all 35 F, plants, indicating that this particular mutant is dominant. For verification, the reciprocal cross was carried out using niml-5as the pollen donor to fertilize Ws-0 plants. In this case, all 18 F, plants were phenotypically nim, confirming the dominance of the mutation.

To determine whether the nim 1-2 through nim 1-6 mutations were allelic to the previously characterized nim -1 mutation, pollen from nim -1 was used to fertilize nim1-2 through niml-6. Because niml-1 carried resistance to kanamycin, F, descendants were identified by antibiotic resistance. In all cases, the kanamycin-resistant F, plants were nim, indicating they were all allelic to the niml-1 mutant. Because the nimi-5 mutant is dominant and apparently homozygous for the mutation, it was necessary to analyze niml-I complementation in the F 2 generation. If niml-1 and niml-5were allelic, then the expectation would be that all F 2 plants have a nim phenotype. If not, then 13 of 16 F 2 plants would have been expected to have a nim phenotype. Of 94 plants, 88 clearly supported fungal growth following INA treatment. Six plants showed an associated phenotype of black specks on the leaves reminiscent of a lesion mimic phenotype and supported little fungal growth following INA treatment. Because niml-5 carries a point mutation in the NIM1 gene (infra), it is considered to be a niml allele.

To determine the relative strength of the different niml alleles, each mutant was analyzed for the growth of P. parasitica under normal growth conditions and following WO 98/26082 PCT/EP97/07012 -18pretreatment with either SA, INA, or BTH. As shown in Table 1, during normal growth, nim nimi-2, nimi-3, nim1-4, and nim1-6 all supported approximately the same rate of fungal growth, which was somewhat faster than the Ws-0 control. The exception was the plants, in which fungal growth was delayed by several days relative to both the other nim mutants and the Ws-0 control, but eventually all of the nim -5 plants succumbed to the fungus. Following SA treatment, the mutants could be grouped into three classes: niml-4 and nim1-6 showed a relatively rapid fungal growth; niml-1, niml-2, nim1-3 plants exhibited a somewhat slower rate of fungal growth; and fungal growth in nimi-5 plants was even slower than in the untreated Ws-0 controls. Following either INA or BTH treatment, the mutants also seemed to fall into three classes where niml-4 was the most severely compromised in its ability to restrict fungal growth following chemical treatment; niml-1, nimi-2, niml-3, and nimi-6were all moderately compromised; and nim1-5 was only slightly compromised. In these experiments, Ws-0 did not support fungal growth following INA or BTH treatment. Thus, with respect to inhibition of fungal growth following chemical treatment, the mutants fall into three classes with nim -4 being the most severely compromised, nim nim1-2, nim1-3 and nim1-6 showing an intermediate inhibition of fungus and nim 1-5 with only slightly impaired fungal resistance.

The accumulation of PR-1 mRNA was also used as a criterion to characterize the different niml alleles. RNA was extracted from plants 3 days after either water or chemical treatment, or 14 days after inoculation with a compatible fungus parasitica isolate Emwa). The RNA gel blot in Figure 3 shows that PR-1 mRNA accumulated to high levels following treatment of wild-type plants with SA, INA, or BTH or infection by P. parasitica. In the niml-1, nim1-2, and niml-3 plants, PR-1 mRNA accumulation was dramatically reduced relative to the wild type following chemical treatment. PR-1 mRNA was also reduced following P. parasitica infection, but there was still some accumulation in these mutants. In the nimi-4 and nimi-6 plants, PR-1 mRNA accumulation was more dramatically reduced than in the other alleles following chemical treatment (evident in longer exposures) and significantly less PR-1 mRNA accumulated following P. parasitica infection, supporting the idea that these could be particularly strong niml alleles. Interestingly, PR-1 mRNA accumulation was elevated in the nim1-5 mutant, but only mildly induced following chemical treatment or P. parasitica infection. Based on both PR-1 mRNA accumulation and fungal infection, the mutants fall into three classes: severely compromised alleles (niml-4 and nim1-6); moderately compromised alleles (nim nim1-2, and nim1-3); and a weakly compromised allele WO 98/26082 PCT/EP97/n7012 -19- Fine Structure Mapping of the niml Mutation To determine a rough map position for NIM1, 74 F 2 nim phenotype plants from a cross between niml-1 (Ws-0) and Landsberg erecta (Le) were identified for their susceptibility to P. parasitica and lack of accumulation of PR-1 mRNA following INA treatment. After testing a number of simple sequence length polymorphism (SSLP) markers (Bell and Ecker 1994), niml was found to lie about 8.2 centimorgans (cM) from nga128 and 8.2 cM from ngall11 on the lower arm of chromosome 1. In subsequent analysis, niml-1 was found to lie between ngal 11 and about 4 cM from the SSLP marker ATHGENEA.

For fine structure mapping, 1138 nim plants from an F 2 population derived from a 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 following INA treatment.

DNA was extracted from these plants and scored for zygosity at both ATHGENEA and ngal 11. As shown in Figures 5A-5D, 93 recombinant chromosomes were identified between ATHGENEA and niml, giving a genetic distance of approximately 4.1 cM (93 of 2276), and 239 recombinant chromosomes were identified between ngal 11 and niml, indicating a genetic distance of about 10.5 cM (239 of 2276). Informative recombinants in the ATHGENEA to ngal 11 interval 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 these were used to construct a low resolution map of the region (Figure 5A). The AFLP markers W84.2 (1 cM from nimi) and W85.1 (0.6 cM from niml) were used to isolate yeast artificial chromosome (YAC) clones from the CIC (for Centre 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 (data not shown) were identified with the W85.1 marker. However, it was determined that there was a gap between the two sets of flanking YAC clones. From this point, bacterial artificial chromosome (BAC) and P1 clones that overlapped CIC12H07 and CIC12F04 were isolated and mapped, and three sequential walking steps were then carried out extending the BAC/P1 contig toward NIM1 (Liu et al., 1995; Chio et al., 1995). At various times during the walk, new AFLPs were developed that were specific for BAC or P1 clones, and these were used to determine whether the NIM1 gene had been crossed. It was determined that NIM1 had been crossed when BAC and P1 clones were isolated that gave rise to both AFLP markers L84.6a and L84.8. The AFLP marker L84.6a found on P1 clones P1-18, P1-17, and P1-21 identified three recombinants and L84.8 found on P1 clones P1-20, P1- 22, P1-23, and P1-24 and BAC clones, BAC-04, BAC-05, and BAC-06 identified one WO 98/26082 PCT/EP97/n7n12 recombinant. Because these clones overlap to form a large contig (>100 kb), and include AFLP markers that flank niml, the gene was located on the contig. The BAC and P1 clones that comprised the contig were used to generate eight additional AFLP markers, which showed that niml was located between L84.Y1 and L84.8, representing a gap 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 P1-18. A cosmid contig was developed using AFLP markers derived from these clones. Physical mapping showed that the physical distance between L84.Y1 and L84.8 was greater than 90 kb, giving a genetic to physical distance of roughly 1 megabase per cM. To facilitate the later identification of the NIM1 gene, the DNA sequence of BAC-04 was determined.

Isolation of the NIM1 Gene To identify which cosmids contained the NIM1 gene, the 12 cosmids listed in Table 4 of the Examples were transformed into nim and transformants were evaluated for their ability to complement the mutant phenotype. Cosmids D5, El, and D7 were all found to complement niml-1, as determined by the ability of the transformants to accumulate PR-1 mRNA following INA treatment. The ends of these cosmids were sequenced and found to be located on the DNA sequence of BAC-04. There were 9,918 base pairs in the DNA region shared by D7 and D5 that contained the NIM1 gene. As shown in Figure 5D, four putative gene regions were identified in this 10-kb sequence. Region 1 could potentially encode a protein of 19,105 D, region 3 could encode a protein of 44,554 D, and region 4 could encode a protein of 52,797 D. Region 2 had four open reading frames of various sizes located close together, suggesting a gene with three introns. Analysis using the NetPlantGene program (Hebsgaard et al., 1996) indicated a high probability that the open reading frames could be spliced together to form a large open reading frame encoding a protein of 66,039 D.

To ascertain which gene region contained the NIM1 gene, gel blots containing RNA isolated from leaf tissue of Ws-0 and the different niml mutants following either water or chemical treatment were probed with DNA derived from each of the four gene regions. In these experiments, care was taken to label probes to high specific activity and autoradiographs were exposed for more than 1 week. In our past experience, these conditions would 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 probe was induced by BTH treatment of wild-type plants, but WO 98/26082 PCT/EP97/07012 -21 not in any of the mutants. Furthermore, RNA accumulation was elevated in all of the plants following P. parasitica infection, indicating that this particular gene is induced following pathogen infection.

To further establish the gene region encoding NIM1, the DNA sequence from each of the four gene regions was determined for each of the nim alleles and compared with the corresponding gene region from Ws-0. No mutations were detected between Ws-0 and the mutant alleles in either gene regions 3 or 4 and only a single change was found in gene region 1 in the nim1-6 mutant. However, a single base pair mutation was found in each of the alleles relative to Ws-0 for region 2. The DNA sequence of 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 that causes a frameshift resulting in a change in seven amino acids and a deletion of 349 amino acids. In niml-2, there is a cytidine-to-thymidine transition at position 3763 that changes a histidine to a tyrosine. In niml-3, a single adenosine is deleted at position 3301 causing a frameshift that altered 10 amino acids and deleted 412 from the predicted protein. Interestingly, both niml-4 and niml-5 have a guanosine-to-adenosine transition at position 4160 changing an arginine to a lysine, and in niml-6, there is a cytosine-to-thymidine transition resulting in a stop codon causing the deletion of 255 amino acids from the predicted protein. Although the mutation in nim1-4 and nim 1-5 alters the consensus donor splice site for the mRNA, RT-PCR analysis indicates that this mutation does not lead to an alteration of mRNA splicing (data not shown).

NIM1 Homologues The gene region 2 DNA sequence was used in a Blast search (Altschul et al., 1990) and identified an exact match with the Arabidopsis expressed sequence tag (EST) T22612 and significant matches to the rice ESTs S2556, S2861, S3060 and S3481 (see Figure 8).

A DNA probe covering base pairs 2081 to 3266 was used to screen an Arabidopsis cDNA library, and 14 clones were isolated that correspond to gene region 2. From the cDNA sequence, we could confirm the placement of the exon/intron borders shown in Figure 6.

Rapid amplification of cDNA ends by polymerase chain reaction (RACE) was carried out using RNA from INA-treated Ws-0 plants and the likely transcriptional start site was determined to be the A at position 2588 in Figure 6.

Using the NIM1 cDNA as a probe, homologs of Arabidopsis NIM1 can be identified and isolated through screening genomic or cDNA libraries from different plants such as, but not limited to following crop plants: rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, WO 98/26082 PCT/EP97/07012 -22spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane. Standard techniques for accomplishing this include hybridization screening of plated DNA libraries (either plaques or colonies; see, e.g. Sambrook et al., Molecular Cloning eds., Cold Spring Harbor Laboratory Press. (1989)) and amplification by PCR using oligonucleotide primers (see, e.g.

Innis et al., PCR Protocols, a Guide to Methods and Applications eds., Academic Press (1990)). Homologues identified are genetically engineered into the expression vectors listed below and transformed into the above listed crops. Transformants are evaluated for enhanced disease resistance using relevant pathogens of the crop plant being tested.

For example, NIM1 homologs in the genomes of cucumber, tomato, tobacco, maize, wheat and barley have been detected by DNA blot analysis. Genomic DNA was isolated from cucumber, tomato, tobacco, maize, wheat and barley, restriction digested with the enzymes BamHI, Hindlll, Xbal, or Sail, electrophoretically separated on 0.8% agarose gels and transferred to nylon membrane by capillary blotting. Following UV-crosslinking to affix the DNA, the membrane was hybridized under low stringency conditions [(1%BSA; 520mM NaPO 4 pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride) at 0 C for 18-24h] with 32 P-radiolabelled Arabidopsis thaliana NIM1 cDNA. Following hybridization the blots were washed under low stringency conditions [6XSSC for 15 min.

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

In addition, expressed sequence tags (EST) identified with similarity to the NIM1 gene such as the rice EST's described above can also be used to isolate homologues. The rice EST's may be especially useful for isolation of NIM1 homologues from other monocots.

Homologues may also be obtained by PCR. In this method, comparisons are made between known homologues rice and Arabidopsis). Regions of high amino acid and DNA similarity or identity are then used to make PCR primers. Once a suitable region is identified, primers for that region are made with a diversity of substitutions in the 3 rd codon position. The PCR reaction is performed from cDNA or genomic DNA under a variety of standard conditions. When a band is apparent, it is cloned and/or sequences to determine if it is a NIM1 homologue.

WO 98/26082 PCT/EP97/07012 -23- Overexpression of NIM1 Confers Disease Resistance In Plants The present invention also concerns the production of transgenic plants that express higher-than-wild-type levels of the NIM1 gene, or functional variants and mutants thereof, and thereby have broad spectrum disease resistance. In a preferred embodiment of the invention, the expression of the NIM1 gene is at a level which is at least two-fold above the expression level of the NIM1 gene in wild-type plants and is preferably tenfold above the wild-type expression level. Overexpression of the NIM1 gene mimics the effects of inducer compounds in that it gives rise to plants with a constitutive immunity (CIM) phenotype.

Several methods are described for producing plants that overexpress the NIM1 gene and thereby have a CIM phenotype. A first method is selecting transformed plants that have high-level expression of NIM1 and therefore a CIM phenotype due to insertion site effect. Table 6 shows the results of testing of various transformants for resistance to fungal infection. As can be seen from this table, a number of transformants showed less than normal fungal growth and several showed no visible fungal growth at all. RNA was prepared from collected samples and analyzed as described in Delaney et al, 1995. Blots were hybridized to the Arabidopsis gene probe PR-1 (Uknes et al, 1992). Three lines showed early induction of PR-1 gene expression in that PR-1 mRNA was evident by 24 or 48 hours following fungal treatment. These three lines also demonstrated resistance to fungal infection.

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

The NIM1 Gene Is A Homolog Of IkBa The NIM1 gene is a key component of the systemic acquired resistance (SAR) pathway in plants (Ryals et al.,1996). The NIM1 gene is associated with the activation of SAR by chemical and biological inducers and, in conjunction with such inducers, is required for SAR and SAR gene expression. The location of the NIM1 gene was determined by molecular biological analysis of the genome of mutant plants known to carry the mutant niml gene, which gives the host plants extreme sensitivity to a wide variety of pathogens and renders them unable to respond to pathogens and chemical inducers of SAR. The WO 98/26082 PCT/EP97/07012 -24 wildtype NIM1 gene of Arapidopsis has been mapped and sequenced (SEQ ID NO:2). The wild-type NIM1 gene product (SEQ ID NO:3) is involved in the signal transduction cascade leading to both SAR and gene-for-gene disease resistance in Arabidopsis (Ryals et al., 1997). Recombinant overexpression of the wild-type form of NIM1 gives rise to plants with a constitutive immunity (CIM) phenotype and therefore confers disease resistance in transgenic plants. Increased levels of the active NIM1 protein produce the same diseaseresistance effect as chemical induction with inducing chemicals such as BTH, INA, and SA.

The sequence of the NIM1 gene (SEQ ID NO:2) was used in BLAST searches, and matches were identified based on homology of one rather highly conserved domain in the NIM1 gene sequence to ankyrin domains found in a number of proteins such as spectrins, ankyrins, NF-KB and IKB (Michaely and Bennett, Trends Cell Biol 2, 127-129 (1992)).

Beyond the ankyrin motif, however, conventional computer analysis did not detect other strong homologies, including homology to IKBa. Despite the failings of the computer programs, pair-wise visual inspections between the NIM1 protein (SEQ ID NO:3) and known ankyrin-containing proteins were carried out, and striking similarities were found to members of the IxBa class of transcription regulators (Baeuerle and Baltimore 1996; Baldwin 1996). As shown in Figure 9, the NIM1 protein (SEQ ID NO:3) shares significant homology with IKBa proteins from mouse, rat, and pig (SEQ ID NOs: 18, 19, and respectively).

NIM1 contains several important structural domains of lKBec throughout the entire length of the protein, including ankyrin domains (indicated by the dashed underscoring in Figure 2 amino-terminal serines (amino acids 55 and 59 of NIM1) a pair of lysines (amino acids 99 and 100 in NIM1) and an acidic C-terminus. Overall, NIM1 and IKBa share identity at 30% of the residues and conservative replacements at 50% of the residues.

Thus, there is homology between IKBa and NIM1 throughout the proteins, with an overall similarity of One way in which IhBa protein functions in signal transduction is by binding to the cytosolic transcription factor NF-KB and preventing it from entering the nucleus and altering transcription of target genes (Baeuerle and Baltimore, 1996; Baldwin, 1996). The target genes of NF-KB regulate (activate or inhibit) several cellular processes, including antiviral, antimicrobial and cell death responses (Baeuerle and Baltimore, 1996). When the signal transduction pathway is activated, IkBa is phosphorylated at two serine residues (amino acids 32 and 36 of Mouse IrBa). This programs ubiquitination at a double lysine (amino acids 21 and 22 of Mouse IKBa). Following ubiquitination, the NF-KB/IKB complex is routed through the proteosome where IKBa is degraded and NF-KB is released to the nucleus.

WO 98/26082 PCT/EP97/07012 The phosphorylated serine residues important in liBa function are conserved in NIM1 within a large contiguous block of conserved sequence from amino acids 35 to 84 (Figure In contrast to IkBa, where the double lysine is located about 15 amino acids toward the N-terminus of the protein, in NIM1 a double lysine is located about 40 amino acids toward the C-terminal end. Furthermore, a high degree of homology exists between NIM1 and IKBa in the serine/threonine rich carboxy terminal region which has been shown to be important in basal turnover rate (Sun et al., Mol. Cell. Biol. 16, 1058-1065 (1996)).

According to the present invention based on the analysis of structural homology and the presence of elements known to be important for IkBa function, NIM1 is expected to function like the IKBa, having analogous effects on plant gene regulation.

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

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

Altered Forms Of The NIM1 Gene In view of the above, the present invention encompasses altered forms of NIM1 that act as dominant-negative regulators of the SAR signal transduction pathway. Plants transformed with these dominant negative forms of NIM1 have the opposite phenotype as niml mutant plants in that the plants transformed with altered forms of NIM1 exhibit constitutive SAR gene expression and therefore a CIM phenotype. Because of the position the NIM1 gene holds in the SAR signal transduction pathway, it is expected that a number of alterations to the gene, beyond those specifically disclosed herein, will result in constitutive expression of SAR genes and, therefore, a CIM phenotype.

WO 98/26082 PCTEP97/07f12 26 Phosphorylation of serine residues in human IKBa is required for stimulus activated degradation of IKBa thereby activating NF-iB. Mutagenesis of the serine residues (S32 and S36) in human IKBa to alanine residues inhibits stimulus-induced phosphorylation, thus blocking IkBa proteosome-mediated degradation (Traenckner et al., 1995; Brown et a., 1996; Brockman et al., 1995; Wang et al., 1996). This altered form of IKBa can function as a dominant-negative form by retaining NF-KB in the cytoplasm thereby blocking downstream signaling events. Based on the amino acid sequence comparison between NIM1 and IKB shown in Figure 9, serines 55 (S55) and 59 (S59) in NIM1 (SEQ ID NO:3) are homologous to S32 and S36 in human IKBa. To construct dominant-negative forms of NIM1, the serines at amino acid positions 55 and 59 are mutagenized to alanine residues. Thus, in a preferred embodiment of the present invention, the NIM1 gene is altered so that the encoded product has alanines instead of serines in the amino acid positions corresponding to positions 55 and 59 of the Arabidopsis NIM1 amino acid sequence. The present invention also encompasses 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 therewith.

Deletion of amino acids 1-36 (Brockman et al, 1995; Sun et al, 1996) or 1-72 (Sun et al., 1996) of human IkBa, which includes ubiquination lysine residues K21 and K22 as well as phosphorylation sites S32 and S36, results in a dominant-negative IkBa phenotype in transfected human cell cultures. An N-terminal deletion of the first 125 amino acids of the NIM1 gene product will remove eight lysine residues which could serve as ubiquination sites as well as the putative phosphorylation sites at S55 and S59 discussed above. Thus, in a preferred embodiment of the present invention, the NIM1 gene is altered so that the encoded product is missing approximately the first 125 amino acids compared to the native Arabidopsis NIM1 amino acid sequence. The present invention also encompasses diseaseresistant 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 therewith.

Deletion of amino acids 261-317 of human IkBa may result in enhanced intrinsic stability by blocking constitutive phosphorylation of serine and threonine residues in the Cterminus. This altered form of IKBa is expected to function as a dominant-negative form. A region rich in serine and threonine is present at amino acids 522-593 in the C-terminus of NIM1. Thus, in a preferred embodiment of the present invention, the NIM1 gene is altered so that the encoded product is missing approximately its C-terminal portion, including amino acides 522-593, compared to the native Arabidopsis NIM1 amino acid sequence. The WO 98/26082 PCT/EPW/7nfl 2 -27present invention also encompasses 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 therewith.

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

The present invention concerns DNA molecules encoding altered forms of the NIM1 gene such as those described above, expression vectors containing such DNA molecules, and plants and plant cells transformed therewith. The invention also concerns methods of activating SAR in plants and conferring to plants a CIM phenotype and broad spectrum disease resistance by transforming the plants with DNA molecules encoding altered forms of the NIM1 gene product. The present invention additionally concerns plants transformed with an altered form of the NIM1 gene.

Disease Resistance The overexpression of the wild-type NIM1 gene in plants and the expression of altered forms of the NIM1 gene in plants results in immunity to a wide array of plant pathogens, which include, but are not limited to viruses or viroids, e.g. tobacco or cucumber mosaic virus, ringspot virus or necrosis virus, pelargonium leaf curl virus, red clover mottle virus, tomato bushy stunt virus, and like viruses; fungi, e.g. Phythophthora parasitica and Peronospora tabacina; bacteria, e.g. Pseudomonas syringae and Pseudomonas tabac, insects such as aphids, e.g. Myzus persicae; and lepidoptera, Heliothus spp.; and nematodes, Meloidogyne incognita. The vectors and methods of the invention are useful against a number of disease organisms including but not limited to downy mildews such as Scleropthora macrospora, Sclerophthora rayissiae, Sclerospora graminicola, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora sacchari and Peronosclerospora maydis; rusts such as Puccinia sorphi, Puccinia polysora 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.

WO 98/26082 PCT/EP97/07012 -28- The methods of the present invention can be utilized to confer disease resistance to a wide variety of plants, including gymnosperms, monocots, and dicots. Although disease resistance can be conferred upon any plants falling within these broad classes, it is particularly useful in agronomically important crop plants, such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.

Transformed cells can be regenerated into whole plants such that the gene imparts disease resistance to the intact transgenic plants. The expression system can be modified so that the disease resistance gene is continuously or constitutively expressed.

Recombinant DNA Technology The NIM1 DNA molecule or gene fragment conferring disease resistance to plants by allowing induction of SAR gene expression or the altered form of the NIM1 gene conferring disease resistance to plants by enhancing SAR gene expression can be incorporated in plant or bacterial cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule comprised within SEQ ID NO:1 or a functional variant thereof or a molecule encoding one of the altered forms of NIM1 described above into an expression system to which the DNA molecule is heterologous not normally present).

The heterologous DNA molecule is inserted into the expression system or vector in proper orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences. A large number of vector systems known in the art can be used, such as plasmids, bacteriophage viruses and other modified viruses. Suitable vectors include, but are not limited to, viral vectors such as lambda vector systems Xgtll, Agtl0 and Charon 4; plasmid vectors such as pBI121, pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101, pCDNAII; and other similar systems. The NIM1 coding sequence and the altered NIM1 coding sequences described herein can be cloned into the vector using standard cloning procedures in the art, 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 that will result in a sufficient expression level or constitutive WO 98/26082 PCT/EP97/07012 -29expression must be present in the expression vector. RNA polymerase normally binds to the promoter and initiates transcription of a gene. Promoters vary in their strength, ability to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters can be used. The components of the expression cassette may be modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications may be employed. Plant cells transformed with such modified expression systems, then, exhibit overexpression or constitutive expression of genes necessary for activation of SAR.

A. Construction of Plant Transformation Vectors Numerous transformation vectors are available for plant transformation, and the genes of this invention can be used in conjunction with any such vectors. The selection of vector will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptll gene which confers resistance to kanamycin and related antibiotics (Messing Vierra. Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bargene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al. Theor. Appl. Genet 79: 625-631 (1990)), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger Diggelmann, Mol Cell Biol 4: 2929- 2931), and the dhfrgene, which confers resistance to methatrexate (Bourouis et al., EMBO J. 1099-1104 (1983)), and the EPSPS gene, which confers resistance to glyphosate Patent Nos. 4,940,935 and 5,188,642).

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

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

a. pCIB200 and pCIB2001: The binary vectors pclB200 and pCIB2001 are used for the construction of recombinant vectors for use with Agrobacterium and are constructed in the following manner. pTJS75kan is created by Narl digestion of pTJS75 (Schmidhauser Helinski, J.

Bacteriol. 164: 446-455 (1985)) allowing excision of the tetracycline-resistance gene, followed by insertion of an Acclfragment from pUC4K carrying an NPTII (Messing Vierra, WO 98/26082 PCT/EP97/07n012 Gene 19: 259-268 (1982): Bevan et al., Nature 304: 184-187 (1983): McBride et al., Plant Molecular Biology 14: 266-276 (1990)). Xhollinkers are ligated to the EcoRVfragment of PCIB7 which contains the left and right T-DNA borders, a plant selectable nos/nptll chimeric gene and the pUC polylinker (Rothstein et al., Gene 53:153-161 (1987)), and the Xhol digested fragment are cloned into Sail-digested pTJS75kan to create pCIB200 (see also EP 0 332 104, example 19). pCIB200 contains the following unique polylinker restriction sites: EcoRI, Sstl, Kpnl, Bglll, Xbal, and Sail. pCIB2001 is a derivative of pCIB200 created by the insertion into the polylinker of additional restriction sites. Unique restriction sites in the polylinker of pCIB2001 are EcoRI, Sstl, Kpnl, Bglll, Xbal, Sail, Mlul, Bcll, Avrll, Apal, Hpal, and Stul. pCIB2001, in addition to containing these unique restriction sites also has plant and bacterial kanamycin selection, left and right T-DNA borders for Agrobacterium-mediated transformation, the RK2-derived trfA function for mobilization between E. co/iand other hosts, and the OriTand OriVfunctions also from RK2. The pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals.

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

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

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

WO 98/26082 PCT/EP97/07012 -31a. pCIB3064: pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques in combination with selection by the herbicide basta (or phosphinothricin). The plasmid pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. coliGUS gene and the CaMV 35S transcriptional terminator and is described in the PCT published application WO 93/07278. The 35S promoter of this vector contains two ATG sequences of the start site. These sites are mutated using standard PCR techniques in such a way as to remove the ATGs and generate the restriction sites Sspl and Pvull. The new restriction sites are 96 and 37 bp away from the unique Sallsite and 101 and 42 bp away from the actual start site. The resultant derivative of pCIB246 is designated pCIB3025. The GUS gene is then excised from pCIB3025 by digestion with Salland Sacl, the termini rendered blunt and religated to generate plasmid pCIB3060. The plasmid pJIT82 is obtained from the John Innes Centre, Norwich and the a 400 bp Smalfragment containing the bargene from Streptomyces viridochromogenes is excised and inserted into the Hpalsite of pCIB3060 (Thompson et al. EMBO J 6: 2519-2523 (1987)). This generated pCIB3064, which comprises the bargene under the control of the CaMV 35S promoter and terminator for herbicide selection, a gene for ampicillin resistance (for selection in E. coll) and a polylinker with the unique sites Sphl, PstI, Hind/ll, and BamHI. This vector is suitable for the cloning of plant expression cassettes containing their own regulatory signals.

b. pSOG19 and is a transformation vector which utilizes the E. col gene dihydrofolate reductase (DFR) as a selectable marker conferring resistance to methotrexate. PCR is used to amplify the 35S promoter (-800 bp), intron 6 from the maize Adhl gene (-550 bp) and 18 bp of the GUS untranslated leader sequence from pSOG10. A 250-bp fragment encoding the E. colidihydrofolate reductase type II gene is also amplified by PCR and these two PCR fragments are assembled with a Sacl-Pstlfragment from pB1221 (Clontech) which comprises the pUC19 vector backbone and the nopaline synthase terminator.

Assembly of these fragments generates pSOG19 which contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene and the nopaline synthase terminator. Replacement of the GUS leader in pSOG19 with the leader sequence from Maize Chlorotic Mottle Virus (MCMV) generates the vector pSOG35. pSOG19 and carry the pUC gene for ampicillin resistance and have Hindill, Sphl, Pstl and EcoRI sites available for the cloning of foreign substances.

WO 98/26082 PCT/EP97/07fTl 12 -32- B. Requirements for Construction of Plant Expression Cassettes Gene sequences intended for expression in transgenic plants are first assembled in expression cassettes behind a suitable high expression level promoter and upstream of a suitable transcription terminator. These expression cassettes can then be easily transferred to the plant transformation vectors described above.

1. Promoter Selection The selection of the promoter used in expression cassettes will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters will express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example) and the selection will reflect the desired location of accumulation of the NIM1 gene product or altered NIM1 gene product. Alternatively, the selected promoter may drive expression of the gene under a light-induced or other temporally regulated promoter.

a. Constitutive Expression, the CaMV 35S Promoter: Construction of the plasmid pCGN1761 is described in the published patent application EP 0 392 225 (example 23) which is hereby incorporated by reference.

pCGN1761 contains the "double" 35S promoter and the tmltranscriptional terminator with a unique EcoRI site between the promoter and the terminator and has a pUC-type backbone.

A derivative of pCGN1761 is constructed which has a modified polylinker which includes Notl and Xholsites in addition to the existing EcoRI site. This derivative is designated pCGN1761ENX. pCGN1761ENX is useful for the cloning of cDNA sequences or gene sequences (including microbial ORF sequences) within its polylinker for the purpose of their expression under the control of the 35S promoter in transgenic plants. The entire promoter-gene sequence-tml terminator cassette of such a construction can be excised by Hindlll, Sphl, Sall, and Xbalsites 5' to the promoter and Xbal, BamHland Bgll sites 3' to the terminator for transfer to transformation vectors such as those described above.

Furthermore, the double 35S promoter fragment can be removed by 5' excision with Hindlll, Sphl, Sail, Xbal, or Pstl, and 3' excision with any of the polylinker restriction sites (EcoRI, Notl or Xhol) for replacement with another promoter.

b. Modification of pCGN1761ENX by Optimization of the Translational Initiation Site: For any of the constructions described herein, modifications around the cloning sites can be made by the introduction of sequences which may enhance translation. This is particularly useful when overexpression is desired.

WO 98/26082 PCT/EP97/n7t12l -33pCGN1761ENX is cleaved with Sphl, treated with T4 DNA polymerase and religated, thus destroying the Sphl site located 5' to the double 35S promoter. This generates vector pCGN1761ENX/Sph-. pCGN1761ENX/Sph- is cleaved with EcoRI, and ligated to an annealed molecular adaptor of the sequence 5'-AATTCTAAAGCATGCCGATCGG-3/5'- AATTCCGATCGGCATGCTTTA-3' (SEQ ID NO's: 12 and 13). This generates the vector pCGNSENX, which incorporates the quasi-optimized plant translational initiation sequence TAAA-C adjacent to the ATG which is itself part of an Sphl site which is suitable for cloning heterologous genes at their initiating methionine. Downstream of the Sphl site, the EcoRI, Notl, and Xholsites are retained.

An alternative vector is constructed which utilizes an Ncol site at the initiating ATG.

This vector, designated pCGN1761NENX is made by inserting an annealed molecular adaptor of the sequence 5'-AATTCTAAACCATGGCGATCGG-3'/5'- AATTCCGATCGCCATGGTTTA-3' (SEQ ID NO's: 14 and 15) at the pCGN1761ENX EcoRI site. Thus the vector includes the quasioptimized sequence TAAACC adjacent to the initiating ATG which is within the Ncolsite. Downstream sites are EcoRI, Noti, and Xhol.

Prior to this manipulation, however, the two Ncol sites in the pCGN1761ENX vector (at upstream positions of the 5' 35S promoter unit) are destroyed using similar techniques to those described above for Sphl or alternatively using "inside-outside" PCR. Innes et al.

PCR Protocols: A guide to methods and applications. Academic Press, New York (1990).

This manipulation can be assayed for any possible detrimental effect on expression by insertion of any plant cDNA or reporter gene sequence into the cloning site followed by routine expression analysis in plants.

c. Expression under a Chemically/Pathogen Regulatable Promoter: The double 35S promoter in pCGN1761ENX may be replaced with any other promoter of choice which will result in suitably high expression levels. By way of example, a chemically regulated PR-1 promoter, which is described in U.S. Patent No. 5,614,395, which is hereby incorporated by reference in its entirety, may replace the double promoter. The promoter of choice is preferably excised from its source by restriction enzymes, but can alternatively be PCR-amplified using primers which carry appropriate terminal restriction sites. Should PCR-amplification be undertaken, then the promoter should be re-sequenced to check for amplification errors after the cloning of the amplified promoter in the target vector. The chemically/pathogen regulatable tobacco PR-la promoter is cleaved from plasmid pCIB1004 (see EP 0 332 104, example 21 for construction which is hereby incorporated by reference) and transferred to plasmid pCGN1761ENX (Uknes et al. 1992). pCIB1004 is cleaved with Ncoland the resultant 3' WO 98/26082 PCT/EP97/07012 -34overhang of the linearized fragment is rendered blunt by treatment with T4 DNA polymerase. The fragment is then cleaved with Hindll/and the resultant PR-la-promotercontaining fragment is gel purified and cloned into pCGN1761ENX from which the double promoter has been removed. This is done by cleavage with Xholand blunting with T4 polymerase, followed by cleavage with Hindlll and isolation of the larger vector-terminator containing fragment into which the pCIB1004 promoter fragment is cloned. This generates a pCGN1761ENX derivative with the PR-la promoter and the tm/terminator and an intervening polylinker with unique EcoRI and Not sites. Selected NIM1 genes can be inserted into this vector, and the fusion products promoter-gene-terminator) can subsequently be transferred to any selected transformation vector, including those described in this application.

Various chemical regulators may be employed to induce expression of the NIM1 coding sequence in the plants transformed according to the present invention. In the context of the instant disclosure, "chemical regulators" include chemicals known to be inducers for the PR-1 promoter in plants, or close derivatives thereof. A preferred group of regulators for the PR-1 promoter is based on the benzo-1,2,3-thiadiazole (BTH) structure and includes, but is not limited to, the following types of compounds: benzo-1,2,3thiadiazolecarboxylic acid, benzo-1,2,3-thiadiazolethiocarboxylic acid, cyanobenzo-1,2,3thiadiazole, benzo-1,2,3-thiadiazolecarboxylic acid amide, benzo-1,2,3-thiadiazolecarboxylic acid hydrazide, benzo-1,2,3-thiadiazole-7-carboxylic acid, benzo-1,2,3-thiadiazole-7thiocarboxylic acid, 7-cyanobenzo-1,2,3-thiadiazole, benzo-1,2,3-thiadiazolecarboxylate in which the alkyl group contains one to six carbon atoms, methyl benzo-1,2,3-thiadiazole-7carboxylate, n-propyl benzo-1,2,3-thiadiazole-7-carboxylate, benzyl benzo-1,2,3-thiadiazole- 7-carboxylate, benzo-1,2,3-thiadiazole-7-carboxylic acid sec-butylhydrazide, and suitable derivatives thereof. Other chemical inducers may include, for example, benzoic acid, salicylic acid polyacrylic acid and substituted derivatives thereof; suitable substituents include lower alkyl, lower alkoxy, lower alkylthio, and halogen. Still another group of regulators for the chemically inducible DNA sequences of this invention is based on the pyridine carboxylic acid structure, such as the isonicotinic acid structure and preferably the haloisonicotinic acid structure. Preferred are dichloroisonicotinic acids and derivatives thereof, for example the lower alkyl esters. Suitable members of this class of regulator compounds are, for example, 2,6-dichloroisonicotinic acid (INA), and the lower alkyl esters thereof, especially the methyl ester.

WO 98/26082 PCT/EP97/07012 d. Constitutive Expression, the Actin Promoter: Several isoforms of actin are known to be expressed in most cell types and consequently the actin promoter is a good choice for a constitutive promoter. In particular, the promoter from the rice Actlgene has been cloned and characterized (McElroy etal.

Plant Cell 2:163-171 (1990)). A 1.3kb fragment of the promoter was found to contain all the regulatory elements required for expression in rice protoplasts. Furthermore, numerous expression vectors based on the Actl promoter have been constructed specifically for use in monocotyledons (McElroy et al. Mol. Gen. Genet. 231: 150-160 (1991)). These incorporate the Act/-intron 1, Adhl5' flanking sequence and Adhl-intron 1 (from the maize alcohol dehydrogenase gene) and sequence from the CaMV 35S promoter. Vectors showing highest expression were fusions of 35S and Actlintron or the Actl5' flanking sequence and the Actl intron. Optimization of sequences around the initiating ATG (of the GUS reporter gene) also enhanced expression. The promoter expression cassettes described by McElroy et al. (Mol. Gen. Genet. 231: 150-160 (1991)) can be easily modified for the expression of cellulase genes and are particularly suitable for use in monocotyledonous hosts. For example, promoter-containing fragments is removed from the McElroy constructions and used to replace the double 35S promoter in pCGN1761 ENX, which is then available for the insertion of specific gene sequences. The fusion genes thus constructed can then be transferred to appropriate transformation vectors. In a separate report the rice Actl promoter with its first intron has also been found to direct high expression in cultured barley cells (Chibbar et al. Plant Cell Rep. 12: 506-509 (1993)).

e. Constitutive Expression, the Ubiquitin Promoter: Ubiquitin is another gene product known to accumulate in many cell types and its promoter has been cloned from several species for use in transgenic plants sunflower Binet et al. Plant Science 79: 87-94 (1991) and maize Christensen et al. Plant Molec.

Biol. 12: 619-632 (1989)). The maize ubiquitin promoter has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926 (to Lubrizol) which is herein incorporated by reference. Taylor et al. (Plant Cell Rep. 12: 491-495 (1993)) describe a vector which comprises the maize ubiquitin promoter and first intron and its high activity in cell suspensions of numerous monocotyledons when introduced via microprojectile bombardment. The ubiquitin promoter is suitable for the expression of cellulase genes in transgenic plants, especially monocotyledons. Suitable vectors are derivatives of or any of the transformation vectors described in this application, modified by the introduction of the appropriate ubiquitin promoter and/or intron sequences.

WO 98/26082 PCT/EP97/07012 -36f. Root Specific Expression: Another pattern of expression for the NIM1 gene of the instant invention is root expression. A suitable root promoter is described by de Framond (FEBS 290: 103-106 (1991)) and also in the published patent application EP 0 452 269 (to Ciba-Geigy) which is herein incorporated by reference. This promoter is transferred to a suitable vector such as pCGN1761ENX for the insertion of a cellulase gene and subsequent transfer of the entire promoter-gene-terminator cassette to a transformation vector of interest.

g. Wound-Inducible Promoters: Wound-inducible promoters may also be suitable for expression of NIM1 genes of the invention. Numerous such promoters have been described Xu et al. Plant Molec. Biol.

22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989), Rohrmeier Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), Warner et al. Plant J. 3:191-201 (1993)) and all are suitable for use with the instant invention. Logemann et al. describe the 5' upstream sequences of the dicotyledonous potato wuni gene. Xu et al. show that a wound-inducible promoter from the dicotyledon potato (pin2) is active in the monocotyledon rice. Further, Rohrmeier Lehle describe the cloning of the maize WiplcDNA which is wound induced and which can be used to isolate the cognate promoter using standard techniques. Similar, Firek et al. and Warner et al.

have described a wound-induced gene from the monocotyledon Asparagus officinalis which is expressed at local wound and pathogen invasion sites. Using cloning techniques well known in the art, these promoters can be transferred to suitable vectors, fused to the NIM1 genes of this invention, and used to express these genes at the sites of plant wounding.

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

WO 98/26082 PCT/EP97/07012 -37i. Leaf-Specific Expression: A maize gene encoding phosphoenol carboxylase (PEPC) has been described by Hudspeth Grula (Plant Molec Biol 12: 579-589 (1989)). Using standard molecular biological techniques the promoter for this gene can be used to drive the expression of any gene in a leaf-specific manner in transgenic plants.

j. Expression with Chloroplast Targeting: Chen Jagendorf Biol. Chem. 268: 2363-2367 (1993) have described the successful use of a chloroplast transit peptide for import of a heterologous transgene. This peptide used is the transit peptide from the rbcS gene from Nicotiana plumbaginifolia (Poulsen et al. Mol. Gen. Genet. 205:193-200 (1986)). Using the restriction enzymes Dral and Sphl. pr Tsp5091 and Sphl the DNA sequence encoding this transit peptide can be excised from the plasmid prbcS-8B and manipulated for use with any of the constructions described above. The Dral-Sphlfragment extends from -58 relative to the initiating rbcS ATG to, and including, the first amino acid (also a methionine) of the mature peptide immediately after the import cleavage site, whereas the Tsp5091-Sphl fragment extends from -8 relative to the initiating rbcS ATG to, and including, the first amino acid of the mature peptide.

Thus, these fragments can be appropriately inserted into the polylinker of any chosen expression cassette generating a transcriptional fusion to the untranslated leader of the chosen promoter 35S, PR-la, actin, ubiquitin etc.), while enabling the insertion of a NIM1 gene in correct fusion downstream of the transit peptide. Constructions of this kind are routine in the art. For example, whereas the Dral end is already blunt, the 5' Tsp5091 site may be rendered blunt by T4 polymerase treatment, or may alternatively be ligated to a linker or adaptor sequence to facilitate its fusion to the chosen promoter. The 3' Sphl site may be maintained as such, or may alternatively be ligated to adaptor of linker sequences to facilitate its insertion into the chosen vector in such a way as to make available appropriate restriction sites for the subsequent insertion of a selected NIM1 gene. Ideally the ATG of the Sphl site is maintained and comprises the first ATG of the selected NIM1 gene. Chen Jagendorf provide consensus sequences for ideal cleavage for chloroplast import, and in each case a methionine is preferred at the first position of the mature protein.

At subsequent positions there is more variation and the amino acid may not be so critical.

In any case, fusion constructions can be assessed for efficiency of import in vitro using the methods described by Bartlett et al. (In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier pp 1081-1091 (1982)) and Wasmann et al. (Mol. Gen. Genet.

WO 98/26082 PCT/EP97/07012 -38- 205: 446-453 (1986)). Typically the best approach may be to generate fusions using the selected NIM1 gene or altered form of the NIM1 gene with no modifications at the amino terminus, and only to incorporate modifications when it is apparent that such fusions are not chloroplast imported at high efficiency, in which case modifications may be made in accordance with the established 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 fragment from prbcS-8B to the cloning vector pCGN1761ENX/Sph-. This plasmid is cleaved with EcoRI and the termini rendered blunt by treatment with T4 DNA polymerase.

Plasmid prbcS-8B is cleaved with Sphl and ligated to an annealed molecular adaptor of the sequence 5'-CCAGCTGGAATTCCG-3'/5'-CGGAATTCCAGCTGGCATG-3' (SEQ ID NO's: 16 and 17). The resultant product is 5'-termirially phosphorylated by treatment with T4 kinase. Subsequent cleavage with Dral releases the transit peptide encoding fragment which is ligated into the blunt-end ex-EcoRI sites of the modified vector described above.

Clones oriented with the 5' end of the insert adjacent 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 rbcS-8A promoter-transit peptide sequence extending from -58 relative to the rbcS ATG to the ATG of the mature protein, and including in that region a unique Sphl site, and a newly created EcoRI site, as well as the existing Notland Xhol sites of pCGN1761ENX.

This new vector is designated pCGN1761/CT. DNA sequences are transferred to pCGN1761/CT in frame by amplification using PCR techniques and incorporation of an Sphl, NSphl, or Nlalllsite at the amplified ATG, which following restriction enzyme cleavage with the appropriate enzyme is ligated into Sphl-cleaved pCGN1761/CT. To facilitate construction, it may be required to change the second amino acid of the product of the cloned gene; however, in almost all cases the use of PCR together with standard site directed mutagenesis will enable the construction of any desired sequence around the cleavage site and first methionine of the mature protein.

A further preferred vector is constructed by replacing the double 35S promoter of pCGN1761ENX with the BamHI-Sphlfragment of prbcS-8A which contains the full-length, light-regulated rbcS-8A promoter from -1038 (relative to the transcriptional start site) up to the first methionine of the mature protein. The modified pCGN1761 with the destroyed Sphl is cleaved with Pstland EcoRI and treated with T4 DNA polymerase to render termini blunt.

prbcS-8A is cleaved with Sphl and ligated to the annealed molecular adaptor of the sequence described above. The resultant product is 5'-terminally phosphorylated by treatment with T4 kinase. Subsequent cleavage with BamHI releases the promoter-transit peptide containing fragment which is treated with T4 DNA polymerase to render the BamHI WO 98/26082 PCT/EP97/n7nii -39terminus blunt. The promoter-transit peptide fragment thus generated is cloned into the prepared pCGN1761ENX vector, generating a construction comprising the rbcS-8A promoter and transit peptide with an Sphl site located at the cleavage site for insertion of heterologous genes. Further, downstream of the Sphl site there are EcoRI (re-created), Notl, and Xholcloning sites. This construction is designated pCGN1761rbcS/CT.

Similar manipulations can be undertaken to utilize other GS2 chloroplast transit peptide encoding sequences from other sources (monocotyledonous and dicotyledonous) and from other genes. In addition, similar procedures can be followed to achieve targeting to other subcellular compartments such as mitochondria.

2. Transcriptional Terminators A variety of transcriptional terminators are available for use in expression cassettes.

These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Appropriate transcriptional terminators are those which are known to function in plants and include the CaMV 35S terminator, the tmlterminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons.

3. Sequences for the Enhancement or Regulation of Expression Numerous sequences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes of this invention to increase their expression in transgenic plants.

Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adhlgene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells. Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al., Genes Develop. 1:1183-1200 (1987)). In the same experimental system, the intron from the maize bronzel gene had a similar effect in enhancing expression. Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.

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

Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the "W-sequence"), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be WO 98/26082 PCT/EP97/07012 effective in enhancing expression Gallie et al. Nucl. Acids Res. 15: 8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990)).

4. Targeting of the Gene Product Within the Cell Various mechanisms for targeting gene products are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterized in some detail. For example, the targeting of gene products to the chloroplast is controlled by a signal sequence found at the amino terminal end of various proteins which is cleaved during chloroplast import to yield the mature protein Comai et al. J. Biol. Chem. 263: 15104-15109 (1988)). These signal sequences can be fused to heterologous gene products to effect the import of heterologous products into the chloroplast (van den Broeck, et al. Nature 313: 358-363 (1985)). DNA encoding for appropriate signal sequences can be isolated from the 5' end of the cDNAs encoding the RUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2 protein and many other proteins which are known to be chloroplast localized.

Other gene products are localized to other organelles such as the mitochondrion and the peroxisome Unger et al. Plant Molec. Biol. 13: 411-418 (1989)). The cDNAs encoding these products can also be manipulated to effect the targeting of heterologous gene products to these organelles. Examples of such sequences are the nuclear-encoded ATPases and specific aspartate amino transferase isoforms for mitochondria. Targeting cellular protein bodies has been described by Rogers et al. (Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).

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

By the fusion of the appropriate targeting sequences described above to transgene sequences of interest it is possible to direct the transgene product to any organelle or cell compartment. For chloroplast targeting, for example, the chloroplast signal sequence from the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused in frame to the amino terminal ATG of the transgene. The signal sequence selected should include the known cleavage site, and the fusion constructed should take into account any amino acids after the cleavage site which are required for cleavage. In some cases this requirement may be fulfilled by the addition of a small number of amino acids between the WO 98/26082 PCT/EP97/07012 -41cleavage site and the transgene ATG or, alternatively, replacement of some amino acids within the transgene sequence. Fusions constructed for chloroplast import can be tested for efficacy of chloroplast uptake by in vitro translation of in vitro transcribed constructions followed by in vitro chloroplast uptake using techniques described by Bartlett et al. In: Edelmann etal. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier pp 1081-1091 (1982) and Wasmann et al. Mol. Gen. Genet. 205: 446-453 (1986). These construction techniques are well known in the art and are equally applicable to mitochondria and peroxisomes.

The above-described mechanisms for cellular targeting can be utilized not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters so as to effect a specific cell-targeting goal under the transcriptional regulation of a promoter which has an expression pattern different to that of the promoter from which the targeting signal derives.

C. Transformation Once 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 tissues, root tissues, meristems, and protoplasts. The present system can be utilized in any plant which can be transformed and regenerated. Such methods for transformation and regeneration are well known in the art. Methodologies for the construction of plant expression cassettes as well as the introduction of foreign DNA into plants is generally described in the art. Generally, for the introduction of foreign DNA into plants, Ti plasmid vectors have been utilized for the delivery of foreign DNA. Also utilized for such delivery have been direct DNA uptake, liposomes, electroporation, micro-injection, and microprojectiles. Such methods had been published in the art. See, for example, Bilang et al. (1991) Gene 100: 247-250; Scheid et al., (1991) Mol. Gen. Genet. 228:104-112; Guerche et al., (1987) Plant Science 52:111-116; Neuhause et al., (1987) Theor. Apl.

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 Weissbach, eds.) Academic Press, Inc. (1988); and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc. (1989). See also US. patent No. 5,625,136 which are incorporated herein by reference in their entirety. It is understood that the method of transformation will depend upon 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, Barnason, and Horsch, R.B. Transformation of WO 98/26082 PCT/EP97/07012 -42- Brassica napus with Agrobacterium tumefaciens based vectors. PI.Cell Rep. 6:321-325, 1987; Block, M.d. Genotype independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens. Theor.appl.genet. 76:767-774, 1988; Deblock, Brouwer, and Tenning, 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, Skene, Cain, and Scott, N.S. Transgenic grapevines: regeneration of shoots expressing beta-glucuronidase. Pl.Cell Rep. 41:1045-1049, 1990; Hinchee, Newell, ConnorWard, Armstrong, Deaton, Sato, 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 transformation. PI.Cell Rep. 10:308-314, 1991; Cousins, Lyon, 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 Transferred Genes. GENE 118:255-260,1992; Christou, Ford, and Kofron, M. The development of a variety-independent gene-transfer method for rice.

Trends.Biotechnol 10:239-246, 1992; D'Halluin, Bossut, Bonne, Mazur, B., Leemans, and Botterman, J. Transformation of sugarbeet (Beta vulgaris and evaluation of herbicide resistance in transgenic plants. Bio/Technol. 10:309-314. 1992; Dhir, Dhir, Savka, Belanger, Kriz, Farrand, and Widholm, J.M.

Regeneration of Transgenic Soybean (Glycine Max) Plants from Electroporated Protoplasts.

Plant Physiol 99:81-88, 1992; Ha, Wu, and Thorne, T. K. Transgenic turf-type tall fescue (Festuca arundinacea Schreb.) plants regenerated from protoplasts. P. Cell Rep.

11:601 -604, 1992; Blechl, A.E. Genetic Transformation The New Tool for Wheat Improvement 78th Annual Meeting Keynote Address. Cereal Food World 38:846-847, 1993; Casas, Kononowicz, Zehr, Tomes, Axtell, Butler, Bressan, and Hasegawa, P.M. Transgenic Sorghum Plants via Microprojectile Bombardrment.

Proc Natacad Sci USA 90:11212-11216, 1993; Christou, P. Philosophy and Practice of Variety Independent Gene Transfer into Recalcitrant Crops. In Vitro Cell Dev Biol-Plant 29P: 119-124, 1993; Damiani, Nenz, Paolocci, and Arcioni, S. Introduction of Hygromycin Resistance in Lotus spp Through Agrobacterium Rhizogenes Transformation.

Transgenic Res 2:330-335, 1993; Davies, Hamilton, and Mullineaux,

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Transformation of Peas. PI.Cell Rep. 12:180-183, 1993; Dong, J.Z. and Mchughen,

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Transgenic Flax Plants from Agrobacterium Mediated Transformation Incidence of Chimeric WO 98/26082 PCT/EP97/n7l-1 -43- Regenerants and Inheritance of Transgenic Plants. Plant Sci 91:139-148, 1993; Fitch, Manshardt, Gonsalves, and Slightom, J.L. Transgenic Papaya Plants from Agrobacterium Mediated Transformation of Somatic Embryos. PI.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, Abraham, Morocz, Bottka, Feher, and Dudits, D. Production of Transgenic Maize Plants by Direct DNA Uptake into Embryogenic Protoplasts. Plant Sci 90:41-52, 1993; Guo, Xu, Wei, 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 electroporationmediated 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, Hagel, Becker, Martin, and Lorz, H. Transgenic Cereal (Tritordeum) Plants Obtained at High Efficiency by Microprojectile Bombardment of Inflorescence Tissue. PLANT J5:583-592, 1994; Becker, Brettschneider, and Lorz, H. Fertile Transgenic Wheat from Microprojectile Bombardment of Scutellar Tissue. Plant J 5:299-307, 1994; Biswas, Iglesias, Datta, and Potrykus, I. Transgenic Indica Rice (Oryza Sativa L) Plants Obtained by Direct Gene Transfer to Protoplasts.

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Biotechnol32: 1-10, 1994; Borkowska, Kleczkowski, Klos, Jakubiec, and Wielgat, B. Transformation of Diploid Potato with an Agrobacterium Tumefaciens Binary Vector System Methodological Approach. Acta Physiol Plant 16:225-230, 1994; Brar, Cohen, Vick, and Johnson, G.W. Recovery of Transgenic 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 Advances. Agro Food Ind Hi Tech 5: 17-27, 1994; Chupeau, Pautot, 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 PI.Cell Rep. 13:582-586,1994; Hartman, Lee, Day, and Tumer, N.E. Herbicide Resistant Turfgrass (Agrostis Palustris Huds) by Biolistic Transformation. Bid-Technology 12:919923, 1994; Howe, G.T., Goldfarb, 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, 1994; Konwar, B.K. Agrobacterium Tumefaciens Mediated Genetic Transformation of Sugar Beet (Beta Vulgaris J Plantbiochem Biotechnol 3:37-41, 1994; Ritala, Aspegren, Kurten, Salmenkalliomarttila, Mannonen, Hannus, R., Kauppinen, Teeri, and Enari, T.M. Fertile Transgenic Barley by Particle WO 98/26082 PCT/EPq7/7ft1 f -44- Bombardment of Immature Embryos. Plant Mol Biol24:317-325, 1994; Scorza, Cordts, Ramming, 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 Opinbiotechnol5:158-162, 1994; Spangenberg, Wang, Nagel, and Potrykus, I. Protoplast Culture and Generation of Transgenic Plants in Red Fescue (Festuca Rubra Plant Sci 97:83-94, 1994; Spangenberg, Wang, Nagel, 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 Numbers of Independently Transformed Fertile Barley Plants. Plant Physiol 104:3748, 1994; Weeks, Anderson, and Blechl, A.E.

Stable Transformation of Wheat (Triticum Aestivum L) by Microprojectile Bombardment.

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Cell Biochem :104, 1994; Ye, Brown, Scorza, Cordts, and Sanford, J.C.

Genetic Transformation of Peach Tissues by Particle Bombardment. Jamer Sochortsci 119:367-373, 1994; Spangenberg, Wang, Nagel, and Potrykus, I. Protoplast Culture And Generation Of Transgenic Plants In Red Fescue (Festuca Rubra Plant Science 1994 97:83-94, 1995. See also, U.S. Patent No. 5,639,949, hereby incorporated by reference in its entirety.

Bacteria from the genus Agrobacterium can be utilized to transform plant cells.

Suitable species of such bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogens. Agrobacterium tumefaciens strains LBA4404 or EHA105) is particularly useful due to its well-known ability to transform plants.

1. Transformation of Dicotyledons Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques which do not require Agrobacterium.

Non-Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described by Paszkowski et al., EMBO J 3: 2717-2722 (1984), Potrykus et al., Mol. Gen.

Genet. 199: 169-177 (1985), Reich etal., Biotechnology 4:1001-1004 (1986), and Klein et al., Nature 327: 70-73 (1987). In each case the transformed cells are regenerated to whole plants using standard techniques known in the art.

Agrobacterium-mediated transformation is a preferred technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species. The many crop species which are alfalfa and poplar (EP 0 317 511 (cotton), EP 0 249 432 (tomato, to Calgene), WO 87/07299 (Brassica, to Calgene), WO 98/26082 PCT/EP97/0712 US 4,795,855 (poplar)). Agrobacterium transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest pCIB200 or pCIB2001) to an appropriate Agrobacterium strain which may depend of the complement of virgenes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e.g.

strain C1B542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5:159-169 (1993)). The transfer of the recombinant binary vector to Agrobacterium is accomplished by a triparental mating procedure using E. co/icarrying the recombinant binary vector, a helper E. co/istrain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Hbfgen Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).

Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols well known in the art. Transformed tissue is regenerated on selectable medium carrying the antibiotic or herbicide resistance marker present between the binary plasmid T- DNA borders.

Another approach to transforming plant cells with a gene involves propelling inert or biologically active particles at plant tissues and cells. This technique is disclosed in U.S.

Patent Nos. 4,945,050; 5,036,006; and 5,100,792 all to Sanford et al. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the desired gene. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles dried yeast cells, dried bacterium or a bacteriophage, each containing DNA sought to be introduced) can also be propelled into plant cell tissue.

2. Transformation of Monocotyledons Transformation of most monocotyledon species has now also become routine.

Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and particle bombardment into callus tissue. Transformations can be undertaken with a single DNA species or multiple DNA species cotransformation) and both these techniques are suitable for use with this invention. Cotransformation may have the advantage of avoiding complete vector construction and of generating transgenic plants with unlinked loci for the gene of interest and the selectable WO 98/26082 PCT/EP97/07012 -46marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable. However, a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al. Biotechnology 4: 1093-1096 (1986)).

Patent Applications EP 0 292 435 ([1280/1281] to Ciba-Geigy), EP 0 392 225 (to Ciba-Geigy) and WO 93/07278 (to Ciba-Geigy) describe techniques for the preparation of callus and protoplasts from an elite inbred line of maize, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts.

Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and Fromm et al. (Biotechnology 8: 833-839 (1990)) have published techniques for transformation of A188-derived maize line using particle bombardment. Furthermore, application WO 93/07278 (to Ciba-Geigy) and Koziel et al. (Biotechnology ii: 194-200 (1993)) describe techniques for the transformation of elite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after pollination and a PDS-1000He Biolistics device for bombardment.

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

Patent Application EP 0 332 581 (to Ciba-Geigy) describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Furthermore, wheat transformation has been described by Vasil et al. (Biotechnology 1j: 667-674 (1992)) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al.

(Biotechnology 11: 1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102: 1077-1084 (1993)) using particle bombardment of immature embryos and immature embryo-derived callus. A preferred technique for wheat transformation, however, involves the transformation of wheat by particle bombardment of immature embryos and includes either a high sucrose or a high maltose step prior to gene delivery. Prior to bombardment, any number of embryos (0.75-1 mm in length) are plated onto MS medium with 3% sucrose (Murashiga Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/I 2,4-D for induction of somatic embryos, which is allowed to proceed in the dark. On the chosen day of bombardment, embryos are removed from the induction medium and placed onto the osmoticum induction medium with sucrose or maltose added at the desired WO 98/26082 PCT/EP97/07012 47 concentration, typically The embryos are allowed to plasmolyze for 2-3 h and are then bombarded. Twenty embryos per target plate is typical, although not critical. An appropriate gene-carrying plasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer size gold particles using standard procedures. Each plate of embryos is shot with the DuPont Biolistics® helium device using a burst pressure of -1000 psi using a standard 80 mesh screen. After bombardment, the embryos are placed back into the dark to recover for about 24 h (still on osmoticum). After 24 hrs, the embryos are removed from the osmoticum and placed back onto induction medium where they stay for about a month before regeneration. Approximately one month later the embryo explants with developing embryogenic callus are transferred to regeneration medium (MS 1 mg/liter NAA, 5 mg/liter GA), further containing the appropriate selection agent (10 mg/l basta in the case of pCIB3064 and 2 mg/I methotrexate in the case of pSOG35). After approximately one month, developed shoots are transferred to larger sterile containers known as "GA7s" which contain half-strength MS, 2% sucrose, and the same concentration of selection agent.

Patent application 08/147,161 describes methods for wheat transformation and is hereby incorporated by reference.

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

Breeding The isolated gene fragment of the present invention or altered forms of the NIM1 gene can be utilized to confer disease resistance to a wide variety of plant cells, including those of gymnosperms, monocots, and dicots. Although the gene can be inserted into any plant cell falling within these broad classes, it is particularly useful in crop plant cells, such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.

The overexpression of the NIM1 gene and mutants thereof necessary for constitutive expression of SAR genes, in combination with other characteristics important for production and quality, can be incorporated into plant lines through breeding. Thus a further embodiment of the present invention is a method of producing transgenic WO 98/26082 PCT/EP97/07012 WO 9826082PCT/EP97O7012 -48descendants of a transgenic parent plant comprising an isolated DNA molecule encoding an altered form of a NIM1 protein according to the invention comprising transforming said parent plant with a recombinant vector molecule according to the invention and transferring the trait to the descendants of said transgenic parent plant involving known plant breeding techniques.

Breeding approaches and techniques are known in the art. See, for example, Welsh J. R., Fundamentals of Plant Genetics and Breeding, John Wiley Sons, NY (1981); Crop Breeding, Wood D. R. American Society of Agronomy Madison, Wisconsin (1983); Mayo The Theory of Plant Breeding, Second Edition, Clarendon Press, Oxford (1987); Singh, Breeding for Resistance to Diseases and Insect Pests, Springer-Verlag,

NY

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

Propagation of genetic properties engineered into the transgenic seeds and plants and maintainance in descendant plants The genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in descendant plants. Generally said maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting. Specialized processes such as hydroponics or greenhouse technologies can also be applied. As the growing crop is vulnerable to attack and damages caused by insects or infections as well as to competition by weed plants, measures are undertaken to control weeds, plant diseases, insects, nematodes, and other adverse conditions to improve yield.

These include mechanical measures such a tillage of the soil or removal of weeds and infected plants, as well as the application of agrochemicals such as herbicides, fungicides, gametocides, nematicides, growth regulants, ripening agents and insecticides.

Use of the advantageous genetic properties of the transgenic plants and seeds according to the invention can further be made in plant breeding which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering. The various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate descendant plants. Depending on the desired properties different breeding measures are taken. The relevant techniques are well known in the art WO 98/26082 PCT/EP97/07012 -49and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc. Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical or biochemical means. Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines. Thus, the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines which for example increase the effectiveness of conventional methods such as herbicide or pestidice treatment or allow to dispense with said methods due to their modified genetic properties.

Alternatively new crops with improved stress tolerance can be obtained which, due to their optimized genetic "equipment", yield harvested product of better quality than products which were not able to tolerate comparable adverse developmental conditions.

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

Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD®), methalaxyl (Apron and pirimiphos-methyl (Actellic If desired these compounds are formulated together with further carriers, surfactants or applicationpromoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests. The protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods of application are also possible such as treatment directed at the buds or the fruit.

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

WO 98/26082 PCT/EP97/07012 The seeds may be provided in a bag, container or vessel comprised of a suitable packaging material, the bag or container capable of being closed to contain seeds. The bag, container or vessel may be designed for either short term or long term storage, or both, of the seed.

Examples of a suitable packaging material include paper, such as kraft paper, rigid or pliable plastic or other polymeric material, glass or metal. Desirably the bag, container, or vessel is comprised of a plurality of layers of packaging materials, of the same or differing type. In one embodiment the bag, container or vessel is provided so as to exclude or limit water and moisture from contacting the seed. In one example, the bag, container or vessel is sealed, for example heat sealed, to prevent water or moisture from entering. In another embodiment water absorbent materials are placed between or adjacent to packaging material layers. In yet another embodiment the bag, container or vessel, or packaging material of which it is comprised is treated to limit, suppress or prevent disease, contamination or other adverse affects of storage or transport of the seed. An example of such treatment is sterilization, for example by chemical means or by exposure to radiation.

Comprised by the present invention is a commercial bag comprising seed of a transgenic plant comprising at least one altered form of a NIM1 protein or a NIM1 protein that is expressed in said transformed plant at higher levels than in a wild type plant, together with a suitable carrier, together with lable instructions for the use thereof for conferring broad spectrum disease resistance to plants.

Disease Resistance Disease Resistance evaluation is performed by methods known in the art. For examples see, Uknes et al, (1993) Molecular Plant Microbe Interactions 6: 680-685; Gorlach et al., (1996) Plant Cell 8:629-643; Alexander et al., Proc. Natl. Acad. Sci. USA 90: 7327- 7331.

A. Phytophthora parasitica (Black shank) Resistance Assay Assays for resistance to Phytophthora parasitica, the causative organism of black shank, are performed on six-week-old plants grown as described in Alexander et al., Proc.

Natl. Acad. Sci. USA 90: 7327-7331. Plants are watered, allowed to drain well, and then inoculated by applying 10 ml of a sporangium suspension (300 sporangia/ml) to the soil.

Inoculated plants are kept in a greenhouse maintained at 23-25°C day temperature, and 22°C night temperature. The wilt index used for the assay is as follows: 0=no symptoms; 1=no symptoms; 1=some sign of wilting, with reduced turgidity; 2=clear wilting symptoms, but no rotting or stunting; 3=clear wilting symptoms with stunting, but no apparent stem rot; WO 98/26082 PCT/EP97/07012 -51 4=severe wilting, with visible stem rot and some damage to root system; 5=as for 4, but plants near death or dead, and with severe reduction of root system. All assays are scored blind on plants arrayed in a random design.

B. Pseudomonas syringae Resistance Assay Pseudomonas syringae pv. tabaci strain #551 is injected into the two lower leaves of several 6-7-week-old plants at a concentration of 106 or 3 x 106 per ml in H 2 0. Six individual plants are evaluated at each time point. Pseudomonas tabaci infected plants are rated on a point disease severity scale, 5=100% dead tissue, 0=no symptoms. A T-test (LSD) is conducted on the evaluations for each day and the groupings are indicated after the Mean disease rating value. Values followed by the same letter on that day of evaluation are not statistically significantly different.

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

D. Peronospora parasitica Resistance Assay Assays for resistance to Peronospora parasitica are performed on plants as described in Uknes et al, (1993). Plants are inoculated with a combatible isolate of P.

parasitica by spraying with a conidial suspension (approximately 5 x 104 spores per milliliter). Inoculated plants are incubated under humid conditions at 17° C in a growth chamber with a 14-hr day/10-hr night cycle. Plants are examined at 3-14 days, preferably 7-12 days, after inoculation for the presence of conidiophores. In addition, several plants from each treatment are randomly selected and stained with lactophenol-trypan blue (Keogh et al., Trans. Br. Mycol. Soc. 74: 329-333 (1980)) for microscopic examination.

WO 98/26082 PCT/EP97/07012 -52- BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 shows the effect of chemical inducers on the induction of SAR gene expression in wild-type and nimi plants. Chemical induction of SAR genes is diminished in niml plants. Water, SA, INA, or BTH is applied to 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 FIGURE 2 depicts PR-1 gene expression in pathogen-infected Ws-O and nim1 plants.

Pathogen induction of PR-1 is diminished in niml plants. Wild type (WT) and niml plants were spray-inoculated with the Emwa race of P. parasitica. Samples were collected at days 0, 1, 2, 4, and 6 and RNA is analyzed by blot hybridization with an A. thaliana PR-1 cDNA probe to measure PR-1 mRNA accumulation.

FIGURE 3 shows the accumulation of PR-1 mRNA in niml mutants and wild-type plants after pathogen infection or chemical treatment. Plants containing niml alleles nim1- 1, and -6 and Ws-O (Ws) were treated with water SA, INA, or BTH 3 days before RNA isolation. The Emwa sample consists of RNA isolated from plants 14 days post-inoculation with the Emwa isolate of P. parasitica. Blots were hybridized using an Arabidopsis PR-1 cDNA as a probe (Uknes et 1992).

FIGURE 4 shows the levels of SA accumulation in Ws-O and niml plants infected with P. syringae. niml plants accumulate SNA following pathogen exposure. Leaves of wild type and niml plantsare infiltrated with PstDC3000(avrRpt2) or carrier medium (10 mM MgCI 2 alone. After 2 days, samples were collected from untreated, MgCI 2 -treated, and DC3000(avrRpt2)-treated plants. Bacteria-treated samples were separated into primary (infiltrated) and secondary (noninfiltrated) leaves. Free SA and total SA following hydrolysis with P-glucosidase were quantified by HPLC. Error bars indicate SD of three replicate samples.

FIGURES 5A-D present a global map at increasing levels of resolution of the chromosomal region centered on NIM1 with recombinants indicated, including, BACs, YACs and Cosmids in NIM1 region.

Map position of NIM1 on chromosome 1. The total number of gametes scored is 2276.

Yeast artificial chromosome (striped), bacterial artificial chromosome (BAC), and P1 clones used to clone NIM1.

Cosmid clones that cover the NIM1 locus. The three cosmids that complement niml-1 are shown as thicker lines.

WO 98/26082 PCT/EP97/07012 -53- The four putative gene regions on the smallest fragment of complementing genomic DNA. The four open reading frames that comprise the NIM1 gene are indicated by the open bars. The arrows indicate the direction of transcription. Numbering is relative to the first base of 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 NIM1 gene product, including changes in the various alleles. This nucleic acid sequence, which is on the opposite strand as the 9.9 kb sequence presented in SEQ ID NO:1, is also presented in SEQ ID NO:2, and the amino acid sequence of the NIM1 gene product is also presented in SEQ ID NO:3.

FIGURE 7 shows the accumulation of NIM1 induced by INA, BTH, SA and pathogen treatment in wild type plants and mutant alleles of nimi. The RNA gel blots in Figure 3 were probed for expression of RNA by using a probe derived from 2081 to 3266 in the sequence shown in Figure 6.

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

FIGURE 9 is a sequence alignment of the NIM1 protein sequence with IKBa from mouse, rat, and pig. Vertical bars above the sequences indicate amino acid identity between NIM1 and the IKBa sequences (matrix score equals double dots above the sequences indicate a similarity score single dots above the sequences indicate a similarity score <0.5 but and a score <0.0 indicates no similarity and has no indicia above the sequences (see Examples). Locations of the mammalian IkBa ankyrin domains were identified according to de Martin et al., Gene 152, 253-255 (1995). The dots within a sequence indicate gaps between NIM1 and IkBc proteins. The five ankyrin repeats in IKBa are indicated by the dashed lines under the sequence. Amino acids are numbered relative to the NIM1 protein with gaps introduced where appropriate. Plus signs are placed above the sequences every 10 amino acids.

WO 98/26082 PCT/EP97/07012 -54-

DEPOSITS

The following vector molecules have been deposited with American Type Culture Collection 12301 Parklawn Drive Rockville, MD 20852, U.S.A. on the dates indicated below: Plasmid BAC-04 was deposited with ATCC on May 8,1996 as ATCC 97543.

Plasmid P1-18 was deposited with ATCC on June 13, 1996 as ATCC 97606.

Cosmid D7 was deposited with ATCC on September 25, 1996 as ATCC 97736.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE USTING SEQ ID NO: 1 9919-bp genomic sequence of NIM1 gene region 2 in Figure SEQ ID NO: 2 5655-bp genomic sequence in Figure 6 (opposite strand from SEQ ID NO:1). comprising the coding region of the wild-type Arabidopsis thaliana NIM1 gene.

SEQ ID NO: 3 AA sequence of wild-type NIM1 protein encoded by cds of SEQ ID N02.

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

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

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

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

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

SEQ ID NO: 9 Rice-3 AA sequence 208-288 from Figure 8.

SEQ ID NO: 10 Rice-4 AA sequence 33-155 from Figure 8.

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

SEQ ID NO: 12 Oligonudeotide.

SEQ ID NO: 13 Oligonudeotide.

SEQ ID NO: 14 Oligonucleotide.

SEQ ID NO: 15 Oligonudeotide.

SEQ ID NO: 16 Oligonudeotide.

SEQ ID NO: 17 Oligonudeotide.

SEQ ID NO: 18 is the mouse IcBa amino acid sequence from Figure 8.

SEQ ID NO: 19 is the rat IKBa amino acid sequence from Figure 8.

SEQ ID NO: 20 is the pig IkBa amino acid sequence from Figure 8.

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

SEQ ID NO's: 22 and 23 are the DNA coding sequence and encoded amino acid sequence, respectively, of a dominant-negative form of the NIM1 protein having alanine residues instead of serine residues at amino acid positions 55 and 59.

WO 98/26082 PCT/EP97/07012 SEQ ID NO's: 24 and 25 are the DNA coding sequence and encoded amino acid sequence, respectively, of a dominant-negative form of the NIM1 protein having an N-terminal deletion.

SEQ ID NO's: 26 and 27 are the DNA coding sequence and encoded amino acid sequence, respectively, of a dominant-negative form of the NIM1 protein having a C-terminal deletion.

SEQ ID NO's: 28 and 29 are the DNA coding sequence and encoded amino acid sequence, respectively, of an altered form of the NIM1 gene having both N-terminal and Cterminal amino acid deletions.

SEQ ID NO's: 30 and 31 are the DNA coding sequence and encoded amino acid sequence, respectively, of the ankyrin domain of NIM1.

SEQ ID NOs:32 through 39 are oligonucleotide primers.

Definitions acct

AFLP:

avrRpt2:

BAC:

BTH:

CIM:

cim: cM: cprl: Col-O: ECs: Emwa:

EMS:

INA:

Ler: Ist.

nahG: catechol NahG: ndr accelerated cell death mutant plant Amplified Fragment Length Polymorphism avirulence gene Rpt2, isolated from Pseudomonas syringae Bacterial Artificial Chromosome benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester Constitutive IMmunity phenotype (SAR is constitutively activated) constitutive immunity mutant plant centimorgans constitutive expresser of PR genes mutant plant Arabidopsis ecotype Columbia Enzyme combinations Peronospora parasitica isolate compatible in the Ws-O ecotype of Arabidopsis ethyl methane sulfonate 2,6-dichloroisonicotinic acid Arabidopsis ecotype Landsberg erecta lesions simulating disease mutant plant salicylate hydroxylase Pseudomonas putida that converts salicylic acid to Arabidopsis line transformed with nahG gene non-race-specific disease resistance mutant plant WO 98/26082 PCT/EP97/07012 nim: NIM1: NIM1: nim 1: Noco:

ORF:

PCs:

PR:

SA:

SAR:

SSLP:

UDS:

Wela: Ws-O:

WT:

YAC:

-56non-inducible immunity mutant plant the wild type gene, involved in the SAR signal transduction cascade Protein encoded by the wild type NIM1 gene mutant allele of NIM1, conferring disease susceptibility to the plant; also refers to mutant Arabidopsis thaliana plants having the niml mutant allele of NIM1 Peronospora parasitica isolate compatible in the Col-O ecotype of Arabidopsis open reading frame Primer combinations Pathogenesis Related salicylic acid Systemic Acquired Resistance Simple Sequence Length Polymorphism Universal Disease Susceptible phenotype Peronospora parasitica isolate compatible in the Weiningen ecotype of Arabidopsis Arabidopsis ecotype Issilewskija wild type Yeast Artificial Chromosome WO 98/26082 PCT/EP97/07n12 -57-

EXAMPLES

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

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

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

A. Characterization of niml Mutants Example 1: Plant Lines and Fungal Strains Arabidopsis thaliana ecotype Isilewskija (Ws-O; stock number CS 2360) and fourthgeneration

(T

4 seeds from T-DNA-transformed lines were obtained from the Ohio State University Arabidopsis Biological Resource Center (Columbus, OH). Second generation

(M-

2) seeds from ethyl methane sulfonate (EMS) mutagenized Ws-O plants were obtained from Lehle Seeds (Round Rock, TX).

Pseudomonas syringae pv. Tomato (Pst strain DC3000 containing the cloned avrRpt2 gene [DC3000(avrRpt2)] 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 fOr Pflanzenbiologie, Zurich, Switzerland; and Noco from J.

Parker, Sainsbury Laboratory, Norwich, England. Fungal cultures were maintained by weekly culturing on Arabidopsis ecotype Ws-O, Weiningen, and Col-O, for P. parasitica pathovars Emwa, Wela, and Noco, respectively.

WO 98/26082 PCT/EP97/07012 -58- Example 2: Mutant Screens

M

2 or T 4 seeds were grown on soil for 2 weeks under 14 hr of light per day, misted with 0.33 mM INA (0.25 mg/ml made from 25% INA in wettable powder; Ciba, Basel, Switzerland), and inoculated 4 days later by spraying a P. parasitica conidial suspension containing 5-10 x 104 conidiospores per 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. Plants were kept under humid conditions at 18 0 C for 1 week and then scored for fungal sporulation. Plants that supported fungal growth after INA treatment were selected as putative mutants.

Following incubation in a high humidity environment, plants with visible disease symptoms were identified, typically 7 days after the infection. These plants did not show resistance to the fungus, despite the application of the resistance-inducing chemical and were thus potential nim (noninducible-immunity) mutant plants. From 360,000 plants, potential nim mutants were identified.

These potential mutant plants were isolated from the flat, placed under low humidity conditions and allowed to set seed. Plants derived from this seed were screened in an identical manner for susceptibility to the fungus Emwa, again after pretreatment with INA.

The descendant plants that showed infection symptoms were defined as nim mutants. Six nim lines were thus identified. One line (niml-1) was isolated from the T-DNA population and five (niml-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 (BTH) are chemicals that, like INA, induce broad spectrum disease resistance (SAR) in wild type plants. Mutant plants were treated with SA, INA, and BTH and then assayed for resistance to Peronospora parasitica. P. parasitica isolate 'Emwa' is a P.p. isolate that is compatible in the Ws ecotype. Compatible isolates are those that are capable of causing disease on a particular host. The P. parasitica isolate 'Noco' is incompatible on Ws but compatible on the Columbia ecotype. Incompatible pathogens are recognized by the potential host, eliciting a host response that prevents disease development.

WO 98/26082 PCT/EP97/07012 -59- Wild-type seeds and seeds for each of the niml alleles (niml-1, -6) were sown onto MetroMix 300 growing media, covered with a transparent plastic dome, and placed at 4 0 C in the dark for 3 days. After 3 days of 4 0 C treatment, the plants were moved to a phytotron for 2 weeks. By approximately 2 weeks post-planting, germinated seedlings had produced 4 true leaves. Plants were then treated with H 2 0, 5mM SA, 300 RM BTH ,or 300 uM INA. Chemicals were applied as a fine mist to completely cover the seedlings using a chromister. Water control plants were returned to the growing phytotron while the chemically treated plants were held in a separate but identical phytotron. At 3 days postchemical application, water and chemically treated plants were inoculated with the compatible 'Emwa' isolate. 'Noco' inoculation was conducted on water treated plants only.

Following inoculation, plants were covered with a clear plastic dome to maintain high humidity required for successful P. parasitica infection and placed in a growing chamber with 19 0 C day/170 C night temperatures and 8h light/16h dark cycles.

To determine the relative strength of the different niml alleles, each mutant was microscopically analyzed at various timepoints after inoculation for the growth of P.

parasitica under normal growth conditions and following pretreatment with either SA, INA, or BTH. Under magnification, sporulation of the fungus could be observed at very early stages of disease development. The percentage of plants/pot showing sporulation at 5d, 6d, 7d, 11d and 14d after inoculation was determined and the density of sporulation was also recorded.

Table 1 shows, for each of the niml mutant plant lines, the percent of plants that showed some surface conidia on at least one leaf after infection with the Emwa race of P.

parasitica. P. parasitica was inoculated onto the plants three days after water or chemical treatment. The table indicates the number of days after infection that the disease resistance was rated.

WO 98/26082 PCT/EP97/07012 60 Table 1 Percent Infection Emwa/Control mutant D-a0 Day 5 Day 6 Day 7 Day 11 Ws WT 0 10 25 100 niml-1 0 75 95 100 100 nim 1-2 0 30 85 100 100 nim 1-3 0 30 90 100 100 niml-4 0 80 100 100 100 nim 1-5 0 0 5 100 100 nim 1-6 0 5 70 80 100 Percent Infection Emwa/SA mutant Day 0 Da 5 Day 6 Day 7 Day 11 Ws WT 0 5 30 70 100 niml-1 0 5 95 100 100 nim 1-2 0 5 95 100 100 nim 1-3 0 10 90 100 100 nim 1-4 0 75 100 100 100 nim 1-5 0 0 20 75 100 niml1-6 0 80 100 100 100 Percent Infection Emwa/INA mutant Day 0 Day 5 Day 6 Day 7 Day 11 Ws WT 0 0 0 0 0 niml-1 0 5 80 100 100 nim 1-2 0 15' 95 100 100 nim 1-3 0 10 60 100 100 nim 1-4 0 80 100 100 100 nim 1-5 0 0 0 5 nim 1-6 0 1 50 90 100 Percent Infection EmwaIBTH mutant Day 0 Day 5 Day 6 Day 7 Day 11 Ws WT 0 0 0 0 0' niml-1 0 1 5 30 100 niml-2 0 0 25 90 100 niml-3 0 15 70 100 100 niml1-4 0 80 100 100 100 0 0 1 1 nim 1-6 0 1 90 100 100 WO 98/26082 PCT/EP97/07012 61- As shown in Table 1, during normal growth, niml-1, niml-2, niml-3, niml-4, and nim 1-6 all supported approximately the same rate of fungal growth, which was somewhat faster than the Ws-0 control. The exception was the nim -5 plants where fungal growth was delayed by several days relative to both the other niml mutants and the Ws-0 control, but eventually all of the niml-5 plants succumbed to the fungus.

Following SA treatment, the mutants could be grouped into three classes: niml-4 and nim1-6 showed a relatively rapid fungal growth; nim niml-2, nim1-3 plants exhibited a somewhat slower rate of fungal growth; and fungal growth in nim 1-5 plants was even slower than in the untreated Ws-0 controls. Following either INA or BTH treatment, the mutants also fell into three classes where nim1-4 was the most severely compromised in its ability to restrict fungal growth following chemical treatment; niml-1, niml-2, niml-3, and nim 1-6 were all moderately compromised; and nim 1-5 was only slightly compromised. In these experiments, Ws-0 did not support fungal growth following INA or BTH treatment.

Thus, with respect to inhibition of fungal growth following chemical treatment, the mutants fell into three classes with niml-4 being the most severely compromised, niml-1, niml-2, niml-3 and nim1-6 showing an intermediate inhibition of fungus and nim 1-5 with only slightly impaired fungal resistance.

Table 2 shows the disease resistance assessment via infection rating of the various niml alleles as well as of NahG plants at 7 and 11 days after innoculation with Peronospora parasitica. WsWT indicates the Ws wild type parent line in which the niml alleles were found. The various niml alleles are indicated in the table and the NahG plant is indicated also.

A description of the NahG plant has been previously published. (Delaney et al., Science 266, pp. 1247-1250 (1994)). NahG Arabidopsis is also described in U.S. Patent Application Serial No. 08/454,876, incorporated by reference herein. nahG is a gene from Pseudomonas putida encoding a salicylate hydroxylase that converts salicylic acid to catechol, thereby eliminating the accumulation of salicylic acid, a necessary signal transduction component for SAR in plants. Thus, NahG Arabidopsis plants do not display normal SAR, and they show much greater susceptibility in general to pathogens. However, the NahG plants still respond to the chemical inducers INA and BTH. NahG plants therefore serve as a kind of universal susceptibility control.

WO 98/26082 PCT/EP97/07012 -62- Table 2 Infection Severity EmnwalWater mutant DaM 7 Day 11 Ws WT 3 3 niml-1 4 nim 1-2 3 4 niml-3 4 4 niml-4 5 nim 1-5 1 nim 1-6 3 NahG 4 Infection Severity EmwaISA mutant Day 7 Day 11 Ws WT 3 4 niml-1 3 nim 1-2 3 4 niml-3 3 4 nim 1-4 4 3 3 nim 1-6 4 NahG 4 Infection Severity Emwa/INA mutant Day7 Day 11 Ws WT 0 0 nil-i 2.5 4 nim 1-2 4 4 nim 1-3 3 nim 1-4 4 1 2 niml1-6 3 NahG 3 3 Infection Severity Emwa/BTH mutant Day 7 Day 11 Ws WT 0 0 nil-i 2.5 4 nim 1-2 3.5 4 niml1-3 3 niml-4 4 nim 1-5 1.5 2 nim 1-6 3 4 NahG 0 0 WO 98/26082 PCT/EP97/07012 -63- From Table 2 it can be seen that the nim1-4 and nim1-6 alleles had the most severe Peronospora parasitica infections; this was most easily observable at the earlier time points.

In addition, the niml-5 allele showed the greatest response to both INA and BTH and therefore was deemed the weakest niml allele. The NahG plants showed very good response to both INA and BTH and looked very similar to the nim1-5 allele. However, at late time points, Day 11 in the Table, the disease resistance induced in the NahG plants began to fade, and there was a profound difference between INA and BTH in that the INAinduced resistance faded much faster and more severely than the resistance induced in the NahG plants by BTH. Also seen in these experiments was that INA and BTH induced very good resistance in Ws to Emwa, and the nimi-1, nim1-2 and other nim1 alleles showed virtually no response to SA or INA with regard to disease resistance.

The nimi plants' lack of responsiveness to the SAR-inducing chemicals SA, INA, and BTH implies that the mutation is downstream of the entry point(s) for these chemicals in the signal transduction cascade leading to systemic acquired resistance.

Example 4: Northern Analysis 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 of interest to investigate whether pathogen infection could induce SAR gene expression in these plants, as it does in wild type plants. Thus, the accumulation of SAR gene mRNA was also used as a criterion to characterize the different niml alleles.

Wild-type seeds and seeds for each of the niml alleles (niml-1, -6) were sown onto MetroMix 300 growing media, covered with a transparent plastic dome, and placed at 4°C in the dark for 3 days. After 3 days of 4°C treatment, the plants were moved to a phytotron for 2 weeks. By approximately two weeks post-planting, germinated seedlings had produced 4 true leaves. Plants were then treated with H 2 0, 5mM SA, 300 p M BTH ,or 300 UM INA. Chemicals were applied as a fine mist to completely cover the seedlings using a chromister. Water control plants were returned to the growing phytotron while the chemically treated plants were held in a separate but identical phytotron. At 3 days post-chemical application, water and chemically treated plants were inoculated with the compatible Emwa isolate. Noco inoculation was conducted on water treated plants only. Following inoculation, plants were covered with a clear plastic dome to maintain high humidity required for successful P. parasitica infection and placed in a growing chamber with 190C day/170C night temperatures and 8h light/16h dark cycles. RNA was extracted from plants 3 days after either water or chemical treatment, or 14 days after inoculation with WO 98/26082 PCT/EP97/07012 -64the compatible P. parasitica Emwa isolate. The RNA was size-fractionated by agarose gel electrophoresis and transferred to GeneScreenPlus membranes (DuPont).

Figures 1-3 present various RNA gel blots that indicate that SA, INA and BTH induce neither SAR nor SAR gene expression in niml plants. In Figure 1, replicate blots were hybridized to Arabidopsis gene probes PR-1, PR-2 and PR-5 as described in Uknes et al.

(1992). In contrast to the case in wild type plants, the chemicals did not induce RNA accumulation from any of these 3 SAR genes in niml-1 plants.

As shown in Figure 2, pathogen infection (Emwa) of wild type Ws-O plants induced PR-1 gene expression within 4 days after infection. In niml-1 plants, however, PR-1 gene expression was not induced until 6 days after infection and the level was reduced relative to the wild type at that time. Thus, following pathogen infection, PR-1 gene expression in niml-1 plants was delayed and reduced relative to the wild type.

The RNA gel blot in Figure 3 shows that PR-1 mRNA accumulates to high levels following treatment of wild-type plants with SA, INA, or BTH or infection by P. parasitica. In the nim nim1-2, and nim1-3 plants, PR-1 mRNA accumulation was dramatically reduced relative to the wild type following chemical treatment. PR-1 mRNA was also reduced following P. parasitica infection, but there was still some accumulation in these mutants. In the nimi-4 and nimi-6 plants, PR-1 mRNA accumulation was more dramatically reduced than in the other alleles following chemical treatment (evident in longer exposures) and significantly less PR-1 mRNA accumulated following P. parasitica infection, supporting the idea that these are particularly strong niml alleles. PR-1 mRNA accumulation was elevated in the nim1-5 mutant, but only mildly induced following chemical treatment or P. parasitica infection. Based on both PR-1 mRNA accumulation and fungal infection, the mutants have been determined to fall into three classes: severely compromised alleles (niml-4 and nimlmoderately compromised alleles (niml-1, niml-2, and niml-3); and a weakly compromised allele Example 5: Determination of SA Accumulation in nimi Plants Infection of wild type plants with pathogens that cause a necrotic reaction leads to accumulation of SA in the infected tissues. Endogenous SA is required for signal transduction in the SAR pathway, as breakdown of the endogenous SA leads to a decrease in disease resistance. This defines SA accumulation as a marker in the SAR pathway (Gaffney et al, 1993, Science 261, 754-756). The phenotype of niml plants indicates a disruption in a component of the SAR pathway downstream of SA and upstream of SAR gene induction.

WO 98/26082 PCT/EP97/07012 niml plants were tested for their ability to accumulate SA following pathogen infection.

Pseudomonas syringae tomato strain DC 3000, carrying the avrRpt2 gene, was injected into leaves of 4-week-old niml plants. The leaves were harvested 2 days later for SA analysis as described by Delaney et al, 1995, PNAS 2, 6602-6606. This analysis showed that the niml plants accumulated high levels of SA in infected leaves, as shown in Figure 4.

Uninfected leaves also accumulated SA, but not to the same levels as the infected leaves, similar to what has been observed in wild-type Arabidopsis. This indicates that the nim mutation maps downstream of the SA marker in the signal transduction pathway.

Furthermore, INA and BTH (inactive in nim1 plants) have been demonstrated to stimulate a component in the SAR pathway downstream of SA (Vernooij et al. (1995); Friedrich, et al.

(1996); and Lawton, et al. (1996)). In addition, as described above, exogenously applied SA did not protect nimi plants from Emwa infection.

Example 6: Genetic Analysis To determine dominance of the various mutants that display the niml phenotype, pollen from wild type plants was transferred to the stigmata of niml-1, If the mutation is dominant, then the niml phenotype will be observed in the resulting F1 plants. If the mutation is recessive, then the resulting F1 plants will exhibit a wild type phenotype.

The data presented in Table 3 show that when niml-1, -4 and -6 were crossed with the wild type, the resulting F1 plants exhibited the wild type phenotype. Thus, these mutations are recessive. In contrast, the niml-5 Xwild type F1 descendants all exhibited the niml phenotype, indicating that this is a dominant mutation. Following INA treatment, no P. parasitica sporulation was observed on wild type plants, while the F1 plants supported growth and some sporulation of P. parasitica. However, the niml phenotype in these F1 plants was less severe than observed when niml-5 was homozygous.

To determine allelism, pollen from the kanamycin-resistant nim1-1 mutant plants was transferred to the stigmata of nim1-2, Seeds resulting from the cross were plated onto Murashige-Skoog B5 plates containing kanamycin at 25 lg/ml to verify the hybrid origin of the seed. Kanamycin resistant (F1) plants were transferred to soil and assayed for the niml phenotype. Because the F1 descendants of the cross of the mutant with the Ws wild type display a nimi phenotype, analysis of niml-5X nim1-1 F2 was also carried out.

As shown in Table 3, all of the resulting F1 plants exhibited the niml phenotype.

Thus, the mutation in the niml-2, -6 was not complemented by the niml-1; these WO 98/26082 PCT/EP97/07012 -66plants all fall within the same complementation group and are therefore allelic. F2 descendants from the niml-5 Xnim-1 cross also displayed the nimi phenotype, confirming that nim-5 is a nimi allele.

Table 3. Genetic Segregation of nim Mutants Phenotype Female Male Wild type a niml b Mutant Generatio n niml-1 Fl F2 niml-2 F1 niml-3 F1 nimi-4 F1 nim 1-5 F1 F1 nim1-6 F1 niml-2 F1 niml-3 F1 nim1-4 F1 F1 F2 niml-6 F1 wild type nim1-1 nim 1-2 niml-3 niml-4 nim 1-5 Wild type niml-6 nim1-2 nim1-3 nim -4 nim1-5 nim1-6 Wild type Wild type Wild type Wild type Wild type niml-1 niml-1 niml-1 niml-1 niml-1 Number of plants with elevated PR-1 mRNA accumulation and absence of P.

parasitica after INA treatment.

Number of plants with no PR-1 mRNA accumulation and presence of P. parasitica after INA treatment.

Wild type denotes the wild type Ws-0 strain.

B. Mapping of the nimi Mutation Mapping of the nim1 mutation is described in exhaustive detail in Applicants' U.S.

Patent Application Serial No. 08/773,559, filed December 27, 1996, which is incorporated by reference herein in its entirety.

Example 7: Identification of Markers in and Genetic Mapping of the NIM1 Locus To determine a rough map position for NIMi, 74 F 2 nim plants from a cross between niml-1 (Ws-0) and Landsberg erecta (Le) were identified for their susceptibility to P.

parasitica and lack of accumulation of PR-1 mRNA following INA treatment. Using simple sequence length polymorphism (SSLP) markers (Bell and Ecker 1994), niml-1 was determined to lie about 8.2 centimorgans (cM) from nga128 and 8.2 cM from ngal 11 on the WO 98/26082 PCT/EP97/07012 -67lower arm of chromosome 1. In addition, niml-1 was determined to lie between ngal 11 and about 4 cM from the SSLP marker ATHGENEA. (Figure For fine structure mapping, 1138 nim plants from an F 2 population derived from a cross between nim -1 and LerDP23 were identified based on both their inability to accumulate PR-1 mRNA and their ability to support fungal growth following INA treatment.

DNA was extracted from these plants and scored for zygosity at both ATHGENEA and ngal 11. As shown in Figure 5A, 93 recombinant chromosomes were identified between ATHGENEA and niml-1, giving a genetic distance of approximately 4.1 cM (93 of 2276), and 239 recombinant chromosomes were identified between nga111 and nim -1, indicating a genetic distance of about 10.5 cM (239 of 2276). Informative recombinants in the ATHGENEA to ngal 11 interval were further analyzed using amplified fragment length polymorphism (AFLP) analysis (Vos et al., 1995).

AFLP markers between ATHGENEA and ngal 11 were identified and were used to construct a low resolution map of the region (Figures 5A and 5B). AFLP markers W84.2 (1 cM from nim and W85.1 (0.6 cM from nim were used to isolate yeast artificial chromosome (YAC) clones from the CIC (for Centre 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) To bridge the gap between the two sets of flanking YAC clones, bacterial artificial chromosome (BAC) and P1 clones that overlapped CIC12H07 and CIC12F04 were isolated and mapped, and sequential walking steps were carried out extending the BAC/P1 contig toward NIM1 (Figure 5C; Liu et al., 1995; Chio et al., 1995). New AFLP's were developed during the walk that were specific for BAC or P1 clones, and these were used to determine whether the NIM1 gene had been crossed.

NIM1 had been crossed when BAC and P1 clones were isolated that gave rise to both AFLP markers L84.6a and L84.8. The AFLP marker L84.6a found on P1 clones P1-18, P1-17, and P1-21 identified three recombinants and L84.8 found on P1 clones P1-20, P1- 22, P1-23, and P1-24 and BAC clones, BAC-04, BAC-05, and BAC-06 identified one recombinant. Because these clones overlapped to form a large contig (>100 kb), and included AFLP markers that flanked niml, the gene was determined to be located on the contig. The BAC and P1 clones that comprised the contig were used to generate additional AFLP markers, which showed that niml was located between L84.Y1 and L84.8, representing a gap of about 0.09 cM.

WO 98/26082 PCT/EP97/07012 -68- C. Isolation of the NIM1 Gene Example 8: Construction of a Cosmid Contig A cosmid library of the NIM1 region was constructed in the Agrobacteriurn-compatible T-DNA cosmid vector pCLD04541 using CsCI-purified DNA from BAC-06, BAC-04, and P1- 18. The DNAs of the three clones were mixed in equimolar quantities and were partially digested with the restriction enzyme Sau3A. The 20-25 kb fragments were isolated using a sucrose gradient, pooled and filled in with dATP and dGTP. Plasmid pCLD04541 was used as T-DNA cosmid vector. This plasmid contains a broad host range pRK290-based replicon, a tetracycline resistance gene for bacterial selection and the nptll gene for plant selection. The vector was cleaved with Xhol and filled in with dCTP and dTTP. The prepared fragments were then ligated into the vector. The ligation mix was packaged and transduced into E. colistrain XL1-blue MR (Stratagene). Resulting transformants were screened by hybridization with the BAC04, BAC06 and P1-18 clones and positive clones isolated. Cosmid DNA was isolated from these clones and template DNA was prepared using the ECs EcoRI/Msel and Hindlll/Msel. The resulting AFLP fingerprint patterns were analyzed to determine the order of the cosmid clones. A set of 15 semi-overlapping cosmids was selected spanning the nim region (Figure 5D). The cosmid DNAs were also restricted with EcoRI, Pstl, BssHII and SgrAI. This allowed for the estimation of the cosmid insert sizes and the verification of the overlaps between the various cosmids as determined by AFLP fingerprinting.

Physical mapping showed that the physical distance between L84.Y1 and L84.8 was kb, giving a genetic to physical distance of -1 megabase per cM. To facilitate the identification of the NIM1 gene, the DNA sequence of BAC04 was determined.

Example 9: Identification of a Clone containing the NIM1 Gene.

Cosmids generated from clones spanning the NIM1 region were moved into Agrobacterium tumefaciens AGL-1 through conjugative transfer in a tri-parental mating with helper strain HB101 (pRK2013). These cosmids were then used to transform a kanamycinsensitive niml-1 Arabidopsis line using vacuum infiltration (Bechtold et al., 1993; Mindrinos et al., 1994). Seed from the infiltrated plants was harvested and allowed to germinate on GM agar plates containing 50 mg/ml kanamycin as a selection agent. Only plantlets that were transformed with cosmid DNA could detoxify the selection agent and survive.

Seedlings that survived the selection were transferred to soil approximately two weeks after WO 98/26082 PCT/EP97/07012 -69plating and tested for the niml phenotype as described below. Transformed plants that no longer had the niml phenotype identified cosmid(s) containing a functional NIM1 gene.

Example 10: Complementation of the nim1 Phenotype Plants transferred to soil were grown in a phytotron for approximately one week after transfer. 300pm INA was applied as a fine mist to completely cover the plants using a chromister. After two days, leaves were harvested for RNA extraction and PR-1 expression analysis. The plants were then sprayed with Peronospora parasitica (isolate Emwa) and grown under high humidity conditions in a growing chamber with 190C day/17°C night temperatures and 8h light/16h dark cycles. Eight to ten days following fungal infection, plants were evaluated and scored positive or negative for fungal growth. Ws and niml plants were treated in the same way to serve as controls for each experiment.

Total RNA was extracted from the collected tissue using a LiCI/phenol extraction buffer (Verwoerd, et al. 1989). RNA samples were run on a formaldehyde agarose gel and blotted to GeneScreen Plus (DuPont) membranes. Blots were hybridized with a 32 P-labeled PR-1 cDNA probe. The resulting blots were exposed to film to determine which transformants were able to induce PR-1 expression after INA treatment. The results are summarized in Table 4, which shows complementation of the niml phenotype by cosmid clones D5, El, and D7.

Table 4 Clone Name of transformants of plants with INA induced A8-- PR-1/ of plants tested A8 3 0/3 All 8 4/18(22%) C2 10 1/10 C7 33 1/32 02 81 4/49 6 5/6 (83%) E1 10 10/10 (100%) D7 129 36/36 (100%) E8 9 0/9 F12 6 0/6 E6 1 0/1 E7 34 0/4 WS-control (wild-type) NA 28/28 (100%) niml-1 phenotype control NA 0/34 NA not applicable WO 98/26082 PCT/EP97/07012 Example 11: Sequencing of the NIM1 Gene Region BAC04 DNA (25 ug, obtained from KeyGene) was the source of DNA used for sequence analysis, as this BAC was the clone completely encompassing the region that complemented the niml mutants. BAC04 DNA was randomly sheared in a nebulizer to generate fragments with an average length of about 2 kb. Ends of the sheared fragments were repaired, and the fragments were purified. Prepared DNA was ligated with EcoRVdigested pBRKanF4 (a derivative of pBRKanF1 (Bhat 1993)). Resulting kanamycin-resistant colonies were selected for plasmid isolation using the Wizard Plus 9600 Miniprep System (Promega). Plasmids were sequenced using dye terminator chemistry (Applied BioSystems, Foster City, CA) with primers designed to sequence both strands of the plasmids (M13-21 forward and T7 reverse, Applied BioSystems). Data was collected on ABI377 DNA sequencers. Sequences were edited and assembled into contigs using 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 Gordon, Washington University, St. Louis, MO). DNA from the cosmids found to complement the niml-1 mutation was sequenced using primers designed by Oligo 5.0 Primer Analysis Software (National Biosciences, Inc., Plymouth, MN).

Sequencing of DNA from Ws-0 and the niml alleles and cDNAs was performed essentially as described above.

A region of approximately 9.9 kb defined by the overlap of cosmids El and D7 was identified by complementation analysis to contain the niml region. Primers that flanked the insertion site of the vector and that were specific to the cosmid backbone were designed using Oligo 5.0 Primer Analysis Software (National Biosciences, Inc.). DNA was isolated from cosmids D7 and El using a modification of the ammonium acetate method (Traynor, 1990. BioTechniques 676.) This DNA was directly sequenced using Dye Terminator chemistry above. The sequence obtained allowed determination of the endpoints of the complementing region. The region defined by the overlap of cosmids El and D7 is presented as SEQ ID NO:1.

A truncated version of the BamHI-EcoRV fragment was also constructed, resulting in a construct that contained none of the "Gene 3" region (Fig. 5D). The following approach was necessary due the presence of Hindlll sites in the Bam-Spe region of the DNA. The BamHI-EcoRV construct was completely digested with Spel, then was split into two separate reactions for double digestion. One aliquot was digested with BamHI, the other Hindlll. A BamHI-Spel fragment of 2816 bp and a Hindlll-Spel fragment of 1588 bp were isolated from agarose gels (QiaQuick Gel extraction kit) and were ligated to BamHI-Hindlll- WO 98/26082 PCT/EP97/07012 -71digested pSGCGO1. DH5a was transformed with the ligation mix. Resulting colonies were screened for the correct insert by digestion with Hindlll following 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 NIM1 Region The 9.9 kb region containing the NIM1 gene was analyzed for the presence of open reading frames in all six frames using Sequencher 3.0 and the GCG package. Four regions containing large ORF's were identified as possible genes (Gene Regions 1-4 in Figure 5D). These four regions were PCR amplified from DNA of the wild-type parent and the six different niml allelic variants niml-1, and Primers for these amplifications were selected using Oligo 5.0 (National Biosciences, Inc.) and were synthesized by Integrated DNA Technologies, Inc. PCR products were separated on agarose gels and were purified using the QIAquick Gel Extraction Kit. The purified genomic PCR products were directly sequenced using the primers used for the initial amplification and with additional primers designed to sequence across any regions not covered by the initial primers. Average coverage for these gene regions was approximately reads/base.

Sequences were edited and were assembled using Sequencher 3.0. Base changes specific to various niml alleles were identified only in the region designated Gene Region 2, as shown below in Table 5, which shows sequence variations among all six of the nimi alleles.

WO 98/26082 -72- Table PCT/EP97/07012 Gene Regior Allele/ ecotype 1 (bases 590- 1090) 2 (NIM1) (bases 1380-4100) Gene Reqion 3 4 (bases 5870 (bases 8140- -6840) 9210) no changes no changes niml-1 nim 1-2 nim 1-3 no changes no changes no changes t inserted at 2981: change of 7AA and premature termination of protein.

g to a at 2799: His to Tyr i no chanoes nn phannao n hl ing

I

deletion of t at 3261: change of 10AA and premature termination of protein.

nim-4 no changes c to t at 2402: nim -5 no changes c to t at 2402: Arg t no changes c to t at 2402: Argt niml-6 g to a at 734: g to a at 2670: Gin asp to lys to lys :o lys to Stop

WS

(compared to Columbia)

RNA

detected no changes a to g at 1607: lie to Leu a to c at 2344: intron t to g at 2480: Gin to Pro g to c at 2894: Ser to Trp ggc deleted at 3449: lose Ala c to t at 3490: Ala to Thr c to t at 3498: Ser to Asn a to t at 3873: non-coding g to a at 3992: non-coding g to a at 4026: non-coding g to a at 4061: non-coding Yes no changes no changes no changes no changes t to a at 5746 a to t at 5751 t to a at 5754 c to t at 6728 a to t at 6815 t to c at 6816 No no changes no changes no changes no changes t to g at 8705 g to t at 8729 g to t at 8739 g to t at 8784 c to a at 8789 c to t at 8812 a to g at 8829 t to g at 8856 a to c at 9004 a to t at 9011 a to g at 8461 No No Positions listed in the table relate to SEQ ID NO:1. All alleles niml-1 to niml-6are WS strain. Columbia-0 represents the wild type It is apparent that the NIM1 gene lies within Gene Region 2, because there are amino acid changes or alterations of sequence within the open reading frame of Gene Region 2 in all six niml alleles. At the same time, at least one of the niml alleles shows no changes in the open reading frames within Gene Regions 1, 3 and 4. Therefore, the only gene region within the 9.9 kb region that could contain the NIM1 gene is Gene Region 2.

The Ws section of Table 5 indicates the changes in the Ws ecotype of Arabidopsis relative to the Columbia ecotype of Arabidopsis. The sequences presented herein relate to the Columbia ecotype of Arabidopsis, which contains the wild type gene in the experiments described herein. The changes are listed as amino acid changes within Gene Region 2 (the NIM1 region) and are listed as changes in base pairs in the other regions.

WO 98/26082 PCT/EP97/07012 -73- The cosmid region containing the niml gene was delineated by a BamH1-EcoRV restriction fragment of -5.3 kb. Cosmid DNA from D7 and plasmid DNA from pBlueScriptll(pBSII) were digested with BamHI and with EcoRV (NEB). The 5.3 kb fragment from D7 was isolated from agarose gels and was purified using the QIAquick gel extraction kit 28796, Qiagen). The fragment was ligated overnight to the Bam-EcoRV-digested pBSII and the ligation mixture was transformed into E. colistrain DH5a. Colonies containing the insert were selected, DNA was isolated, and confirmation was made by digestion with Hindlll. The Bam-EcoRV fragment was then engineered into a binary vector (pSGCGO1) for transformation into Arabidopsis.

Example 13: Northern Analysis of the Four Gene Regions Identical Northern blots were made from RNA samples isolated from water-, SA-, BTH- and INA-treated Ws and niml lines as previously described in Delaney, et al. (1995).

These blots were hybridized with PCR products generated from the four gene regions identified in the 9.9 kb NIM1 gene region (SEQ ID NO:1). Only the gene region containing the NIM1 gene (Gene Region 2) had detectable hybridization with the RNA samples, indicating that only the NIM1 region contains a detectable transcribed gene (Figure 5D and Table Example 14: Complementation with Gene Region 2 Gene Region 2 (Fig. 5D) was also demonstrated to contain the functional NIM1 gene by doing additional complementation experiments. A BamHI/Hindlll genomic DNA fragment containing Gene Region 2 was isolated from cosmid D7 and was cloned into the binary vector pSGCGO1 containing the gene for kanamycin resistance. The resulting plasmid was transformed into the Agrobacterium strain GV3101 and positive colonies were selected on kanamycin. PCR was used to verify that the selected colony contains the plasmid. Kanamycin-sensitive niml-1 plants were infiltrated with this bacteria as previously described. The resulting seed was harvested and planted on GM agar containing kanamycin. Plants surviving selection were transferred to soil and tested for complementation. Transformed plants and control Ws and niml plants were sprayed with 300m INA. Two days later, leaves were harvested for RNA extraction and PR-1 expression analysis. The plants were then sprayed with Peronospora parasitica (isolate Emwa) and grown as previously described. Ten days following fungal infection, plants were WO 98/26082 PCT/EP97/07012 -74evaluated and scored positive or negative for fungal growth. All of the 15 transformed plants, as well as the Ws controls, were negative for fungal growth following INA treatment, while the nimi controls were positive for fungal growth. RNA was extracted and analyzed as described above for these transformants and controls. Ws controls and all transformants showed PR-1 gene induction following INA treatment, while the nimi controls did not show PR-1 induction by INA.

Example 15: Isolation of a NIM1 cDNA An Arabidopsis cDNA library made in the IYES expression vector (Elledge et al, 1991, PNAS 88, 1731-1735) was plated and plaque lifts were performed. Filters were hybridized with a 32P-labeled PCR product generated from Gene Region 2 (Figure 5D). 14 positives were identified from a screen of approximately 150,000 plaques. Each plaque was purified and plasmid DNA was recovered. cDNA inserts were digested out of the vector using EcoRI, agarose-gel-purified and sequenced. Sequence obtained from the longest cDNA is indicated in SEQ ID NO:2 and Figure 6. To confirm that the 5' end of the cDNA had been obtained, a Gibco BRL 5' RACE kit was used following manufacturer's instructions. The resulting RACE products were sequenced and found to include the additional bases indicated in Figure 6. The transcribed region present in both cDNA clones and detected in RACE is shown as capital letters in Figure 6. Changes in the alleles are shown above the DNA strand. Capitals indicate the presence of the sequence in a cDNA clone or detected after RACE PCR.

The same RNA samples produced in the induction studies (Figure 3) were also probed with the NIM1 gene using a full-length cDNA clone as a probe. In Figure 7 it can be seen that INA induced the NIM1 gene in the wild type Ws allele. However, the niml-1 mutation allele showed a lower basal level expression of the NIM1 gene, and it was not inducible by INA. This was similar to what was observed in the niml-3 allele and the niml-6 allele. The nim 1-2 allele showed approximately normal levels in the untreated sample and showed similar induction to that of the wild type sample, as did the niml-4 allele. The nim1- 5 allele seemed to show higher basal level expression of the NIM1 gene and much stronger expression when induced by chemical inducers.

WO 98/26082 PCT/EP97/07012 D. NIM1 Homologues Example 16: BLAST Search with the NIM1 Sequence A multiple sequence alignment was constructed using Clustal V (Higgins, Desmond G.

and Paul M. Sharp (1989), Fast and sensitive multiple sequence alignments on a microcomputer, CABIOS 5:151-153) as part of the DNA* (1228 South Park Street, Madison Wisconsin, 53715) Lasergene Biocomputing Software package for the Macintosh (1994).

Certain regions of the NIM1 protein are homologous in amino acid sequence to 4 different rice cDNA protein products. The homologies were identified using the NIM1 sequences in a GenBank BLAST search. Comparisons of the regions of homology in NIM1 and the rice cDNA products are shown in Figure 8 (See also, SEQ ID NO:3 and SEQ ID NO's: 4-11).

The NIM1 protein fragments show from 36 to 48% identical amino acid sequences with the 4 rice products.

Example 17: Isolation of Homologous Genes from Other Plants Using the NIM1 cDNA as a probe, homologs of Arabidopsis NIM1 are identified through screening genomic or cDNA libraries from different crops such as, but not limited to those listed below in Example 22. Standard techniques for accomplishing this include hybridization screening of plated DNA libraries (either plaques or colonies; see, e.g.

Sambrook et al., Molecular Cloning eds., Cold Spring Harbor Laboratory Press. (1989)) and amplification by PCR using oligonucleotide primers (see, e.g. Innis et al., PCR Protocols, a Guide to Methods and Applications eds., Academic Press (1990)). Homologs identified are genetically engineered into the expression vectors herein and transformed into the above listed crops. Transformants are evaluated for enhanced disease resistance using relevant pathogens of the crop plant being tested.

NIM1 homologs in the genomes of cucumber, tomato, tobacco, maize, wheat and barley have been detected by DNA blot analysis. Genomic DNA was isolated from cucumber, tomato, tobacco, maize, wheat and barley, restriction digested with the enzymes BamHI, Hindll, Xbal, or Sail, electrophoretically separated on 0.8% agarose gels arid transferred to nylon membrane by capillary blotting. Following UV-crosslinking to affix the DNA, the membrane was hybridized under low stringency conditions [(1%BSA; 520mM NaPO 4 pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride) at 55 0 C for 18-24h] with 32 P-radiolabelled Arabidopsis thaliana NIM1 cDNA. Following hybridization the blots were washed under low stringency conditions [6XSSC for 15 min.

-76- (X3) 3XSSC for 15 min. (X1) at 550C; 1XSSC is 0.15M NaCI, 15mM Na-citrate (pH7.0)] and exposed to X-ray film to visualize bands that correspond to NIM1.

In addition, expressed sequence tags (EST) identified with similarity to the NIM1 gene such as the rice EST's described in Example 16 can also be used to isolate homologues.

The rice EST's may be especially useful for isolation of NIM1 homologues from other monocots.

Homologues may be obtained by PCR. In this method, comparisons are made between known homologues rice and Arabidopsis). Regions of high amino acid and DNA similarity or identity are then used to make PCR primers. Regions rich in M and W are best followed by regions rich in F, Y, C, H, Q, K and E because these amino acids are encoded by a limited number of codons. Once a suitable region is identified, primers for that region are made with a diversity of substitutions in the 3 rd codon position. This diversity of substitution in the third position may be constrained depending on the species that is being targeted. For example, because maize is GC rich, primers are designed that utilize a 15 G or a C in the 3 r position, if possible.

The PCR reaction is performed from cDNA or genomic DNA under a variety of °standard conditions. When a band is apparent, it is cloned and/or sequenced to determine if it is a NIM1 homologue.

E. Overexpression of NIM1 Confers Disease Resistance In Plants Overexpression of the NIM1 gene in transgenic plants to confer a CIM phenotype is also described in Applicants' WO 97/49822 (which corresponds to US Patent Application Serial No. 08/773,559, filed December, 27, 1996) which is incorporated by reference herein in its entirety.

Example 18: Overexpression Expression of NIM1 Due To Insertion Site Effect To determine if any of the transformants described above in Example 10/Table 4 had overexpression of NIM1 due to insertion site effect, primary transformants containing the D7, D5 or El cosmids (containing the NIM1 gene) were selfed and the T2 seed collected.

Seeds from one El line, four D5 lines and 95 D7 lines were sown on soil and grown as described above. When the T2 plants had obtained at least four true leaves, a single leaf was harvested separately for each plant. RNA was extracted from this tissue and analyzed for PR-1 and NIM1 expression. Plants were then inoculated with P. parasitica (Emwa) and ,e dalyzed for fungal growth at 3-14 days, preferably 7-12 days, following infection. Plants WO 98/26082 PCT/EP97/07012 -77showing higher than normal NIM1 and PR-1 expression and displaying fungal resistance demonstrated that overexpression of NIM1 confers a CIM phenotype.

Table 6 shows the results of testing of various transformants for resistance to fungal infection. As can be seen from the table, a number of transformants showed less than normal fungal growth and several showed no visible fungal growth at all. RNA was prepared from collected samples and analyzed as previously described (Delaney et al, 1995). Blots were hybridized to the Arabidopsis gene probe PR-1 (Uknes et al, 1992).

Lines D7-74, D5-6 and E1-1 showed early induction of PR-1 gene expression, whereby PR- 1 mRNA was evident by 24 or 48 hours following fungal treatment. These three lines also demonstrated resistance to fungal infection.

Table 6 Line P.parasitica Line P.parasitica Line P.parasitica growth growth growth D7-2 negative 52 90 3 53 91 9 54 92 11 56 93 12 57 94 13 58 95 14 59 96 17 60 97 18 61 98 19 62 100 63 101 21 64 102 22 66 103 23 67 104 24 68 106 69 107 28 70 108 29 71 114 31 72 115 32 73 118 33 74 negative 119 34 75 122 77 123 36 78 124 38 79 125 39 80 126 42 81 128 43 82 129 46 83 130 WO 98/26082 PCT/EP97/07012 -78- 47 84 D5-1 48 85 2 49 86 4 87 6 51 89 negative E1-1 negative Plants were treated with P. parasitica isolate Emwa and scored 10 days later.

normal fungal growth less than normal fungal growth negative, no visible fungal growth Example 19: NIM1 Overexpression Under Its Native Promoter Plants constitutively expressing the NIM1 gene were generated from transformation of Ws wild type plants with the BamHI-Hindlll NIM1 genomic fragment (SEQ ID NO: 2 bases 1249-5655) containing 1.4 kb of promoter sequence. This fragment was cloned into pSGCG01 and transformed into the Agrobacterium strain GV3101 (pMP90, Koncz and Schell (1986) Mol. Gen. Genet. 204:383-396). Ws plants were infiltrated as previously described. The resulting seed was harvested and plated on GM agar containing 50 pg/ml kanamycin. Surviving plantlets were transferred to soil and tested as described above for resistance to Peronospora parasitica isolate Emwa. Selected plants were selfed and selected for two subsequent generations to generate homozygous lines. Seeds from several of these lines were sown in soil and 15-18 plants per line were grown for three weeks and tested again for Emwa resistance without any prior treatment with an inducing chemical. Approximately 24 hours, 48 hours, and five days after fungal treatment, tissue was harvested, pooled and frozen for each line. Plants remained in the growth chamber until ten days after inoculation when they were scored for resistance to Emwa.

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

A subset of the resistant lines were also tested for increased resistance to the bacterial pathogen Pseudomonas syringae DC3000 to evaluate the spectrum of resistance evident as described by Uknes et al. (1993). Experiments were done essentially as described by Lawton et al. (1996). Bacterial growth was slower in those lines that also WO 98/26082 PCT/EP97/07012 -79demonstrated constitutive resistance to Emwa. This shows that plants overexpressing the NIM1 gene under its native promoter have constitutive immunity against pathogens.

To assess additional characteristics of the CIM phenotype in these lines, unifected plants are evaluated for free and glucose-conjugated salicylic acid and leaves are stained with lactophenol blue to evaluate for the presence of microscopic lesions. Resistance plants are sexually crossed with SAR mutants such as NahG and ndrl to establish the epistatic relationship of the resistance phenotype to other mutants and evaluate how these dominant negative mutants of NIM1 may influence the salicylic acid-dependent feedback loop.

Example 20: 35S Driven Overxpression of NIM1 The full-length NIM1 cDNA (SEQ ID NO: 21) was cloned into the EcoRI site of pCGN1761 ENX (Comai et al. (1990) Plant Mol. Biol. 15, 373-381). From the resulting plasmid, an Xbalfragment containing an enhanced CaMV 35S promoter, the NIM1 cDNA in the correct orientation for transcription, and a tml 3' terminator was obtained. This fragment was cloned into the binary vector pCIB200 and transformed into GV3101. Ws plants were infiltrated as previously described. The resulting seed was harvested and plated on GM agar containing 50 pg/ml kanamycin. Surviving plantlets were transferred to soil and tested as described above. Selected plants were selfed and selected for two subsequent generations to generate homozygous lines. Nine of the 58 lines tested demonstrated resistance when they were treated with Emwa without prior chemical treatment. Thus, overexpression of the NIM1 cDNA also results in disease-resistant plants.

Example 21: High Level Expression of NIM1 in Crop Plants Those constructs conferring a CIM phenotype in Col-0 or Ws-0 and others are transformed into crop plants for evaluation. Although the NIM1 gene can be inserted into any plant cell falling within these broad classes, it is particularly useful in crop plant cells, such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane. Transformants are evaluated for enhanced disease resistance.

In a preferred embodiment of the invention, the expression of the NIM1 gene is at a level WO 98/26082 PCT/EP97/07012 which is at least two-fold above the expression level of the NIM1 gene in wild type plants and is preferably ten-fold above the wild type expression level.

F. Other Uses of nim Phenotype Plants Generally Example 22: The Use of nim Mutants in Disease Testing nim mutants are challenged with numerous pathogens and found to develop larger lesions more quickly than wild-type plants. This phenotype is referred to as UDS (i.e.

universal disease susceptibility) and is a result of the mutants failing to express SAR genes to effect the plant defense against pathogens. The UDS phenotype of nim mutants renders them useful as control plants for the evaluation of disease symptoms in experimental lines in field pathogenesis tests where the natural resistance phenotype of so-called wild type lines may vary to different pathogens and different pathotypes of the same pathogen).

Thus, in a field environment where natural infection by pathogens is being relied upon to assess the resistance of experimental lines, the incorporation into the experiment of nim mutant lines of the appropriate crop plant species would enable an assessment of the true level and spectrum of pathogen pressure, without the variation inherent in the use of nonexperimental lines.

Example 23: Assessment of the Utility of Transgenes for the Purposes of Disease Resistance nim mutants are used as host plants for the transformation of transgenes to facilitate their assessment for use in disease resistance. For example, an Arabidopsis nim mutant line, characterized by its UDS phenotype, is used for subsequent transformations with candidate genes for disease resistance thus enabling an assessment of the contribution of an individual gene to resistance against the basal level of the UDS nim mutant plants.

Example 24: nim Mutants as a Tool in Understanding Plant-Pathogen Interactions nim mutants are useful for the understanding of plant pathogen interactions, and in particular for the understanding of the processes utilized by the pathogen for the invasion of plant cells. This is so because nim mutants do not mount a systemic response to pathogen attack, and the unabated development of the pathogen is an ideal scenario in which to study its biological interaction with the host.

WO 98/26082 PCT/EP97/07012 -81 Of futher significance is the observation that a host nim mutant may be susceptible to pathogens not normally associated with that particular host, but instead associated with a different host. For example, an Arabidopsis nim mutant such as niml-1, or-6 is challenged with a number of pathogens that normally only infect tobacco, and found to be susceptible. Thus, the nim mutation causing the UDS phenotype leads to a modification of pathogen-range susceptibility and this has significant utility in the molecular, genetic and biochemical analysis of host-pathogen interaction.

Example 25: nim Mutants for Use in Fungicide Screening nim mutants are particularly useful in the screening of new chemical compounds for fungicide activity. nim mutants selected in a particular host have considerable utility for the screening of fungicides using that host and pathogens of the host. The advantage lies in the UDS phenoytpe of the mutant that circumvents the problems encountered by the host being differentially susceptible to different pathogens and pathotypes, or even resistant to some pathogens or pathotypes. By way of example, nim mutants in wheat could be effectively used to screen for fungicides to a wide range of wheat pathogens and pathotypes as the mutants would not mount a resistance response to the introduced pathogen and would not display differential resistance to different pathotypes that might otherwise require the use of multiple wheat lines, each adequately susceptible to a particular test pathogen. Wheat pathogens of particular interest include (but are not limited to) Erisyphe graminis (the causative agent of powdery mildew), Rhizoctonia solani (the causative agent of sharp eyespot), Pseudocercosporella herpotrichoides (the causative agent of eyespot), Puccinia spp. (the causative agents of rusts), and Septoria nodorum.

Similarly, nim mutants of corn would be highly susceptible to corn pathogens and therefore useful in the screening for fungicides with activity against corn diseases.

nim mutants have further utility for the screening of a wide range of pathogens and pathotypes in a heterologous host i.e. in a host that may not normally be within the host species range of a particular pathogen and that may be particularly easily to manipulate (such as Arabidopsis). By virtue of its UDS phenotype the heterologous host is susceptible to pathogens of other plant species, including economically important crop plant species.

Thus, by way of example, the same Arabidopsis nim mutant could be infected with a wheat pathogen such as Erisyphe graminis (the causative agent of powdery mildew) or a corn pathogen such as Helminthosporium maydis and used to test the efficacy of fungicide candidates. Such an approach has considerable improvements in efficiency over currently used procedures of screening individual crop plant species and different cultivars of species WO 98/26082 PCT/EP97/07012 -82with 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 and the possibility of thereby undertaking more tests with limited resources of space.

Example 26: NIM1 Is A Homolog Of IkBa A multiple sequence alignment between the protein gene products of NIM1 and IkB was performed by which it was determined that the NIM1 gene product is a homolog of IKB a (Figure Sequence homology searches were performed using BLAST (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The multiple sequence alignment was constructed using Clustal V (Higgins et al., CABIOS 5,151-153 (1989)) as part of the Lasergene Biocomputing Software package from DNASTAR (Madison, WI). The sequences used in the alignment were NIM1 (SEQ ID NO:3), mouse IKBa (SEQ ID NO:18, GenBank Accession 1022734), rat iKBa (SEQ ID NO:19, GenBank accession Nos. 57674 and X63594; Tewari et al., Nucleic Acids Res. 20, 607 (1992)), and pig licBa (SEQ ID NO:20, GenBank accession No.

Z21968; de Martin et al., EMBO J. 12, 2773-2779 (1993); GenBank accession No. 517193, de Martin et al., Gene 152, 253-255 (1995)). Parameters used in the Clustal analysis were gap penalty of 10 and gap length penalty of 10. Evolutionary divergence distances were calculated using the PAM250 weight table (Dayhoff et al., "A model of evolutionary change in proteins. Matrices for detecting distant relationships." In Atlas of Protein Sequence and Structure, Vol. 5, Suppl. 3, Dayhoff, ed (National Biomedical Research Foundation, Washington, pp. 345-358 (1978)). Residue similarity was calculated using a modified Dayhoff table (Schwartz and Dayhoff, "A model of evolutionary change in proteins." In Atlas of Protein Sequence and Structure, M.O. Dayhoff, ed (National Biomedical Research Foundation, Washington, pp. 353-358 (1979); Gribskov and Burgess, Nucleic Acids Res. 14, 6745-6763 (1986)).

Homology searches indicate similarity of NIM1 to ankyrin domains of several proteins including: ankyrin, NF-KB and IKB. The best overall homology is to IKB and related molecules (Figure NIM1 contains 2 serines at amino acid positions 55 and 59, the serine at position 59 is in a context (D/ExxxxxS) and position (N-terminal) consistent with a role in phosphorylation-dependent, ubiquitin-mediated, inducible degradation. All IKBs have these N-terminal serines and they are required for inactivation of IKB and subsequent release of NF-KB. NIM1 has ankyrin domains (amino acids 262-290 and 323-371). Ankyrin domains are believed to be involved in protein-protein interactions and are a ubiquitous feature for IKB and NF-KB molecules. The C-termini of IKB's can be dissimilar. NIM1 has (I WO 98/26082 PCT/EP97/07012 -83some homology to a QL-rich region (amino acids 491-499) found in the C-termini of some IK Bs.

Example 27: Generation Of Altered Forms Of NIM1 Changes Of Serine Residues 55 and 59 To Alanine Residues Phosphorylation of serine residues in human I B is required for stimulus-activated degradation of IKBa thereby activating NF-KB. Mutagenesis of the serine residues (S32- S36) in human IKBa to alanine residues inhibits stimulus-induced phosphorylation thus blocking IKBa proteosome-mediated degradation Britta-Mareen Traenckner et al., EMBO J. 14: 2876-2883 (1995); Brown et al., Science 267:1485-1488 (1996); Brockman et al., Molecular and Cellular Biology 15: 2809-2818 (1995); Wang et al., Science 274:784-787 (1996)).

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

This can be done by any method known to those skilled in the art, such as, for example, by using the QuikChange Site Directed Mutagenesis Kit (#200518:Strategene).

Using a full length NIM1 cDNA (SEQ ID NO:21) including 42 bp of 5' untranslated sequence (UTR) and 187 bp of 3' UTR, the mutagenized construct can be made per the manufacturer's instructions using the following primers (SEQ ID NO:21, positions 192-226): CAG CTT CGA AGC CGT CTT TGA CGC GCC GGA TG-3' (SEQ ID NO:32) and CAT CCG GCG CGT CAA AGA CGG CTT CGA AGC TGT TG-3' (SEQ ID NO:33), where the underlined bases denote the mutations. The strategy is as follows: The NIM1 cDNA cloned into vector pSE936 (Elledge et al., Proc. Nat. Acad. Sci. USA 88:1731-1735 (1991)) is denatured and the primers containing the altered bases are annealed. DNA polymerase (Pfu) extends the primers by nonstrand-displacement resulting in nicked circular strands.

DNA is subjected to restriction endonuclease digestion with Dpnl, which only cuts methylated sites (nonmutagenized template DNA). The remaining circular dsDNA is transformed into E.colistrain XL1-Blue. Plasmids from resulting colonies are extracted and sequenced to verify the presence of the mutated bases and to confirm that no other mutations occurred.

The mutagenized NIM1 cDNA is digested with the restriction endonuclease EcoRI and cloned into pCGN1761 under the transcriptional regulation of the double 35S promoter of WO 98/26082 PCT/EP97/07012 -84the cauliflower mosaic virus. The transformation cassette including the 35S promoter, NIM1 cDNA and tmlterminator is released from pCGN1761 by partial restriction digestion with Xbal and ligated into the Xbal and ligated into the Xbal site of dephosphorylated pCIB200.

SEQ ID NO's:22 and 23 show the DNA coding sequence and encoded amino acid sequence, respectively, of this altered form of the NIM1 gene.

The present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under the following conditions to the coding sequence set forth in SEQ ID NO:22: hybridization in 1%BSA; 520mM NaPO4, pH7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 550C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C. In these embodiments, alleles of NIM1 hybridizing to SEQ ID NO: 22 under these conditions are altered so that the encoded product has alanines instead of serines in the amino acid positions that correspond to positions 55 and 59 of SEQ ID NO: 22.

Example 28: Generation Of Altered Forms Of NIM1 N-terminal Deletion Deletion of amino acids 1-36 (Brockman et al.; Sun et al.) or 1-72 (Sun et al.) of human IcBa, which includes K21, K22, S32 and S36, results in a dominant-negative IkBa phenotype in transfected human cell cultures. An N-terminal deletion of approximately the first 125 amino acids of the encoded product of the NIM1 cDNA removes eight lysine residues that may serve as potential ubiquitination sites and also removes putative phosphorylation sites at S55 and S59 (see Example This altered gene construct may be produced by any means known to those skilled in the art. For example, using the method of Ho et al., Gene 77:51-59 (1989), a NIM1 form may be generated in which DNA encoding approximately the first 125 amino acids is deleted. The following primers produce a 1612bp PCR product (SEQ ID NO: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 EcoRI linker sequence is in lower case. Amplification of fragments utilizes a reaction mixture comprising 0.1 to 100 ng of template DNA, 10mM Tris pH 8.3/50mM KCI/2 mM MgCI 2 /0.001% gelatin/0.25 mM each dNTP/0.2 mM of each primer and 1 unit rTth DNA polymerase in a final volume of mL and a Perkin Elmer Cetus 9600 PCR machine. PCR conditions are as follows: 940C 3min: 35x (94°C 30 sec: 520C 1 min: 720C 2 min): 72°C 10 min. The PCR product is cloned directly into the pCR2.1 vector (Invitrogen). The PCR-generated insert in the PCR vector is released by restriction endonuclease digestion using EcoRI and ligated into the EcoRI site of dephosphorylated pCGN1761, under the transcriptional regulation of the double WO 98/26082 PCT/EP97/07012 promoter. The construct is sequenced to verify the presence of the synthetic starting ATG and to confirm that no other mutations occurred during PCR. The transformation cassette including the 35S promoter, modified NIM1 cDNA and tml terminator is released from pCGN1761ENX by partial restriction digestion with Xbal and ligated into the Xbal site of pCIB200. SEQ ID NO's:24 and 25 show the DNA coding sequence and encoded amino acid sequence, respectively, of an altered form of the NIM1 gene having an N-terminal amino acid deletion.

The present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under the following conditions to the coding sequence set forth in SEQ ID NO:24: hybridization in 1%BSA; 520mM NaPO 4 pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55°C for 18- 24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C. In these embodiments, alleles of NIM1 hybridizing to SEQ ID NO:24 under these conditions are altered so that the encoded product has an N-terminal deletion that removes lysine residues that may serve as potential ubiquitination sites in addition to the serines at amino acid positions corresponding to positions 55 and 59 of the wild-type gene product.

Example 29: Generation Of Altered Forms Of NIM1 C-terminal Deletion The deletion of amino acids 261-317 of human hcBa is believed to result in enhanced intrinsic stability by blocking the constitutive phosphorylation of serine and threonine residues in the C-terminus. A region rich in serine and threonine is present at amino acids 522-593 in the C-terminus of NIM1. The C-terminal coding region of the NIM1 gene may be modified by deleting the nucleotide sequences which encode amino acids 522-593. Using the method of Ho et al. (1989), the C-terminal coding region and 3' UTR of the NIM1 cDNA (SEQ ID NO:21: 1606-2011) is deleted by PCR, generating a 1623 bp fragment using the following primers: 5'-cggaattcGATCTCTTAATTTGTGAATTT C-3' (SEQ ID NO:36) and 5'-ggaattcTCAACAGTT CATAATCTGGTCG-3' (SEQ ID NO:37) in which a synthetic stop codon is underlined (TGA on complementary strand) and EcoRI linker sequences are in lower case. PCR reaction components are as previously described and cycling parameters are as follows: 94°C 3 min: 30x (94°C 30 sec: 520C 1 min: 720C 2 min); 720C 10 min]. The PCR product is cloned directly into the pCR2.1 vector (Invitrogen). The PCR-generated insert in the PCR vector is released by restriction endonuclease digestion using EcoRI and ligated into the EcoRI site of dephosphorylated pCGN1761, which contains the double 35S promoter. The construct is sequenced to verify the presence of the synthetic in-frame stop codon and to confirm that no other mutations occurred during WO 98/26082 PCT/EP97/07012 -86- PCR. The transformation cassette including the promoter, modified NIM1 cDNA, and tm/ terminator is released from pCGN1761 by partial restriction digestion with Xbaland ligated into the Xbal site of dephosphorylated pCIB200. SEQ ID NO's:26 and 27 show the DNA coding sequence and encoded amino acid sequence, respectively, of an altered form of the NIM1 gene having a C-terminal amino acid deletion.

The present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under the following conditions to the coding sequence set forth in SEQ ID NO:26: hybridization in 1%BSA; 520mM NaPO4, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55 0 C for 18- 24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 550C. In these embodiments, alleles of NIM1 hybridizing to SEQ ID NO:26 under the above conditions are altered so that the encoded product has a C-terminal deletion that removes serine and threonine residues.

Example 30: Generation Of Altered Forms Of NIM1 N-terminal/C-terminal Deletion Chimera An N-terminal and C-terminal deletion form of NIM1 is generated using a unique Kpnl restriction site at position 819 (SEQ ID NO:21). The N-terminal deletion form (Example 28) is restriction endonuclease digested with EcoRI/Kpnl and the 415 bp fragment corresponding to the modified N-terminus is recovered by gel electrophoresis. Likewise, the C-terminal deletion form (Example 29) is restriction endonuclease digested with EcoRI/Kpnl and the 790 bp fragment corresponding to the modified C-terminus is recovered by gel electrophoresis. The fragments are ligated at 15°C, digested with EcoRlto eliminate EcoRI concatemers and cloned into the EcoRI site of dephosphorylated pCGN1761. The N/Cterminal deletion form of NIM1 is under the transcriptional regulation of the double promoter. Similarly, a chimeric form of NIM1 is generated which consists of the S55/S59 mutagenized putative phosphorylation sites (Example 27) fused to the C-terminal deletion (Example 29). The construct is generated as described above. The constructs are sequenced to verify the fidelity of the start and stop codons and to confirm that no mutations occurred during cloning. The respective transformation cassettes including the promoter, NIM1 chimera and tm/terminator are released from pCGN1761 by partial restriction digestion with Xbal and ligated into the Xbal site of dephosphorylated pCIB200.

SEQ ID NO's:28 and 29 show the DNA coding sequence and encoded amino acid sequence, respectively, of an altered form of the NIM1 gene having both N-terminal and Cterminal amino acid deletions.

WO 98/26082 PCT/EP97/07012 -87- The present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under the following conditions to the coding sequence set forth in SEQ ID NO:28: hybridization in 1%BSA; 520mM NaPO 4 pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55°C for 18- 24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C. In these embodiments, alleles of NIM1 hybridizing to SEQ ID NO:28 under the above conditions are altered so that the encoded product has both an N-terminal deletion, which removes lysine residues that may serve as potential ubiquitination sites in addition to the serines at amino acid positions corresponding to positions 55 and 59 of the wild-type gene product, as well as a C-terminal deletion, which removes serine and threonine residues.

Example 31: Generation Of Altered Forms Of NIM1 Ankyrin Domains NIM1 exhibits homology to ankyrin motifs at approximately amino acids 103-362.

Using the method of Ho et al. (1989), the DNA sequence encoding the putative ankyrin domains (SEQ ID NO:2: 3093-3951) is PCR amplified (conditions: 94 0 C 3 min:35x (94°C sec: 62 0 C 30 sec: 72°C 2 min): 72°C 10 min) from the NIM1 cDNA (SEQ ID NO:21: 349- 1128) using the following primers: 5'-ggaattcaATGGACTCCAACAACACCGCCGC-3'

(SEQ

ID NO:38) and 5' ggaattcTCAACCTTCCAAAGTTGCTTCTGATG-3' (SEQ ID NO:39). The resulting product is restriction endonuclease digested with EcoRI and then spliced into the EcoRI site of dephosphorylated pCGN1761 under the transcriptional regulation of the double 35S promoter. The construct is sequenced to verify the presence of the synthetic start codon (ATG), an in-frame stop codon (TGA) and to confirm that no other mutations occurred during PCR. The transformation cassette including the 35S promoter, ankyrin domains, and tmlterminator is released from pCGN1761 by partial restriction digestion with Xbaland ligated into the Xbalsite of dephosphorylated pCIB200. SEQ ID NO's:30 and 31 show the DNA coding sequence and encoded amino acid sequence, respectively, of the ankyrin domain of NIM1.

The present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under the following conditions to the coding sequence set forth in SEQ ID NO:30: hybridization in 1%BSA; 520mM NaPO 4 pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55°C for 18- 24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C. In these embodiments, alleles of NIM1 hybridizing to SEQ ID NO:30 under the above conditions are altered so that the encoded product consists essentially of the ankyrin domains of the wildtype gene product.

WO 98/26082 PCT/EP97/07012 -88- Example 32: Construction Of Chimeric Genes To increase the likelihood of appropriate spatial and temporal expression of altered NIM1 forms, a 4407 bp Hindlll/BamHI fragment (SEQ ID NO:2: bases 1249-5655) and/or a 5655 bp EcoRV/BamHI fragment (SEQ ID NO:2: bases 1-5655) containing the NIM1 promoter and gene is used for the creation of the altered NIM1 forms in Examples 27-31 above. Although the construction steps may differ, the concepts are comparable to the examples previously described herein. Strong overexpression of the altered forms may potentially be lethal. Therefore, the altered forms of the NIM1 gene described in Examples 27-31 may be placed under the regulation of promoters other than the endogenous NIM1 promoter, including but not limited to the nos promoter or small subunit of Rubisco promoter. Likewise, the altered NIM1 forms may be expressed under the regulation of the pathogen-responsive promoter PR-1 Pat. No. 5,614,395). Such expression permits strong expression of the altered NIM1 forms only under pathogen attack or other SARactivating conditions. Furthermore, disease resistance may be evident in the transformants expressing altered NIM1 forms under PR-1 promoter regulation when treated with concentrations of SAR activator compounds BTH or INA) which normally do not activate SAR, thereby activating a feedback loop (Weymann et al., (1995) Plant Cell 7: 2013-2022).

Example 33: Transformation Of Altered Forms Of The NIM1 Into Arabidopsis thaliana The constructs generated (Examples 27-32) are moved into Agrobacterium tumefaciens by electroporation into strain GV3101. These constructs are used to transform Arabidopsis ecotypes Col-0 and Ws-0 by vacuum infiltration (Mindrinos et al., Cell78, 1089- 1099 (1994)) or by standard root transformation. Seed from these plants is harvested and allowed to germinate on agar plates with kanamycin (or another appropriate antibiotic) as selection agent. Only plantlets that are transformed with cosmid DNA can detoxify the selection agent and survive. Seedlings that survive the selection are transferred to soil and tested for a CIM (constitutive immunity) phenotype. Plants are evaluated for observable phenotypic differences compared to wild type plants. WO 98/26082 PCT/EP97/07012 -89- Example 34: Assessment Of CIM Phenotype In Plants Transformed With Altered Forms Of NIM1 A leaf from each primary transformant is harvested, RNA is isolated (Verwoerd et al., 1989, Nuc Acid Res, 2362) and tested for constitutive PR-1 expression by RNA blot analysis (Uknes et al., 1992). Each transformant is evaluated for an enhanced disease resistance response indicative of constitutive SAR expression analysis (Uknes et al., 1992).

Conidial suspensions of 5-10x104 spores/mi from two compatible P. parasitica isolates, Emwa and Noco these fungal strains cause disease on wildtype Ws-O and Col-0 plants, respectively), are prepared, and transformants are sprayed with the appropriate isolate depending on the ecotype of the transformant. Inoculated plants are incubated under high humidity for 7 days. Plants are disease rated at day 7 and a single leaf is harvested for RNA blot analysis utilizing a probe which provides a means to measure fungal infection.

Transformants that exhibit a CIM phenotype are taken to the T1 generation and homozygous plants are identified. Transformants are subjected to a battery of disease resistance tests as described below. Fungal infection with Noco and Emwa is repeated and leaves are stained with lactophenol blue to identify the presence of fungal hyphae as described in Dietrich et al., (1994). Transformants are infected with the bacterial pathogen Pseudomonas syringae DC3000 to evaluate the spectrum of resistance evident as described in Uknes et al. (1993). Uninfected plants are evaluated for both free and glucose-conjugated SA and leaves are stained with lactophenol blue to evaluate for the presence of microscopic lesions. Resistant plants are sexually crossed with SAR mutants such as NahG Pat. No. 5,614,395) and ndrl to establish the epistatic relationship of the resistance phenotype to other mutants and evaluate how these dominant-negative mutants of NIM1 may influence the SA-dependent feedback loop.

Example 35: Isolation Of NIM1 Homologs Using the NIM1 cDNA (SEQ ID NO:21) as a probe, homologs of Arabidopsis NIM1 are identified through screening genomic or cDNA libraries from different crops such as, but not limited to those listed below in Example 36. Standard techniques for accomplishing this include hybridization screening of plated DNA libraries (either plaques or colonies; see, e.g.

Sambrook et Molecular Cloning eds., Cold Spring Harbor Laboratory Press. (1989)) and amplification by PCR using oligonucleotide primers (see, e.g. Innis et al., PCR Protocols, a Guide to Methods and Applications eds., Academic Press (1990)). Homologs WO 98/26082 PCT/EP97/07012 identified are genetically engineered into the expression vectors herein and transformed into the above listed crops. Transformants are evaluated for enhanced disease resistance using relevant pathogens of the crop plant being tested.

NIM1 homologs in the genomes of cucumber, tomato, tobacco, maize, wheat and barley have been detected by DNA blot analysis. Genomic DNA was isolated from cucumber, tomato, tobacco, maize, wheat and barley, restriction digested with the enzymes BamHI, Hindlll, Xbal, or Sail, electrophoretically separated on 0.8% agarose gels and transferred to nylon membrane by capillary blotting. Following UV-crosslinking to affix the DNA, the membrane was hybridized under low stringency conditions [(1%BSA; 520mM NaPO 4 pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride) at 550C for 18-24h] with 32 P-radiolabelled Arabidopsis thaliana NIM1 cDNA. Following hybridization the blots were washed under low stringency conditions [6XSSC for 15 min.

(X3) 3XSSC for 15 min. (X1) at 550C; 1XSSC is 0.15M NaCI, 15mM Na-citrate (pH7.0)] and exposed to X-ray film to visualize bands that correspond to NIM1.

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

CABIOS

5:151-153) as part of the DNA* (1228 South Park Street, Madison Wisconsin, 53715) Lasergene Biocomputing Software package for the Macintosh (1994). Certain regions of the NIM1 protein are homologous in amino acid sequence to 4 different rice cDNA protein products. The homologies were identified using the NIM1 sequences in a GenBank BLAST search. Comparisons of the regions of homology in NIM1 and the rice cDNA products are shown in Figure 8 (See also, SEQ ID NO:3 and SEQ ID NO's:4-11). The NIM1 protein fragments show from 36 to 48% identical amino acid sequences with the 4 rice products.

These rice EST's may be especially useful for isolation of NIM1 homologues from other monocots.

Homologues may be obtained by PCR. In this method, comparisons are made between known homologues rice and Arabidopsis). Regions of high amino acid and DNA similarity or identity are then used to make PCR primers. Regions rich in amino acid residues M and W are best followed by regions rich in amino acid residues F, Y, C, H, Q, K and E because these amino acids are encoded by a limited number of codons. Once a suitable region is identified, primers for that region are made with a diversity of substitutions in the 3 r codon position. This diversity of substitution in the third position may be -91 constrained depending on the species that is being targeted. For example, because maize is GC rich, primers are designed that utilize a G or a C in the 3 rd position, if possible.

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

Example 36: Expression Altered Forms Of NIM1 In Crop Plants Those constructs conferring a CIM phenotype in Col-O or Ws-0 are transformed into crop plants for evaluation. Alternatively, altered native NIMI genes isolated from crops in the preceding example are put back into the respective crops. Although the NIM1 gene can be inserted into any plant cell falling within these broad classes, it is particularly useful in crop plant cells, such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, •15 asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane. Transformants are evaluated for enhanced '2 disease resistance. In a preferred embodiment of the invention, the expression of the .o 20 altered form of the NIM1 gene is at a level which is at least two-fold above the expression level of the native NIM1 gene in wild type plants and is preferably ten-fold above the wild type expression level.

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

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia WO 98/26082 PCT/EP97/07012 -92-

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L. (1994). Arabidopsis mutants simulating disease resistance response. Cell 77, 565-577.

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WO 98/26082 PCT/EP97/07012 -94- Elledge, Mulligan, Ramer, Spottswood, and Davis, R. (1991). I Yes: A multiunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations. Proc. Natl. Acad. Sci. 88, 1731-1735.

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Increased disease susceptibility of transgenic tobacco plants with suppressed levels of preformed phenylpropanoid products. Proc. Natl. Acad. Sci. USA 91, 7802-7806.

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WO 98/26082 PCT/EP97/07012 -97- SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Novartis AG STREET: Schwarzwaldallee 215 CITY: Basel COUNTRY: Switzerland POSTAL CODE (ZIP): 4002 TELEPHONE: +41 61 69 11 11 TELEFAX: 41 61 696 79 76 TELEX: 962 991 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (ii) TITLE OF INVENTION: METHODS OF USING THE NIM1 GENE TO CONFER DISEASE RESISTANCE IN PLANTS (iii) NUMBER OF SEQUENCES: 39 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 9919 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) WO 98/26082 WO 9826082PCT/EP97/07012 98 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID No:l: TGATCATGAA TTGCGTGTAG GGTTGTGTTT

GGACTGGTGT

GTATACGTTT

AGAACATGGA

AGCCGGAAAA

AACCCACTCT

GGCAGCAGAA

CTGCATTTCA

TGAATTTGCA

ACACTGTAGA

ATTGATGATT

TACCAAAGAA

AACACAAAGG

TCCATTAGAG

AAATGCATCA

CTTGTTAAGA

CTTCAACTTG

AACAGCAGAG

CAGGAAGTAA

CTCATCTAAT

TCCTTCGGTG

CCTCTGATCA

TCCTCAGGAC

GTGTCCACTG

AAAGACGTTT

GGCAGCAAAA

GATGGAAATG

TAACTAAGTG

TAATTTGCAG

TTGAAAAGTT

TCAAAGATCA

GGGCTACTTG

GCGCGTTTTG

TAGTGGATTC

TCGACGCTGA

ATAGTTGGCG

GCCTCGTCTC

TAAAGATAGG

GTGTGTAGTA

CAATTGGTCG

TAGTTGGTCC

CAGAAGAGAT

TGGTGACATG

GAGTTTCAGA

TGGACTGCAA

GGCGTTTCCA

AAAAGACTTG

TGATTGCTTT

TTGGTTTCTC

CAGTCCTCAC

6ATGAGCTGA

CAAGAGATTG

CGTCGGGCAG

ACATACTTGT

TGAGTGTCTG

CTTAAAACTT

GTATTGCCTA

TATGAGCTTT

CAGTCCATTG

AACGGCAAGT

TTCCCGCTTG

ACTAATATCA

CCGGACATAG

AGAAGGCGGT

AGAAGGACGA

ATTGAATAGA

TGTTCTATTA

ATCAGGGTAC

CAAAGCTGCG

AACTAATTGG

TCCCTAATCC

AAGGGTTTCA

ACCCTATGAA

CCTTTCCGCT

GAGAGAAGGT

TTGCTGTTAT

120 180 240 300 360 420 480 540 600 660 720 780 TAACGAACCC GGATCACTGT GGCAAGAACC TTGGGTCTAT CACAGGTTCT GTCTGGATTG WO 98/26082 PCT/EP97/07012 99 TTTTTGCTTA CAATTCCATG ATATTTTTGG AGACTACAAC CTGGTGAGCC TTGTGAAGCA GGCTGGATCC ACAAGTCAGA GGACTCAGAA GCTCGGAAAT TGACCAGTGG TCGGAGATAT GTGAAAGCTT TCAGTCTCTT GTTTCAATCG AAGAAGTTTC GTGTCATTCA CAAATGTTGG ACTCAGTGTT CTCTTATCTC AGGTTAAAGG AAGAATTAGG CTTGAATATT CTTTTGGTTC TTCACACTTT GATTCTAGCA ATATATATTA

CAAATCTGTA

ACCTGCAGCA ATAATACACA AGACATAAAC AGTTACAGTC TAAGAGGCAA GAGTCTCACC GGTTGATTTC GATGTGGAAG GTCCTCACTA AAGGCCTTCT

AGGAAGAATT

GGTTAGCCCA

GGAGTCATGA

GGTCTATCAG

CATGTATGCA

ATGCAATGGA

TAGACCTGAA

AAGATACATA

TTGGTGGAAA

ACTCTGTAAC

TACCATTGGT

GGATGCAAAA

ATCATGAAAA

GACGACGATG

AAGTCGAATC

TTAGTGTCTC

TGATTCCTAC

ATTCCCTCAA

CGATAGAAAC

TGACAGCGAA

CCCAGAAATG

GCTGACTAGG

ACTAACTTGC

TAACATGAAT

ATATCCAACA

ACCATCATGG

TCAAATTGTT

CGAAGAGCGA

CAGAATTATA

AGAGAGTTTA

TGTCAGGGAC

TTGTATTTcC

ATAAAGGACA

AATCAGTGGG

AGAAGATTTG

CGCACCTGCA

GTGCAAAGGA

AGAATGCACC

TGTGTAATTC

GTTGCCAGAA

GATGAACAAT

GTTATTGTTG

ACAACATTTG

AACTATATGA

TGGTACAGCA

CGGTTAGACC

GAATTTCCTA

ATGTACCTTT

TCAAAAAGAA

CTCTGGCTCA

AGGGCAATTT

CATGTGGGAA

TTGTTAACTT

TTACACGCCC

GAGTTACAAA

GTTCAGGGAA

TTGACATTAT

ATGTACATAA

TTTGAAGCAC

CGCCAACGAT

AAAATTACAC

TCTTTCCACC

ATTCCAAATT

GCTTCTTTTG

960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 TAGTCGTTTC TCAGCAGGT CGTCTTCTCC GCAAGCCAGT TGAGTCAAGT CCTCACAGTT WO 98/26082 PCT/EP97/07012 -100- CATAATCTGG TCGAGCACTG CCGAACAGCG CGGGAAGAAT CGTTTCCCGA GTTCCACTGA TGATAAAAAA AACAAGGTCA GACAGCAAGT TTGTTTTTTG TGATAAGGAG TCCGATGAAG CGCTTTTAGT CTACTTTGAT GCTCTTCTAG TGATGTTCTC TTCGTACCAG TGAGACGGTC TGTTCCCTTC ATTTCGGCGA TCTCCATTGC AGCAA.GTGCA ACTAAACAGT GGACGACACA CCTAATAGAG AGGACATAAA TTTAATTCAA TCTATTTTCA AGATCGAGCA GCGTCATCTT AGGAACATCT CTAGGAATTT

GTTCTCGTTT

TCGGCCTTTG AGAGAATGCT TGCATTGCTC GGCTTGTTTT GCGATCATGA GTGCGGTTCT ACCTTTTTCC AATAGAGATA GTATCAATTG CACCGTATAT CCCCTCGGAT TCCTATGGTT TGTTGCGGTC TTCACATTGC

AATATGCAA.C

ATTGGTGTGA TCCTCTTTCA AAAGCAACTT CTTATGTACA TTCGAGACAT

GTTTCTTTAC

TCTATCAATT ATCTCTTTAA CAAGCTCTTC

AACAAAACCA

TGGGTGAGAA

GATTCTGAAA

AGGCTCGAGG

AGCTTGTGCT

AAGAATAGTT

ACATATAAGA

CAATTCATCG

GTCTTCTTGC

CGGGATATTA

ACCTTCCAAA

TGGCTCCTTC

GACATCGGCA

AGCGAAATGA

GACTAACTCA

TTTAGGTACC

CGGCAATGAC

TGTTTAAAGA

TCCATACCGG

GGTGCTATCT

CTAGTCACTA

TCCGTTGGAA

ATCATTAGTT

AATAAGACTT

GCCGCCACTG

TCTAGTATTT

TTACATTCAA

GTTGCTTCTG

CGCATCGCAG

AGATCAAGTT

AGAGCACACG

ATATCATCCG

TCCAAACCAA

TTTTCAAGAC

TCATTTAGTT

TTTTAGAAAG

TTACACCCGG

TGAACTCACA

AAAGACGTTG

CACTCAGTTT

GATAGATACC

CAAAAGAGGG

CTACACATAG

CCGCCATAGT

ATGCACTTGC

CAACATGAAG

TTAAAAGATC

CATCATCTAG

AGTCAAGTGC

GCTCTTTACG

TAACCATATC

1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 WO 98/26082 WO 9826082PCT/EP97/07012 101

TACATTAGAC

ACATATATTA

TTGACAATAA

GCAAGCTTGA

GTCCAATAAG TGCCTCTGAA GTGTAACCAT

AGCTTAATGC

TCTTGAAGAT GAAAGCCAAA CCACGTGGCA

GCAATTCTCG

TGCTGCTGTA

AACATAAGCC

TGGCAATCTC CTTAAGCTCG TAGCGGCGGC

TAAAGCGCTC

AAGAAACTTC

CCGGCCGTCG

AGTCAAJAGAC

GGATTCGAAG

TGAGTACTTG

TTCGGCGGCC

AACTAGTGCT

GCTGATTTCA

GGTTCCGATG

AATTGAAATT,

TATCAACTGG

ATTTGGTTAA

CAACGAGAGT

GGTGGTGAGA

AAACCGGTAA ATTTCCAGGA J AGGAAGCTAA ACCGGTTCGG C

TCTCTTTACA

GTATAACCAA

ATACAAATAC

AGATGGTGTT

TAGAGAACT

TCTGCGCATT

AAAACAGTCA

AGCTTCACGG

TTGAAGAAAG

GAGAGAACAA

TGTTGGAGA

kGATAAACAA rAAGAATCGG

'ACAAATTAA

kGATCGAAGA

'ATGAAGAAG

LAGGAATCTT 9J ;TTATAAATG I)

TCTATCCAAT

TGTGTCCTCT

AAGTACTCAA

TTACCTGATA

CCAACATGAA

CAGAAACTCC

CAACCGAATC

CGGCGGTGTT

AGCTTCTCGC

GCTTAGCGTC

GCAATTGCAG

TAGAGGAGTC

CGAATCCATC

A.GAGATCTCT

rAACCATTGA

MATCCTCGTC

~GTCAGAGAT

TAGTATTTA

AGCTTCATAC

ATAACAACTT

GTAAGAACAT

GAGAGTAATT

ATCCACCGCC

TTTAGGCGGC

GAAACCGACT

GTTGGAGTCT

TGACAAAACG

GCTGTAGAAA

AGCAGATACA

GGTGTTATCG

AATGGTGGTG

GCTAATCAAC

CGAGCAGAGC

:CACGGTTTAC

:TTTTTTAAA M'CCGGAGAC

AAGCTTTACC

TGTCTACAAC

ATTCATGAAT

AATTCAGGGA

GGCCGGCAAG

GGTCTCACTc

TCGTAATCCT

TTCTCCTTCT

CACCGGTGGA

TCATCCGGCG

TCAGGTCCGG

GTAGCGACGA

rCCATCAACA 3-AAGAGACCT

:AAGTCAAGT

'ATTTCACCA

LAGATATAAC

LTTTTGTGTT

3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 WO 98/26082 PCTEP97/07O12 102 GCTAATTTTT

GTATATGAGI

TTGGAGATGA

TATAAATATI

TATATAAATA

TTATTTTTTI

TTTATTTTCT

TTGAAGTAAP

CATAAGTTTT

GTAATGTATA

AGCATGTTTA CACTATAATT TATAGCAAAT

GAAAACGCTC

TAAAAACATG AAAGTCATAA TATTGTATAC

ATATAAAAGA

TGCGATAAAT

CAGCTTTTTC

CAAATTTGGC

TCATCCTTCA

AGAAGAAGAA GGAATCTGCA GGATCGAAAA

CGATCGAGAT

ACAACGTAAG

ATTATGTTGA

TAAATTATTG

CTTTCCGCGT

k. AGTTCAATC(

STAAAATTTA'

AATTTAAGA4

LATGATAAAGC

TTATATTTTI

AAATCAAGTC

TTCACAACAA

TGA.ATATATT

CTTCTTTAGT

AATAACTACG

CTTGATTGGT

TAATCACCTC

CAGAAAATGA

ATAAACAGAG

TTTTTACTTT

AGTAAATTTA

ATTATCTTTT

-GGTTCGGTAA

r' TTTTCATCCG

STTAGATTTAC

;GAACGTATAT

CGTTTATTGA

GAATATTTCC

AATCATTATA

TTTTTAATTA

TATTTGCCTT

ACGTAAAAGC

GTTTTCCGGA

TTGGTTCCTC

AAAGATAGGT

TACTTTATAT

TGTATTTCTT

CAATAACTCTC

GAACAATTTT

GCCCCTGAAC

GTTCGTTATT

ATGTGAAAGT

TAAGTTTCAT

AAAAGTAATT

TGGAACTATT

GATATAGGAA

GGATTTGATT

CAACTTCTCG

AAATTCATAA

CTCGATGTTG

ACCGGTAGAC

rAAAGATGCC kGGAGTTATA I kAATGATAAG 'i 'TATAACTCA CTGAAATAGT T

CAAACTAGAT

TTCATATAAA

TACATTTCTG

GCTTTATTCA

TTCAGTGTTC

CTCCTTGTTC

TAAATTACAT

TAAAAACAAT

TTCTGAATCA

CACGTCTAAA

CTGGAAACTG

TCATTTTGTT

LATGAATACA

~TAAGGTAAA

'TAAATTAGG

LTAGCATCAC

'TTCTTTTAA

4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100

ATAAGATTTG

ATATTTAATT

TATGATTTTA

AATTTTACTA

TTAATTTTTT AAAATGATAT ATTATAAAAT TTAATTGAAT CAATCTGATA

TAATTTTTTT

5160 1. j.

WO 98/26082 WO 9826082PCT/EP97/07012 -103- ATCTTCTACC ATCTATTAT; TGCCAAATAT TTAATAAAT'I GACAGCATAC CGTACATATA GTAAGCTATA AATATATGTA TGCTTTAGAT ATTATGTATA TTAAGAGAGA AAATTGGGAC AACCCTTGAT ATA.ATAAAAT TTTGGTATTA TTACATAAAC AACAATTAAA CATCACTAGA AGACTTGAGA TCCTTGTCAA TTAAAGTGTC

GGCCCGTTGC

CGGAAAACAA ACGAAAGAAc CTCTGCGTTT ATGTTGTAGA GCTTGGAGTC

CACGTAATCA

CGACTTTTCA

AAACTGCTCT

TGTCCACTGA

CGAATAGCAC

AGAGTTCGGA

GTATGGAAGA

GTTGATAWA

TTGTGTACCI

AACAATAGTP.

TCTAATATAI

ACTATTTTCT

AACTAGCCAA

CTAAGAAACA

TATATTTATG

CTACGTTTGC

TTCTTCCGTT

AATCAGATCC

GGCAACCACG

AATCTACTCC

TTAAAACCCA

CTAGCTTCCC

AGGACCAAGT

PATTGTGATAA

iTGCGTTTTTT

LGCTTATAAAA

LAGATATTACG

TAAAATATCT

ATACAGTAAC

AAAATCAGAT

AAATTCAATA

CCCCACAATG

ATTTGTCGGC

CAGATCkACC

CTCTTTTTTT

ATCACTACTA

AATGCTTTTA

TAACTCGTGA

TTCGTATCTG

TTCGGTTTTG I

ACTTTAGATA

TTGGAGAATA

CATAGATACG

TGTTGTGTCT

TTTATTAACT

TGTTTTCAAC

TAAATATTCA

TTCCTTTTTA

AGCGAGCCAA

CCATTTTTTT

CTCTCGTAAT

GCATAAACTA

CGAAACAATA

A.TATCTTTCA

kCATCTTCTT k.CTAATCCTG ~GACAAMACC G

AACACCCAAT

TATATACGTG

GGTTATATTG

AAATATGTGT

AATATATTAT

TATAAACAGG

TAAAACAATG

CCTTATAAAA

TTGAGACTTG

TATTTTTTTT

CAGAACAAAA

kATTCAACTT

:AACGTCGTT

:TTTAACCCA

'ATCTTTGTT

~GAAAACATC

;GATCACATT

5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 GTTGTTCCGT GATATCCAAT GCAAGAACCC CGAAACTTGT ATCGGGTTGG

AAAAAATTAA

WO 98/26082 PCT/EP97/07012 104 TCTGTCTGTT TTTGGTAGAC GCAAATTTTC TAATCTCTTC CAGGTAAACG AATCAGAATC GAAAACTTCG CACATAAAAG ACGCCGAGAC TGCGAAAGCC TAAACCCAAT GACATATCCC AGTGGTTGGT TTGAAGACTA TATCCCATGA CTTGCATCAC GGAGACTCCA TTGTTTTGTA TCCTTTTACT ATGTAGTGTT AAGATTGACA AACCATGACT TTTTCCGATT TTTGACGCCG CACCGGAGAA CTCATGATCC ATGAAAAAAT CAGGATCGGT TCTTTGTTTA AGGAAAGTAT ATTAACCA AGATAAAATC TTTATCTTTA GACTTTTAAC TTTTTTTTTT ACCCAACAAT CAACTCATTA TATAATAATG CCAGCTTGTA TAATAATCCA

TTCTGTGATT

TTTGTATTTT

AACCCTTCAC

TGTATCCACG

AAGCTTCGAT

AATCACTCCT

GTATGAAGTA

CGTAGCTTCT

GAAGATAAGA

TGTCGGGAAT

AGAGAACAAC

CAAAATTTGC

TTTCATGATT

CAAATCTACA

CTTTGGATTT

TTTCTATCAT

CAAGTCAATT

CAAATGGTAG

ATACCGGAAA

TTCTGGCTTT

TGATGGTTTT

CTTTATCATT

CTCATGGACA

TCCCGAAATA

CTTGTTGCAC

AAGAAATTCT

AAAGAGATGA

TTATGATGAA

CTTTTTCTTC

AATGTTACTT

GTAATTTAAT

TAATTGTTTT

AATTGACAAT

TCACCATTTT

ATACCCCGAG

GGGTTCA.ATC

GGTATGACCT

GTATACTTAA

CCGGGTGGCA

AAACTGGTTC

CGATTGGTTC

TCTTTATTCA

TGGATCATGT

GCACGATCAC

TAAAGTGTTT

GCTAGTCCTA

GTGATACCTT

TGCTAGACTT

TTTTTCTACT

TCTTTCTTTT

GGCCAATTTA

ACATACACAT

CGATTACCGC

GATACTGTTT

CACAAAGCAA

GAAAGTCGAT

GAAGTTCGTG

TAAGGAGATT

GGAGCCTGAA

CTTGATTTAT

TGAATGAGAA

ATATATCCTT

AAACAAACAA

AAGCCAAAAC

AGGAAACAAC

AATAGATTAA

TAATAAACAT

TTTTCTTATA

6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 WO 98/26082 PCT/EP97/07012 105 AAAATTAGCA CAAAAAAGAT TATCATTGTT TAGCAGATTT AATTTCTAAT TAACTTACGT AATTTCCATT TTCCATAGAT TTAGTTTCCT TAGTAATTTT AACTCTAGTT ATACTTTTGT AATTACAATA TATAAACACT TACTATAGTT AGGGGAAGAT AATGAAAACT TATAATCTCA CGTGTTTTGT CCATATATTT GCTTTTAAGA AGCACAAAAC AAAATTAAAT ATTCCTACAG CTAGTTTGCA TTTGTCGGTC ACAAAAGGGG AAACAAACGT CTCTGTGTTT AAGTTGTAGA GCTTGGTATC

CACGTAATAT

CGACTTTTCA

AAACTGCTCT

TATCCAGTGA CGAATAACAC AACGTTTGCA GTATGTAAGA

TTATCTTTCT

AAATTTTAAG

TAAATGTTTC

GAAGAAAGTT

TCTGATTTAA

ATAACATACA

GTTGC'rTATA

CATATAACAA

CCTTGTTGAT

CATTTCTTCT

AAGAGGCAAA

GGCAAACATG

GCTCTACTCC

TTAAAACCCA

CTAGCTTCCC

AAGACCAAGT

TTTTATTTCC

ATAATATATT

ATCACACTAA

TTTGGCCCAC

AGGATACCAA

TACGTGTTAC

AATATATTCA

AATTAAATAT

TATTTTATGC

TCCGTCCAGA

ATCCTTGTTT

ATCCCAACTA

AATGCTTTCA

TAATCTGTGA

TTCGTAGCTG

TTCGGTTTTG

TTAGTTATCT

GAAATTAAAA

CTAATAATTT

ACTTTTTTGG

AAATGACTAG

TGAACAATAG

TATAACAATG

TCCTATCCCT

CCTACGTTGA

TCAACCCTCT

GTATGAACTA

GAAAGCATTA

ATATCTTTCA

ACATCTTCTT

ACTAACTCTG

GGACATAACC

TAGTACTTTC

GAAGAAAAAA

TTTTTAGTTA

GATCAATTAG

TTAGGACATG

TAACATCTTA

TTTGCATTAA

ACCAAAAAAA

GCCTTGTTGA

CGTAATCAGA

AGTTTA.ACTT

CGACGTCGTT

CTTTTTCCCA

GATTGTTGTT

GGAATAAACC

GGATCACATT

7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 GTGGTTCCAT GATCTCCAAT GCAAGAACCC TGAAGCTTGT ACCGGGTTTG AAAGAATTAG WO 98/26082 WO 9826082PCT/EP97/07012 106- ACCGTCTGTT CTCGGTAGAC AAAAACTTCG CACGCAAAAG AAATATTTTA TACCGGAAAG ACTCCTCACT TTTGGGTTTG GTATTCCGGT ATTGGTCTTG AGCTTTGATC TTTACCTCTC ATCTCTCCTC TCATGGAAAA GAAGTATCCA GAGATATTGT GCTTCTCTTG TTGCACTCTT ATAAGAAAGA ACCTCTTGGA GGAAAAAAGA AAGAAAGAGA GGTAGAGAAC CGACGATGAT CTTCGCTAGT CCCAAAACAA TTTTCTTCGC CAGTCCCAGA ACCTTGAGCC CAAAGTTTCT GACTTAGAAA ACAACATTTT GTTTCTATAG TGTTTTTTTT

GCAAATTTTT

TTCTGAGATT

GCTGCAATCC

GTATGATCTG

TATCATTATA

CTTGTGGCAG

AACTGGTATC

TGGTTCGATG

TATTGATGAG

TCGTGTCCTG

TGAGCACGAT

GAATATACAA

GCAAATTAAC

TAAAAATATA

CTTTTGACTT

GTATATATAT

CTTATTCTAA

TAATCTCTTC

CCGAGTCATA

GGTTACCGTT

ATACTGTTTT

ACAAAGCAAT

AAAATCGATG

AAGTTTGTAT

GAGATTTAGG

CCTCAATTTT

ATTTATCACC

CAGTGAATGA

GTGTTTATAA

CAAAGATAAA

TATAAAATAT

TTAACCAAAT

TCTTTGACAT

TAGATTACCA

CACATAAACG

CCAGGCGATT

AGACCTAATG

GTTGTTGGTT

ATCCCATGAC

GAGACTCCTT

CCTCTTTCGT

TTGACAAACC

CCGATTTCGG

GGAGAACTCA

GATATATAGA

GTATCACAAA

ATCTTCATTA

TTCATTAGGT

TAACAGTAAT

CAAAATTCAA

CTCATTATAT

AATCGGAATC

TCGAAAGCCT

ACTTATCACA

TGCAGACTAT

GTGCATCACA

TGTTATCCAA

AGCGTTCTAG

AAGACTCGTA

ACCCCCGAAG

TGATCTTATT

AATCAGGATT

TTGCCTTTTT

ATGTTTTCCT

TACTTGTAGT

TTAATAGCTA

CAATCTTTGG

CATATACAAA

8460 8520 8580 8640 8700 8760 8820 8880 8940 9000 9060 9120 9180 9240 9300 9360 9420 9480 GTGTTTCCTT TTCAATCAAC ATCCATTTTC TTTAAAAATT AGCAAGTTTG TTCTTATATC WO 98/26082 WO 9826082PCT/EP97/07012 -107 ATCATTCAGC AGATTTCTTA ATTAAACTTA GTGATTTCCA CTTTCTTAGT TTAGTACTTT AAATTTTCAT ATATATAATT ACTCCAGTTT AACTTATGTT AAATGTTTCA TCACACTAAA TTTTAGCTTT ATGAAAkAA ATATCAAATC ACTGAAGACA TTTTTATTTG GCCAATTAGT AATAGACTAA TAGTAACTCA GGCGAAACGA ATATTCTGAT TCTAAAGATA GTAAAAATGA ATTTCACACA CCTAGAAAGA GTAAGGTAGA AACCTTTTTT TCAAGAAGTT CTCATCGAT INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 5655 base pairs TYPE: nucleic acid STR.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: No

TTTTGCACCT

TATTAAAATT

AGAGCATTAA

TTTGTTGGCC

TATGATATCT

ATTTTGATGA

TTTTTGGTCA

ATATGTTTCT

AAAAGTAAAA

GTAATAAATA

TATACTCTAT

CTCTAATTCT

AGGGAATACT

GATTCTTGTA

9540 9600 9660 9720 9780 9840 9900 9919 (iX) FEATURE: NAME/KEY: exon LOCATION: 2787. .3347 OTHER INFORMATION: /product= "1st. exon of NIMi" 4. t.

WO 98/26082 PCZTIEP97/07012 108 (ix) FEATURE: NAME/KEY: exon LOCATION: 3427. .4162 OTHER INFORMATION: /product= 1-2nd exon of NIMI" (ix) FEATURE: NAME/KEY: exon LOCATION: 4271. .4474 OTHER INFORMATION: /product= 113rd exon of NIMi", (ix) FEATURE: NAME/KEY: exon LOCATION: 4586. .4866 OTHER INFORMATION: /product= "4th exon of NIM11' (ix) FEATURE: NAME/KEY: CDS LOCATION: join(2787. .3347, 3427. .4162, 4271. .4474, 4586. .4866) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: TGTGATGCAA GTCATGGGAT ATTGCTTTGT GTTAAGTATA ATAGTCTTCA AACCAACCAC TAAACAGTAT CAGGTCATAC TGGGATATGT CATTGGGTTT AGCGGTAATC GGATTGAACC AGGCTTTCGC AGTCTCGGCG TATGTGTATG TCTCGGGGTA ACTTTTATGT GCGAAGTTTT CGATTCTGAT TCGTTTACCT CGTCTACCAA~ AAACAGACAG ATTAATTTTT TCCAACCCGA CATTGGATAT CACGGAACAA CAATGTGATC CGGTTTTGTC

CAAAACCATC

CAAAGCCAGA

CTTTCCGGTA

TCTACCATTT

GGAAGAGATT

TACAAGTTTC

TCAAAACCGA

ACGTGGATAC

AGTGAAGGGT

TAAAATACAA

GAATCACAGA

AGAAAATTTG

GGGGTTCTTG

AACTTGGTCC

120 180 240 300 360 420 WO 98/26082 WO 9826082PCTIEP97/07012 109 TTCTTCCATA CTCCGAACTC GGTGCTATTC GTCAGTGGAC AAGAGCAGTT TTGAAAAGTC TTGATTACGT GGACTCCAAG CTCTACAACA TAAACGCAGA TGTTCTTTCG TTTGTTTTCC AGCAACGGGC CGACACTTTA GTTGACAAGG ATCTCAAGTC ATCTAGTGAT GTTTAATTGT GGTTTATGTA ATAATACCAA TATTTTATTA TATCAAGGGT TGTCCCAATT TTCTCTCTTA ATATACATAA TATCTAALAGC TTACATATAT TTATAGCTTA ATATATGTAC

GGTATGCTGT

AAATTTATTA AATATTTGGC CTATAATAGA

TGGTAGAAGA

TGATGTTTTC

AAACAAAGAT

GTGGGTTAAA~

CAACGACGTT

GAAGTTGAAT

GTTTTGTTCT

AAAAAAAAAT

TCAAGTCTCA

TTTTTATAAG

ACATTGTTTT

TCCTGTTTAT

AATAATATAT

AACACATATT

CCAATATAAC

CCACGTATAT

AATTGGGTGT

TAAAAAAATT

TCAGGATTAG

CAAGAAGATG

GTGAAAGATA

GTATTGTTTC

TTAGTTTATG

GATTACGAGA

AAAAAAAATG

ATTGGCTCGC

GTAAAAAGGA

ATGAATATTT

AGTTGAAAAC

TAGTTAATAA

TAGACACAAC

CCGTATCTAT

ATATTCTCCA

TTATCTAAAG

ATATCAGATT

TCAGATACGA

TTCACGAGTT

TTAAAAGCAT

GTAGTAGTGA

CAAAAAAAGA

GGGTTGATCT

GGCCGACAAA

TCATTGTGGG

ATATTGAATT

AATCTGATTT

AGTTACTGTA

AAGATATTTT

ACGTAATATC

GTTTTATAAG

AAAAAAACGC

TTTATCACAA

GATTCAATTA

AGGGAAGCTA

ATGGGTTTTA

TGGAGTAGAT

TCGTGGTTGC

GGGATCTGAT

GAACGGAAGA

TGCAAACGTA

GCATAAATAT

TTGTTTCTTA

TTTGGCTAGT

TAGAAAATAG

AATATATTAG

TTACTATTGT

CTTTTATACA

ATGGTACACA

TATTTATCAA

AATTTTATAA

480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 TATATCATTT TAAAAAATTA ATTAAAAGAA'AACTATTTCA TAAALATTGTT CAAAAGATAA WO 98/26082 WO 9826082PCT/EP97/07012 -110- TTAGTAAAAT TAATTAAATA TTAAAATCAT ACAAATCTTA AACGCGGAAA GCAATAATTT TTCAACATAA TCTTACGTTG GATCTCGATC GTTTTCGATC ATGCAGATTC CTTCTTCTTC GTGAAGGATG AGCCAAATTT TGAAAAAGCT GATTTATCGC GTCTTTTATA TGTATACAAT ATTATGACTT TCATGTTTTT AGAGCGTTTT CATTTGCTAT TAATTATAGT GTAAACATGC ATATACATTA CAAAACTTAT TTTTACTTCA AAGAAAATAA TTAAAAAATA ATATTTATAT ATATATTTAT ATCATCTCCA TTCTCATATA CAAAAATTAG CCCGAACCGG TTTAGCTTCC

TGTGATGCTA

TCCTAATTTA

ATTTACCTTA

TTGTATTCAT

CAACAAAATG

TCAGTTTCCA

GTTTAGACGT

ATGATTCAGA

AATTGTTTTT

AATGTAATTT

AGAACAAGGA

TGAACACTGA

GTGAATAAAG

ACAGAAATGT

ATTTATATGA

AATCTAGTTT

CAACACAAAA

TGTTATATCT

TTGAGTTATA

ACTTATCATT

TTATAACTCC

AGGCATCTTT

AGTCTACCGG

GCAACATCGA

GTTATGAATT

ACGAGAAGTT

AAATCAAATC

ATTCCTATAT

GAATAGTTCC

AAATTACTTT

CATGAAACTT

AACTTTCACA

AAATAACGAA

GGTTCAGGGG

TGTCTCCGGT

TTTTAAAAAA

GAGAGTTATT

TAAGAAATAC

TATATAAAGT

AACCTATCTT

TGAGGAACCA

GTCCGGAAAA

TGCTTTTACG

GAAGGCAAAT

CTAATTAAAA

CTATAATGAT

AGGAAATATT

TTCAATAAAC

AATATACGTT

TGTAAATCTA

CCGGATGAAA

CTTACCGAAC

ATAAATACTA

GATCTCTGAC

GTAAATTTAC

AAALAGTAAAA

ACTCTGTTTA

TTCATTTTCT

AGAGGTGATT

CACCAATCAA

TCGTAGTTAT

AACTAAAGAA

AAATATATTC

TTTGTTGTGA

CGACTTGATT

GAAAAATATA

CCCTTTATCA

ATTCTTAAAT

AATAAATTTT

CGGATTGAAC

ACATTTATAA

AAAGATTCCT

1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 WO 98/26082 PCT/EP97/07012 TTCCTGGAA. TTTACCGGTT TTGGTGAAAT GTAAACCGTG GGACGAGGAT GCTTCTTC!AT ATCTCACCAC CACTCTCGTT GACTTGACTT GGCTCTGOTC GTCAATGGTT ATCTTCGATC TTTAACCAAA TCCAGTTGAT AAGGTCTCTT CGTTGATTAG CAGAGATCTC TTTAATTTGT 2640 2700 2760 2813 GAATTTCAAT TCATCGGAAC CTGTTG ATG Met GAC ACC ACC ATT GAT GGA TTC GCC Asp Thr Thr Ile Asp Gly Phe Ala GAT TCT TAT GAA ATC AGC AGO ACT AGT TTC GTC GCT ACC GAT AAC ACC 2861 Asp Ser Tyr Giu Ile GAC TOO TOT ATT GTT Asp Ser Ser Ile Val Ser Thr Ser Phe Val 20 Ala Thr Asp Asn Thr TAT CTG GCC GCC GAA Tyr Leu Ala Ala Giu 35 CAA GTA OTC ACC GGA CCT Gin Val Leu Thr Gly Pro 2909 GAT GTA TOT GOT Asp Val Ser Ala GAC TOG COG GAT Asp Ser Pro Asp CTG CAA TTG CTC TCC AAC AGC TTC GAA TCC GTC TTT Leu Gin Leu Leu Ser Asn Ser Phe Giu Ser Val Phe 50 GAT TTO TAO AGO GAO GCT AAG OTT GTT OTO TCC GAC Asp Phe Tyr Ser Asp Ala Lys Leu Val Leu Ser Asp 65 2957 3005 GGO OGG Gly Arg GAA GTT TOT TTO CAC Glu Val Ser Phe His 80 OGG TGC GTT TTG TCA GOG AGA AGO TOT Arg Cys Val Leu Ser Ala Arg Ser Ser 3053

TTC

Phe TTO AAG AGO GOT TTA Phe Lys Ser Ala Leu 95 GOC GOC GOT AAG AAG Ala Ala Ala Lys Lys 100 GAG AAA GAO TOO AAC Glu Lys Asp Ser Asn 105 3101 AAO ACC GOC GOC GTG AAG OTO GAG OTT AAG GAG ATT GOC AAG GAT TAO 3149 WO 98/26082 WO 9826082PCT/EP97/07012 112- Asn Thr Ala Ala Val Lys Leu Giu Leu 110 Giu Ile Ala Lys Asp Tyr 120

GAA

Glu GTC GGT TTC Val Gly Phe 125 GAT TCG GTT GTG Asp Ser Val Val ACT GTT TTG GCT TAT GTT TAC AGC Thr Val Leu Ala Tyr Vai Tyr Ser 130 135 GGA GTT TCT GAA TGC GCA GAC GAG Gly Val Ser Glu Cys Ala Asp Glu 150 3197 3245 AGC AGA GTG Ser Arg Val 140 AGA CCG CCG CCT AAA Arg Pro Pro Pro Lys 145 AAT TGC Asn Cys 155 TGC CAC GTG GCT TGC Cys His Val Ala Cys 160 CGG CCG GCG GTG GAT TTC ATG TTG GAG Arg Pro Ala Val Asp Phe Met Leu Glu 165 3293

GTT

Val 170 CTC TAT TTG GCT TTC Leu Tyr Leu Ala Phe 175 ATC TTC AAG ATC CCT Ile Phe Lys Ile Pro 180 GAA TTA ATT ACT CTC Giu Leu Ile Thr Leu 185 3341 TAT CAG Tyr Gin GTAAAACACC ATCTGCATTA AGCTATGGTT ACACATTCAT GAATATGTTC 3397 TTACTTGAGT ACTTGTATTT GTATTTCAG AGG CAC TTA Arg His Leu 190 TTG GAC GTT GTA GAC Leu Asp Val Val Asp 195 3450 AAA GTT GTT ATA GAG GAC ACA TTG Lys Val Val Ile Giu Asp Thr Leu 200 GTT ATA Val Ile 205 CTC AAG CTT GCT AAT ATA Leu Lys Leu Ala Asn Ile 210 3498 TGT GGT AAA GCT Cys Gly Lys Ala 215 TGT ATG AAG CTA TTG Cys Met Lys Leu Leu 220 GAT AGA TGT AAA GAG ATT ATT Asp Arg Cys Lys Glu Ile Ile 225 3546 GTC AAG TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA 3594 1' 1.

WO 98/26082 -113- Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys 230 235 PCT/EP97/07012 Ser Leu Pro Glu 240 GAG CTT Glu Leu 245 GTT AAA GAG ATA ATT Val Lys Glu Ile Ile 250 GAT AGA CGT AAA Asp Arg Arg Lys GAG CTT GGT TTG GAG Glu Leu Gly Leu Glu 255 3642

GTA

Val 260 CCT AAA GTA AAG AAA Pro Lys Val Lys Lys 265

CAT

His GTC TCG AAT Val Ser Asn GTA CAT AAG GCA CTT GAC Val His Lys Ala Leu Asp 270 275 TTG AAA GAG GAT CAC ACC Leu Lys Glu Asp His Thr 290 3690 3738 TCG GAT GAT ATT GAG Ser Asp Asp Ile Glu 280 TTA GTC AAG TTG CTT Leu Val Lys Leu Leu 285' AAT CTA GAT GAT GCG TGT GCT CTT CAT Asn Leu Asp Asp Ala Cys Ala Leu His 295 300 TTC GCT GTT GCA TAT TGC AAT Phe Ala Val Ala Tyr Cys Asn 305 3786 GTG AAG Val Lys CAT AGG His Arg 325 ACC GCA ACA GAT CTT Thr Ala Thr Asp Leu 310 AAT CCG AGG GGA TAT Asn Pro Arg Gly Tyr 330 TTA AAA CTT GAT CTT GCC GAT GTC AAC Leu Lys Leu Asp Leu Ala Asp Val Asn 315 320 ACG GTG CTT CAT GTT GCT GCG ATG CGG Thr Val Leu His Val Ala Ala Met Arg 335 3834 3882

AAG

Lys 340 GAG CCA CAA TTG ATA CTA TCT CTA TTG GAA Glu Pro Gin Leu Ile Leu Ser Leu Leu Glu 345 350 AAA GGT GCA AGT GCA Lys Gly Ala Ser Ala 355 3930 TCA GAA GCA Ser Glu Ala ACT TTG Thr Leu 360 GAA GGT AGA ACC GCA Glu Gly Arg Thr Ala 365 CTC ATG ATC GCA AAA CAA Leu Met Ile Ala Lys Gin 370 3978 GCC ACT ATG GCG GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT 4026 WO 98/26082 WO 9826082PCT/EP97/07012 -114- Ala Thr Met Ala 375 Val Giu Cys Asn Ile Pro Giu Gin Cys Lys His 385 TCT CTC AAA Ser Leu Lys 390 GGC CGA CTA TGT GTA Gly Arg Leu Cys Val 395 GAA ATA CTA GAG CAA GAA GAC AAA Glu Ile Leu Giu Gin Glu Asp Lys 400 4074 CGA GAA Arg Giu 405 CAA ATT CCT AGA GAT Gin Ile Pro Arg Asp 410 GTT CCT CCC TCT TTT GCA GTG GCG GCC Vai Pro Pro Ser Phe Ala Val Ala Ala 415 4122 4162

GAT

Asp 420 GAA TTG AAG ATG ACG CTG Giu Leu Lys Met Thr Leu 425 CTC GAT Leu Asp CTT GAA AAT AGA G Leu Glu Asn Arg 430 GTATCTATCA AGTCTTATTT CTTATATGTT TGAATTAAAT TTATGTCCTC TCTATTAGGA 4222 4278 AACTGAGTGA ACTAATGATA ACTATTCTTT GTGTCGTCCA CTGTTTAG TT GCA CTT Vai Ala Leu 435 GCT CAA CGT CTT TTT CCA ACG GAA OCA CAA GCT GCA ATG GAG ATC GCC 4326 Ala Gin Arg Leu GAA ATG AAG GGA Giu Met Lys Gly 455 Phe 440 Pro Thr Giu Ala Ala Ala Met Glu Ile Ala 450 ACA TGT GAG TTC ATA Thr Cys Giu Phe Ile 460 GTG ACT AGC CTC GAG CCT GAC Val Thr Ser Leu Giu Pro Asp 465 4374 CGT CTC ACT Arg Leu Thr 470 GGT ACG AAG AGA ACA Gly Thr Lys Arg Thr 475 TCA CCG GGT GTA AAG ATA GCA CCT Ser Pro Gly Val Lys Ile Ala Pro 480 4422 TTC AGA ATC CTA GAA GAG CAT Phe Arg Ile Leu Giu Giu His 485 490 CAA AGT AGA CTA AAA GCG CTT TCT AAA Gin Ser Arg Leu Lys Ala Leu Ser Lys 4470 WO 98/26082 PCT/EP97/07012 -115- ACC G GTATGGATTC TCACCCACTT CATCGGACTC CTTATCACAA AAAACAAAAC Thr 500 TAAATGATCT TTAAA.CATGG TTTTGTTACT TGCTGTCTGA CCTTGTTTTT TTTATCATCA G TG GAA CTC GGG AAA CGA TTC TTC! CCG CGC TGT TCG GCA GTG CTC Val Giu Leu Gly Lys Arg Phe Phe Pro Arg CyS Ser Ala Val Leu 4524 4584 4629 GAC CAG ATT ATG AAC Asp Gin Ile Met Asn 520 GAC GAC ACT GCT GAG Asp Asp Thr Ala Giu 535 TGT GAG GAC TTG ACT Cys Giu Asp Leu Thr 525 AAA CGA CTA CAA AAG Lys Arg Leu Gin Lys 540 CAA CTG GCT TGC GGA GAA Gin Leu Ala Cys Gly Giu 530 AAG CAA AGG TAC ATG GAA Lys Gin Arg Tyr Met Giu 545 4677 4725 ATA CAA GAG Ile Gin Giu 550 ACA CTA AAG AAG GCC Thr Leu Lys Lys Ala 555 TTT AGT GAG GAC AAT TTG GAA TTA Phe Ser Giu Asp Asn Leu Giu Leu 560 4773

GGA

Gly

AAT

Asn 565 TCG TCC CTG ACA GAT Ser Ser Leu Thr Asp 570 TCG ACT TCT TCC ACA TCG AAA TCA ACC Ser Thr Ser Ser Thr Ser Lys Ser Thr 575 4821

GGT

Gly 580 GGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT CGT CGG TGA Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg Arg 585 590 GACTCTTGCC TCTTAGTGTA ATTTTTGCTG TACCATATAA TTCTGTTTTC ATGATGACTG TAACTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG 4866 4926 4986 5046 WO 98/26082 PCT/EP97/07012 -116- ATTTGTAATA TATATTTATG TACATCAACA ATAACCCATG ATGGTGTTAC AGAGTTGCTA

GAATCAAAGT

AAAAGAATAT

TTCTTCCTTT

AAGAGAACAC

ATTTGTGAAT

TTCTTCGATT

ACTGAAAGCT

TCCGACCACT

GTGAAATAAT

TCAAGTTCCC

AACCTTTTGT

TGAGTGGGCG

GACACAAGTT

GAAACTTCCC

TTCACAAATT

GGTCATGAGC

GTCAAATTGT

TGAACTTCTG

AACTCGAATT

TGTAAGGTGC

AACAATCCTT

ACATGTGCAG

GCCCTCAAAT

CAGAGCCCAC

TCATCTGTTG

GCAACATTCA

ACACAGCAAG

ATTCTCCTAG

TGCACCATTT

GTGCGTTCGC

CTTCTGTTTC

TGATTTTGAG

GATATTTTCC

TGTTATATGT

TTAGTTTCAG

TCAGCTCCAT

CTGGGTGCAT

TGTCACTGAT

TATCGTCATG

GGAATTGGGC

ACCAAGAACC

ATCTTCCTA.A

GTCTAGAGAT

TGCATCCAAC

ACATGGAAAC

AGACCAAGAG

ACTCCATATC

TAACCATTTC

5106 5166 5226 5286 5346 5406 5466 5526 5586 5646 5655 CGAGCTTCTG AGTCCTTCTT TTTGATGTCC TTTATGTAGG, AATCAAATTC TTCCTTCTGA

CTTGTGGAT

INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 594 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Asp Thr Thr Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser WO 98/26082 WO 9826082PCTEP97/07012 117- Ser Phe Val Ala Giu Gin Ala Val1 Al a Thr Asp Asn Thr 25 Leu Thr Gly Pro 40 Giu Ser Vai Phe Leu Ser Asn 10 Asp Ser Ser Asp Val Ser Asp Ser Pro Gly Arg Glu 75 Ile Val Tyr Leu Ala Leu Gin Leu Asp Ser Phe Asp Phe Tyr 55 Leu Ser Arg Asp Ala Lys Leu Vai 70 Al a Ser Asp Val Ser Phe His Ala Cys Val Leu Ser Glu Arg Ser Ser Phe Lys Ser Ala Ala Ala Lys Lys 100 Glu Lys Asp Ser Asn 105 Tyr Thr Ala Ala Giu Leu Lys 115 Val Thr Val Ile Ala Lys Asp 120 Giu Val Gly Phe 125 Arg Val Lys Leu 110 Asp Ser Val Pro Pro Pro Leu Ala Tyr 130 Lys 145 Arg Gly Vai Ser Giu Val Tyr Ser 135 Ala Asp Giu Met Leu Giu Ser Arg Asn Cys 155 Val Leu Val1 140 Cys His Val Ala Cys 160 Phe Pro Ala Val Lys Ile Pro 180 Val Asp Lys Asp 165 Glu 170 Tyr Tyr Leu Ala Phe Ile 175 Leu Ile Thr Leu 185 Asp Gin Arg His Leu Leu Asp 190 Leu Lys Leu Val Val Val Ile Glu Thr Leu Val Ile WO 98/26082 WO 9826082PCTIEP97/07012 -118- 200 Cys Ala Asn 210 Giu Ile Cys Gly Lys Ala 215 Asn Met Lys Leu Leu 220 Arg Cys Lys Ile Val Lys 225 Leu Ser 230 Val Val Asp Met Pro Giu Giu Leu 245 Pro Lys Giu Ile Ile 250 His Val Ser Leu 235 Asp Arg Arg Val Ser Asn Giu Lys Ser 240 Lys Giu Leu 255 Gly Leu Giu Ala Leu Asp 275 Asp His Thr Val 260 Ser Lys Val Lys Asp Asp Ile Giu 280 Ala Val Lys Leu Leu 285 Phe Val His Lys 270 Leu Lys Giu Ala Val Ala Asn Leu Asp 290 Cys Asp 295 Ala Cys Ala Leu His 300 Lys Asn Val Lys Thr 310 Asn Thr Asp Leu Leu 315 Thr Leu Asp Leu Ala 320 Val Asn His Arg 325 Glu Pro Arg Gly Val Leu His Val Ala 335 Ala Met Arg Ala Ser Ala 355 Ala Lys Gin 370 Lys 340 Ser Pro Gin Leu Ile 345 Glu Ser Leu Leu Giu Ala Thr Gly Arg Thr Ala 365 Ile Giu Lys Gly 350 Leu Met Ile Pro Giu Gin Ala Thr Met Ala 375 Glu Cys Asn Asn 380 Glu Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val li Le GuGn Ile Leu Glu Gln WO 98/26082 WO 9826082PCT/EP97/07012 119- Giu Asp Lys Arg Giu Gln Ile Pro Arg 405 Val Ala Ala Asp Giu Leu Lys Met Thr 420 425 Val Ala Leu Ala Gin Arg Leu Phe Pro 435 440 Giu Ile Ala Glu Met Lys Gly Thr Cys 450 455 Glu Pro Asp Arg Leu Thr Gly Thr Lys 465 470 Ile Ala Pro Phe Arg Ile Leu Giu Giu Asp 410 Val Pro Pro Ser Phe Al a 415 Leu Leu Asp Leu Giu Asn Arg 430 Ala Ala Met Thr Giu Ala Glu Phe Ile 460 Val Thr Ser Leu Arg Thr 475 Ser Pro Giy Vai 485 His Gin Ser Armr T.eu 490 Lys Ala 495 Leu Ser Lys Thr 500 Val Glu Leu Gly Lys 505 Arg Phe Phe Pro Arg Cys Ser 510 Gin Leu Ala Ala Val Leu 515 Asp Gin Ile Met Asn 520 Cys Giu Asp Leu Thr 525 Cys Gly 530 Glu Asp Asp Thr Giu Lys Arg Leu Gin 540 Lys Lys Gin Arg Tyr 545 Met Glu Ile Gin Giu 550 Thr Leu Lys Lys Ala 555 Phe Ser Giu ASP Asn 560 Leu Giu Leu Gly Asn Ser Ser Leu Thr Asp 565 570 Ser Thr Ser Ser Thr Ser 575 Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg WO 98/26082 PCT/EP97/07012 -120- 585 Arg INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 41 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Ile Arg Arg Met Arg Arg Ala Leu 1 5 Lys Leu Met Val Met Gly Glu Gly Asp Ala Ala Asp Ile Glu Leu Val 10 Leu Asp Leu Asp Asp Ala Leu Ala 25 Val His Tyr Ala Val Gin His Cys Asn INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 38 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant WO 98/26082 PCT/EP97/07012 121 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Pro Thr Gly Lys Thr Ala Leu His Leu Ala Ala Glu Met Val Ser Pro 1 5 10 Asp Met Val Ser Val Leu Leu Asp His His Ala Asp Xaa Asn Phe Arg 25 Thr Xaa Asp Gly Val Thr INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 41 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Ile Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile Glu Leu Val 1 5 10 Lys Leu Met Val Met Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala WO 98/26082 PCT/EP97/07012 -122- Val His Tyr Ala Val Gin His Cys Asn INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 27 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Arg Arg Pro Asp Ser Lys Thr Ala Leu His Leu Ala Ala Glu Met Val Ser Pro Asp Met Val Ser Val Leu Leu Asp Gin INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 41 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide WO 98/26082 PCT/EP97/07012 -123- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Ile Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile Glu Leu Val 1 5 10 Lys Leu Met Val Met Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala 20 25 Val His Tyr Ala Val Gin His Cys Asn INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 27 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Arg Arg Pro Asp Ser Lys Thr Ala Leu His Leu Ala Ala Glu Met Val 1 5 10 Ser Pro Asp Met Val Ser Val Leu Leu Asp Gin INFORMATION FOR SEQ ID

II

I

WO 98/26082 PCT/EP97/07012 -124- SEQUENCE CHARACTERISTICS: LENGTH: 41 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile Glu Leu Val 10 Lys Leu Met Val Met Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala 25 Val His Tyr Ala Val Gln His Cys Asn INFORMATION FOR SEQ ID NO:ll: SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide WO 98/26082 PCT/EP97/07012 -125- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Pro Thr Gly Lys Thr Ala Leu His Leu Ala Ala Glu Met Val Ser Pro 1 5 10 Asp Met Val INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: AATTCTAAAG CATGCCGATC GG 22 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide" WO 98/26082 PCT/EP97/07012 -126- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: AATTCCGATC GGCATGCTTT A 21 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: AATTCTAAAC CATGGCGATC GG 22 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide" WO 98/26082 PCT/EFP97/n7012 -127- (xi) SEQUENCE DESCRIPTION: SEQ ID AATTCCGATC GCCATGGTTT A 21 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 15 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CCAGCTGGAA TTCCG INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide" WO 98/26082 PCT/EP97/07012 -128- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CGGAATTCCA GCTGGCATG INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 314 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Met Phe Gin Pro Ala Gly His Gly Gin Asp 1 5 10 Arg Asp Gly Leu Lys Lys Glu Arg Leu Val 25 Gly Leu Asp Ser Met Lys Asp Glu Glu Tyr 40 Leu Arg Glu Ile Arg Leu Gin Pro Gin Glu Trp Ala Met Glu Gly Pro Asp Asp Arg Glu Gin Met Leu His Asp Ser Val Lys Glu Ala Ala Glu Ala Pro Trp Lys Gin Gin 55 Leu Thr Pro Ser Glu Asp Gly Asp Phe Leu His Leu

C.

WO 98/26082 PCT/EP97/07012 129 Ala Ile Ile His Giu Giu Lys Pro Leu Thr Met Giu Val Ile Giy Gin Val Lys Gly Asp 100 Leu Ala Phe Leu Asn 105 Phe Gin Asn Asn Leu Gin Gin Ile Ala Giu Thr Pro Leu 115 His Leu Aia Vai Ile Thr Asn Gin Pro 120 Gly 125 Ala Leu 130 Leu Lys Ala Gly Cys 135 Asp Pro Giu Leu Asp Phe Arg Gly Asn 145 Thr Pro Leu His Leu Ala Cys Giu Gin 150 Giy 155 Cys Leu Ala Ser Val 160 Aia Vai Leu Thr Gin 165 Thr Cys Thr Pro Gin 170 His Leu His Ser Vai Leu 175 Gin Ala Thr His Gly Tyr 195 Asn 180 Tyr Asn Giy His Cys Leu His Leu Aia Ser Thr 190 Gly Ala Asp Leu Ala Ile Val Giu 200 His Leu Val Thr Val Asn 210 Ala Gin Giu Pro Cys 215 Asn Gly Arg Thr Al a 220 Leu His Leu Ala Val 225 Asp Leu Gin Asn Pro 230 Asp Leu Val Ser Leu 235 Leu Leu Lys Cys Gly 240 Ala Asp Val Asn Arg 245 Val Thr Tyr Gin Giy 250 Tyr Ser Pro Tyr Gin Leu 255 Thr Trp Giy Arg Pro Ser Thr Arg Ile Gin Gin Gin Leu Gly Gin Leu WO 98/26082 PCT/EP97/07012 -130- 260 265 270 Thr Leu Glu Asn Leu Gin Met Leu Pro Glu Ser Glu Asp Glu Glu Ser 275 280 285 Tyr Asp Thr Glu Ser Glu Phe Thr Glu Asp Glu Leu Pro Tyr Asp Asp 290 295 300 Cys Val Phe Gly Gly Gin Arg Leu Thr Leu 305 310 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 314 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: Met Phe Gin Pro Ala Gly His Gly Gin Asp Trp Ala Met Glu Gly Pro 1 5 10 Arg Asp Gly Leu Lys Lys Glu Arg Leu Val Asp Asp Arg His Asp Ser 25 Gly Leu Asp Ser Met Lys Asp Glu Asp Tyr Glu Gin Met Val Lys Glu 40 11 WO 98/26082 Leu Arg PCT/EP97/07012 131 Giu Ile Arg Leu Pro Gin Giu Ala Pro Leu Ala Ala Giu Pro Trp Lys Gin Gin Leu 70 Thr Giu Asp Gly Asp 75 Ser Phe Leu His Leu Ala Ile Ile His Giu Glu Lys Thr Leu Met Giu Val Ile Gly Gin Val Lys Gly Thr Pro Leu 115 Asp 100 Leu Aia Phe Leu Asn 105 Phe Gin Asn Asn Leu Gin Gin 110 Ile Aia Giu His Leu Aia Val Thr Asn Gin Pro Gly 125 Ala Leu 130 Leu Lys Ala Gly Cys 135 Asp Pro Giu Leu Arg Asp Phe Arg Gly 140 Asn 145 Thr Pro Leu His Leu 150 Ala Cys Giu Gin Gly 155 Cys Leu Ala Ser Ala Val Leu Thr Gin 165 Thr Cys Thr Pro Gin 170 His Leu His Ser Val Leu 175 Gin Aia Thr Asn 180 Tyr Asn Gly His Thr Cys Leu His Leu 185 Ala Ser Ile 190 Gly Aia ASP His Gly Tyr 195 Leu Gly Ile Val Giu 200 His Leu Val Thr Vai Asn Ala Gin Giu Pro 210 Cys 215 Asn Gly Arg Thr Ala 220 Leu His Leu Ala Val 225 Asp Leu Gin Asn Pro 230 Asp Leu Val Ser Leu 235 Leu Leu Lys Cys WO 98/26082 PCT/EP97/07012 -132- Ala Asp Val Asn Arg 245 Pro Val Thr Tyr Gin Gly 250 Gin Tyr Ser Pro Tyr Gin Leu 255 Thr Trp Gly Thr Leu Glu 275 Tyr Asp Thr Arg 260 Asn Ser Thr Arg Ile 265 Pro Gin Gin Leu Gly Gin Leu 270 Glu Glu Ser Leu Gin Thr Glu Ser Glu Asp 285 Pro Glu Ser Glu 290 Cys Val 305 Phe 295 Arg Glu Asp Glu Leu 300 Tyr Asp Asp Phe Gly Gly Gin 310 Leu Thr Leu INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 314 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Phe Gin Pro Ala Glu Pro Gly Gin Glu Trp Ala Met Glu Gly Pro 1 5 10 Arg Asp Ala Leu Lys Lys Glu Arg Leu Leu Asp Asp Arg His Asp Ser WO 98/26082 WO 9826082PCT/EP97/07012 -133- Ser Gly Leu Asp 25 Met Lys Asp Giu Glu Tyr Giu 40 Arg Leu Giu Pro Gin Giu Ala 55 Gin Leu Thr Giu Asp Giy Asp Gin Met Pro Arg Val Lys Giu Giy Ala Giu Leu Pro Arg Giu Ile Trp, Lys Gin Ile Ile His Ser Phe Leu His Giu Giu 75 Met Leu Lys Ala Leu Giu Vai Val Leu Arg Gin Vai Lys Gly Thr Pro Leu 115 Ala Leu Leu Asp 100 His Ala Phe Leu Asn 105 Thr Gin Asn Asn Leu Ala Val Asn Gin Pro Giu 125 Asp Leu Gin Gin 110 Ile Ala Giu Phe Arg Gly Giu Ala Gly 130 Asn Thr Cys 135 Ala Pro Giu Leu Arg 140 Cys Pro Leu His 145 Gly Leu 150 Pro Cys Glu Gin Gly 155 His Leu Ala Ser Val Leu Thr Gin 165 Tyr Arg Gly Thr Gin 170 Cys Leu His Ser Ile Leu 175 Gin Ala Thr His Gly Tyr 195 Vai Asn Ala Asn 180 Asn Gly His Thr 185 Leu Leu His Leu Leu Gly Ile Val Giu 200 Gin Giu Pro Cys Asn Leu Val Ser Leu 205 Leu Ala Ser Ile 190 Gly Ala Asp His Leu Ala Gly Arg Thr Ala WO 98/26082 WO 9826082PCTIEP97/07012 134 210 Val Asp Leu Gin Asn 225 Ala Asp Val Asn Arg 245 Thr Trp Gly Arg Pro 260 Thr Leu Giu Asn Leu 275 Tyr Asp Thr Glu Ser 215 Asp Pro 230 Val Leu Val Ser Leu 235 Tyr Leu Lys Cys Ser Thr Tyr Gin Thr Arg Ile 265 Met Leu Pro Gly 250 Gin Ser Pro Tyr Gin Leu 255 Gin Gin Leu Gly Gin Leu 270 Glu Glu Ser Gin Giu Ser Glu 290 Cys Val 305 Gly Glu Phe 295 Gin Arg 310 280 Thr Glu Asp Glu Asp 285 Pro Leu 300 Tyr Asp Asp Leu Gly Leu Thr Leu INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 2011 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Arabidopsis thaliana (ix) FEATURE: NME/KEY: misc-feature WO 98/26082 PCT/EP97/07012 135 LOCATION: 1. .2011 OTHER INFORMATION: /note= "NIMi cDNA sequence" (ix) FEATURE: NAME/KEY: CDS LOCATION: 43. .1824 OTHER INFORMATION: /product= "1NIMi protein" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC Met Asp Thr Thr 1 ATT GAT GGA TTC GCC GAT TCT TAT GAA ATC AGC AGC

ACT

Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Thr AGT TTC GTC Ser Phe Val

GCT

Ala

GTA

Val ACC GAT AAC ACC GAC TCC TCT ATT GTT Thr Asp Asn Thr Asp Ser Ser Ile Val 30 CTC ACC GGA CCT GAT GTA TCT GCT CTG Leu Thr Gly Pro Asp Val Ser Ala Leu 45 TAT CTG GCC GCC GAA CAA Tyr Leu Ala Ala Glu Gin CAA TTG CTC TCC AAC AGC Gin Leu Leu Ser Asn Ser 150 198

TTC

Phe GAA TCC Glu Ser 55 GTC TTT GAC TCG CCG Val Phe Asp Ser Pro 60 1 30 GAT GAT TTC TAC AGC GAC GCT AAG Asp Asp Phe Tyr Ser Asp Ala LYS GTT TCT TTC CAC CGG TGC GTT TTG Val Ser Phe His Arg Cy s Vai Leu CTT GTT Leu Val CTC TCC GAC Leu Ser ASP GGC CGG GAA Gly Arg Giu 75 TCA GCG AGA AGC TCT TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAG WO 98/26082 PCT/EP97/07012 -136- Ser Ala Arg Ser Ser GAG AAA GAC TCC AAC Glu Lys Asp Ser Asn 105 ATT GCC AAG GAT TAC Ile Ala Lys Asp Tyr 120 Phe Lys Ser Ala Leu 95 Ala Ala Ala Lys Lys 100 AAC ACC GCC GCC GTG Asn Thr Ala Ala Val 110 AAG CTC GAG CTT AAG GAG Lys Leu Glu Leu Lys Glu 115 GCT TAT GTT Ala Tyr Val 135 TAC AGC Tyr Ser GAA GTC GGT TTC Glu Val Gly Phe 125 AGC AGA GTG AGA Ser Arg Val Arg 140 GAT TCG GTT GTG ACT GTT TTG Asp Ser Val Val Thr Val Leu 130 CCG CCG CCT AAA GGA GTT TCT Pro Pro Pro Lys Gly Val Ser 145 390 438 486 GAA TGC Glu Cys 150 GCA GAC GAG AAT TGC Ala Asp Glu Asn Cys 155 TGC CAC GTG GCT TGC CGG CCG GCG GTG Cys His Val Ala Cys Arg Pro Ala Val 160

GAT

Asp 165 TTC ATG TTG GAG GTT Phe Met Leu Glu Val 170 CTC TAT TTG GCT TTC Leu Tyr Leu Ala Phe 175 GAA TTA ATT ACT Glu Leu Ile Thr CTC TAT CAG AGG CAC TTA Leu Tyr Gin Arg His Leu 185 190 GAC ACA TTG GTT ATA CTC Asp Thr Leu Val Ile Leu 205 ATC TTC AAG ATC CCT Ile Phe Lys Ile Pro 180 GAC GTT GTA GAC AAA Asp Val Val Asp Lys 195 CTT GCT AAT ATA TGT Leu Ala Asn Ile Cys 210 582 630 678

GTT

Val GTT ATA GAG Val Ile Glu 200

GGT

Gly AAA GCT Lys Ala 215 TGT ATG AAG CTA TTG Cys Met Lys Leu Leu 220 GAT AGA TGT AAA Asp Arg Cys Lys GAG ATT ATT GTC Glu Ile Ile Val 225 726 AAG TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA GAG WO 98/26082 PCT/EP97/07012 137- Lys Ser 230 Asn Val Asp Met Ser Leu Glu Lys Ser 240 Leu Pro Glu Glu

CTT

Leu 245 GTT AAA GAG ATA ATT Val Lys Glu Ile Ile 250 GAT AGA CGT AAA GAG Asp Arg Arg Lys Glu 255 CTT GGT TTG GAG GTA Leu Gly Leu Glu Val 260 CCT AAA GTA AAG AAA CAT GTC Pro Lys Val Lys Lys His Val 265 TCG AAT GTA CAT AAG GCA CTT Ser Asn Val His Lys Ala Leu 270 GAC TCG Asp Ser 275 GAT GAT ATT GAG TTA GTC AAG TTG CTT Asp Asp Ile Glu Leu Val Lys Leu Leu 280 285 CTA GAT GAT GCG TGT GCT CTT CAT TTC Leu Asp Asp Ala Cys Ala Leu His Phe 295 300 TTG AAA GAG GAT CAC ACC AAT Leu Lys Glu Asp His Thr Asn 290 GCT GTT GCA TAT TGC AAT GTG Ala Val Ala Tyr Cys Asn Val 305 918 966 AAG ACC Lys Thr 310 GCA ACA GAT CTT TTA Ala Thr Asp Leu Leu 315 AAA CTT GAT CTT GCC GAT GTC AAC CAT Lys Leu Asp Leu Ala Asp Val Asn His 320 1014

AGG

Arg 325 AAT CCG AGG GGA TAT Asn Pro Arg Gly Tyr 330 ACG GTG CTT CAT GTT Thr Val Leu His Val 335 TCT CTA TTG GAA AAA Ser Leu Leu Glu Lys 350 GCT GCG ATG CGG AAG Ala Ala Met Arg Lys 340 GGT GCA AGT GCA TCA Gly Ala Ser Ala Ser 355 GAG CCA CAA TTG ATA Glu Pro Gin Leu Ile 345 1062 1110 1158 GAA GCA ACT TTG GAA GGT AGA ACC GCA Glu Ala Thr Leu Glu Gly Arg Thr Ala 360 365 CTC ATG ATC Leu Met Ile GCA AAA CAA GCC Ala Lys Gin Ala 370 ACT ATG GCG GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT TCT 1206 WO 98/26082 PCT/EP97/07012 -138- Thr Met Ala 375 Val Glu Cys Asn Asn 380 Ile Pro Glu Gin Cys Lys His Ser 385 GAA GAC AAA CGA Glu Asp Lys Arg CTC AAA Leu Lys 390 GGC CGA CTA TGT GTA Gly Arg Leu Cys Val 395 GAA ATA CTA GAG CAA Glu Ile Leu Glu Gin 400 1254

GAA

Glu 405 CAA ATT CCT AGA GAT Gin Ile Pro Arg Asp 410 GTT CCT CCC TCT TTT Val Pro Pro Ser Phe 415 CTC GAT CTT GAA AAT Leu Asp Leu Glu Asn 430 GCA GTG GCG GCC GAT Ala Val Ala Ala Asp 420 AGA GTT GCA CTT GCT Arg Val Ala Leu Ala 435 1302 1350 GAA TTG AAG ATG Glu Leu Lys Met CAA CGT CTT TTT Gin Arg Leu Phe 440 CCA ACG GAA GCA CAA Pro Thr Glu Ala Gin 445 GCT GCA ATG GAG ATC GCC GAA Ala Ala Met Glu Ile Ala Glu 450 1398 ATG AAG Met Lys CTC ACT Leu Thr 470 GGA ACA TGT GAG TTC Gly Thr Cys Glu Phe 455 GGT ACG AAG AGA ACA Gly Thr Lys Arg Thr 475 ATA GTG ACT AGC CTC GAG CCT GAC CGT Ile Val Thr Ser Leu Glu Pro Asp Arg 460 465 TCA CCG GGT GTA AAG ATA GCA CCT TTC Ser Pro Gly Val Lys Ile Ala Pro Phe 480 1446 1494

AGA

Arg 485 ATC CTA GAA GAG CAT Ile Leu Glu Glu His 490 CAA AGT AGA CTA AAA Gin Ser Arg Leu Lys 495 TTC TTC CCG CGC TGT Phe Phe Pro Arg Cys 510 GCG CTT TCT AAA ACC Ala Leu Ser Lys Thr 500 TCG GCA GTG CTC GAC Ser Ala Val Leu Asp 515 1542 1590 GTG GAA CTC GGG AAA Val Glu Leu Gly Lys 505 CAG ATT ATG AAC TGT GAG GAC TTG ACT CAA CTG GCT TGC GGA GAA GAC 1638 WO 98/26082 PCT/EP97/07012 -139- Gln Ile Met Cys Giu Asp Leu Gin Leu Ala Cys Gly Giu Asp 530 GAC ACT GCT Asp Thr Ala 535 GAG AAA CGA CTA CAA Glu Lys Arg Leu Gin 540 AAG AAG CAA AGG TAC ATG GAA ATA Lys Lys Gin Arg Tyr Met Giu Ile 545 1686 CAA GAG Gin Glu 550 ACA CTA AAG AAG GCC Thr Leu Lys Lys Ala 555 TTT AGT GAG GAC AAT TTG GAA TTA GGA Phe Ser Giu Asp Asn Leu Giu Leu Gly 560 1734

AAT

Asn 565 TTG TCC CTG ACA GAT Leu Ser Leu Thr Asp 570 TCG ACT TCT TCC ACA Ser Thr Ser Ser Thr 575 TCG AAA TCA ACC GGT Ser Lys Ser Thr Gly 580 1782 GGA AAG AGG TCT AAC Gly Lys Arg Ser Asn 585 CGT AAA CTC TCT CAT CGT CGT CGG TGA Arg Lys Leu Ser His Arg Arg Arg 590 GACTCTTGCC TCTTAGTGTA ATTTTTGCTG TACCATATAA TTCTGTTTTC ATGATGACTG TAACTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG 1824 1884 1944 2004 2011

ATTTGTA

INFORMATION FOR SEQ ID NO:22: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 2011 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 98/26082 PCT/EP97/07012 -140- (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 43..1824 OTHER INFORMATION: /product= "altered form of NIM1" /note= "Serine residues at amino acid positions 55 and 59 in wild-type NIM1 gene product have been changed to Alanine residues." (ix) FEATURE: NAME/KEY: misc_feature LOCATION: 205..217 OTHER INFORMATION: /note= "nucleotides 205 and 217 changed from T's to G's compared to wild-type sequence." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC Met Asp Thr Thr 1

ATT

Ile GAT GGA TTC Asp Gly Phe GCC GAT Ala Asp 10 TCT TAT Ser Tyr GAA ATC AGC AGC ACT AGT TTC GTC Glu Ile Ser Ser Thr Ser Phe Val 15 102 GCT ACC GAT AAC Ala Thr Asp Asn GTA CTC ACC GGA Val Leu Thr Gly 40 GAC TCC TCT Asp Ser Ser ATT GTT Ile Val TAT CTG GCC GCC GAA CAA Tyr Leu Ala Ala Glu Gin 150 198 CCT GAT GTA TCT Pro Asp Val Ser GCT CTG CAA TTG CTC TCC AAC AGC Ala Leu Gin Leu Leu Ser Asn Ser 45 WO 98/26082 141 PCT/EP97/07012 -141- GAT GAT TTC TAC AGC GAC GCT AAG 24 Asp Asp Phe Tyr Ser Asp Ala Lys TTC GAA GCC GTC TTT GAC GCG CCG Phe Glu Ala Val Phe Asp Ala Pro 60 CTT GTT Leu Val CTC TCC GAC GGC CGG Leu Ser Asp Gly Arg 75 GAA GTT TCT TTC CAC CGG TGC GTT TTG Glu Val Ser Phe His Arg Cys Val Leu 294

TCA

Ser GCG AGA AGC TCT TTC Ala Arg Ser Ser Phe 90 TTC AAG AGC GCT TTA Phe Lys Ser Ala Leu 95 GCC GCC GCT AAG AAG Ala Ala Ala Lys Lys 100 GAG AAA GAC TCC AAC Glu Lys Asp Ser Asn 105 AAC ACC GCC GCC GTG Asn Thr Ala Ala Val- 110 GAA GTC GGT TTC GAT Glu Val Gly Phe Asp 125 AAG CTC GAG CTT AAG GAG Lys Leu Glu Leu Lys Glu 115 TCG GTT GTG ACT GTT TTG Ser Val Val Thr Val Leu 130 342 390 438 ATT GCC AAG GAT Ile Ala Lys Asp 120 GCT TAT GTT TAC Ala Tyr Val Tyr 135 AGC AGC AGA GTG Ser Ser Arg Val 140 AGA CCG CCG CCT AAA GGA GTT TCT Arg Pro Pro Pro Lys Gly Val Ser 145 486 GAA TGC Glu Cys 150 GCA GAC GAG AAT TGC Ala Asp Glu Asn Cys 155 TGC CAC GTG GCT TGC CGG CCG GCG GTG Cys His Val Ala Cys Arg Pro Ala Val 160

GAT

Asp 165 TTC ATG TTG GAG GTT Phe Met Leu Glu Val 170 CTC TAT TTG GCT TTC Leu Tyr Leu Ala Phe 175 ATC TTC AAG ATC CCT Ile Phe Lys Ile Pro 180 GAC GTT GTA GAC AAA Asp Val Val Asp Lys 195 GAA TTA ATT ACT Glu Leu Ile Thr CTC TAT CAG AGG CAC TTA Leu Tyr Gln Arg His Leu 185 190 WO 98/26082 WO 9826082PCTIEP97/07012 -142 GTT GTT ATA GAG GAG ACA TTG GTT Val Val GGT AAA Gly Lys Ile Glu 200 Asp Thr Leu Val

ATA

Ile 205 CTC AAG CTT GCT AAT ATA TGT Leu Lys Leu Ala Asn Ile Cys 210 GCT TGT ATG AAG CTA Ala Cys Met Lys Leu 215 AAT GTA GAT ATG GTT Asn Val Asp Met Val 235 TTG GAT AGA TGT AAA GAG ATT ATT GTC Leu Asp Arg Cys Lys Glu Ile Ile Val 220 225 AGT CTT GAA AAG TCA TTG CCG GAA GAG Ser Leu Glu Lys Ser Leu Pro Giu Glu 240 678 726 774 AAG TCT Lys Ser 230

CTT

Leu 245 GTT AAA GAG ATA ATT Vai Lys Glu Ile Ile 250 GAT AGA CGT AAA GAG Asp Arg Arg Lys Giu 255 GTC TCG AAT GTA CAT Val Ser Asn Val His 270 CTT GGT TTG GAG GTA Leu Gly Leu Glu Vai 260 AAG GCA CTT GAG TCG Lys Ala Leu Asp Ser 275 CCT AAA GTA AAG Pro Lys Vai Lys GAT GAT ATT GAG Asp Asp Ile Glu 280 TTA GTC AAG TTG CTT Leu Val Lys Leu Leu 285 TTG AAA GAG GAT CAC ACC AAT Leu Lys Giu Asp His Thr Asn 290 918 CTA GAT GAT Leu Asp Asp 295 GCG TGT GCT CTT CAT Ala Cys Ala Leu His 300 TTC GCT GTT GCA TAT TGC AAT GTG Phe Ala Val Ala Tyr Cys Asn Val 305 966 AAG ACC Lys Thr 310 GCA ACA GAT CTT TTA AAA CTT GAT CTT GCC GAT GTC AAC CAT Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Vai Asn His 315 320 1014 AGG AAT Arg Asn 325 CCG AGG, GGA TAT Pro Arg Gly T'yr 330 ACG GTG CTT CAT GTT Thr Val Leu His Val 335 GCT GCG ATG CGG AAG Ala Ala Met Arg Lys 340 1062 WO 98/26082 PCT/EP97/07012 -143- GAG CCA CAA TTG ATA CTA TCT CTA TTG Glu Pro Gin Leu GAA GCA ACT TTG Glu Ala Thr Leu 360 ACT ATG GCG GTT Thr Met Ala Val 375 Leu Ser Leu Leu

GAA

Glu 350 AAA GGT GCA AGT GCA TCA Lys Gly Ala Ser Ala Ser 355 GAA GGT AGA ACC GCA CTC ATG ATC GCA AAA CAA GCC Glu Gly Arg Thr Ala Leu Met Ile Ala Lys Gin Ala 365 370 GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT TCT Glu Cys Asn Asn Ile Pro Glu Gin Cys Lys His Ser 380 385 1110 1158 1206 CTC AAA Leu Lys 390 GGC CGA CTA TGT GTA Gly Arg Leu Cys Val 395 GAA ATA CTA GAG CAA GAA GAC AAA CGA Glu Ile Leu Glu Gin Glu Asp Lys Arg 400 1254 1302

GAA

Glu 405 CAA ATT CCT AGA GAT GTT CCT CCC TCT TTT Gin Ile Pro Arg Asp Val Pro Pro Ser Phe 410 415 GCA GTG GCG GCC GAT Ala Val Ala Ala Asp 420 GAA TTG AAG ATG ACG Glu Leu Lys Met Thr 425 CTG CTC GAT CTT GAA Leu Leu Asp Leu Glu 430 AAT AGA GTT GCA CTT GCT Asn Arg Val Ala Leu Ala 435

CAA

Gin CGT CTT TTT Arg Leu Phe 440 ACG GAA GCA Thr Glu Ala CAA GCT GCA ATG GAG ATC GCC GAA Gin Ala Ala Met Glu Ile Ala Glu 445 450 GTG ACT AGC CTC GAG CCT GAC CGT Val Thr Ser Leu Glu Pro Asp Arg 465 1350 1398 1446 ATG AAG GGA Met Lys Gly 455 ACA TGT GAG TTC ATA Thr Cys Glu Phe Ile 460 CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAG ATA GCA CCT TTC 1494 Leu Thr 470 Gly Thr Lys Arg Thr 475 Ser Pro Gly Val Lys 480 Ile Ala Pro Phe WO 98/26082 PTE9/71 PCT/EP97/07012 144-

AGA

Arg 485 ATC CTA GA. GAG Ile Leu Glu Giu CAA AGT AGA CTA AAA Gin Ser Arg Leu Lys 495 GCG CTT TCT AAA ACC Ala Leu Ser Lys Thr 500 TCG GCA GTG CTC GAC Ser Ala Val Leu Asp 515 GTG GAA CTC GGG AAA Val Glu Leu Gly Lys 505 CAG ATT ATG AAC TGT Gin Ile Met Asn Cys 520 CGA TTC TTC CCG CGC Arg Phe Phe Pro Arg 510 1542 1590 1638 1686 GAC ACT GCT Asp Thr Ala 535 GAG AAA Giu Lys GAG GAC TTG ACT Glu Asp Leu Thr 525 CGA CTA CAA AAG Arg Leu Gin Lys 540 CAA CTG GCT TGC GGA GAA GAC Gin Leu Ala Cys Gly Giu Asp 530 AAG CAA AGG TAC ATG GAA ATA Lys Gin Arg Tyr Met Glu Ile 545 CAA GAG Gin Giu 550 ACA CTA AAG AAG GCC Thr Leu Lys Lys Ala 555 TTT AGT GAG GAC AAT TTG GAA TTA GGA Phe Ser Giu Asp Asn Leu Giu Leu Gly 560 1734

AAT

Asn 565 TTG TCC CTG ACA GAT Leu Ser Leu Thr Asp 570 TCG ACT TCT TCC ACA Ser Thr Ser Ser Thr 575 TCG AAA TCA ACC GGT Ser Lys Ser Thr Gly 580 1782 GGA AAG AGG TCT AAC Gly Lys Arg Ser Asn 585 CGT AAA CTC TCT CAT CGT CGT CGG TGA Arg Lys Leu Ser His Arg Arg Arg* 590 GACTCTTGCC TCTTAGTGTA ATTTTTGCTG TACCATATAA TTCTGTTTTC! ATGATGACTG 1824 1884 1944 2004 2011 TAACTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA. TTTGAACCAA TGGTATACAG

ATTTGTA

WO 98/26082-14 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 594 amino acids TYPE: amino acid TOPOLOGY: linear PCT/EP97/07012 (ii) MOLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ ID NO:23: Met Thr Asp Thr Thr Ser Phe Val Ala Glu Gin Ile Asp Gly Phe Ala Asp 10 Ser Tyr Giu Ile Ser Ser Ala Thr Asp Asn Thr 25 Pro Asp Ser Ser Ile Ala Val Leu Thr Leu Ser Asn Gly 40 Val Asp Val Ser Ala Asp Val Tyr Leu Leu Gin Leu Asp Phe Tyr Ser Phe Glu Ala 55 Leu Phe Asp Ala Pro Glu Ser Arg Asp Ala Lys Leu Val 70 Ser Asp Gly Val Ser Phe His Cys Val Leu Ser Glu Ala Arg Ser Ser Phe Phe Lys Ser Ala 90 Asn Thr Ala Ala Val 110 Leu Ala Lys Leu Ala Ala Lys Lys 100 Lys Asp Ser Glu Leu LYS Glu Ile Ala LYS ASP Tyr Glu Val Gly Phe Asp Ser Val 115 125 WO 98/26082 PTE9/71 PCT/EP97/07012 146 Val Thr 130 Val Leu Ala Tyr Tyr Ser Ser Arg Val Arg Pro Pro Pro 140 Lys Gly Val Ser Glu 145 Ala Asp Glu Asn Cys 155 Cys His Val Ala Arg Pro Ala Val Asp 165 Phe Met LTeu Glu Val 170 Leu Tyr Leu Ala Phe Ile 175 Phe Lys Ile Val Val Asp 195 Pro 180 Glu Leu Ile Thr Tyr Gln Arg His Leu Leu Asp 190 Leu Lys Leu Lys Val Val Ile Glu 200 Asp Thr Leu Val Ala Asn 210 Ile Cys Gly Lys Ala 215 Met Lys Leu Asp Arg Cys Lys Glu 225 Ile Ile Val Lys Ser 230 Asn Val Asp Met Val 235 Ser Leu Glu Lys Leu Pro Glu Glu Leu 245 Val Lys Glu Ile Ile 250 Asp Arg Arg LYS Glu Leu 255 Gly Leu Glu Ala Leu Asp 275 Val1 260 Pro Lys Val Lys His Val Ser Asn Val His Lys 270 Leu Lys Glu Ser Asp Asp Ile Glu 280 Leu Val Lys Leu Leu 285 Asp His 290 Thr Asn Leu Asp Asp 295 Ala Cys Ala Leu His 300 Phe Ala Val Ala Tyr Cys 305 Asn Val Lys Thr Ala Thr Asp Leu Leu 315 Lys Leu Asp Leu WO 98/26082 WO 9826082PCT/EP97/07012 147 Asp Val Asn His Arg Asn Pro Arg Gly 325 Tyr 330 Thr Val Leu His Val Ala 335 Ala Met Arg Glu Pro Gin Leu Ile 345 Leu Ser Leu Leu Giu Lys Gly 350 Leu Met Ile Ala Ser Ala 355 Ser Glu Ala Thr Giu Gly Arg Thr Ala 365 Ala Lys 370 Gin Ala Thr Met Ala 375 Val Giu Cys Asn Asn 380 Ile Pro Giu Gin Cys 385 Lys His Ser Leu Lys 390 Gly Arg Leu Cys Giu Ile Leu Giu Gin 400 Giu Asp Lys Arg Giu 405 Gin Ile Pro Arg Asp 410 Vai Pro Pro Ser Phe Ala 415 Val Ala Ala Asp 420 Giu Leu Lys Met Leu Leu Asp Leu Giu Asn Arg 430 Ala Ala Met Val Ala Leu 435 Ala Gin Arg Leu Phe 440 Pro Thr Giu Ala Gin 445 Giu Ile 450 Ala Giu Met Lys Thr Cys Giu Phe Val Thr Ser Leu Giu 465 Pro Asp Arg Leu Thr 470 Giy Thr Lys Arg Thr 475 Ser Pro Giy Val Lys 480 Ile Ala Pro Phe Ile Leu Giu Giu His Gin Ser Arg Leu 490 Lys Aia 495 Leu Ser Lys Thr 500 Vai Giu Leu Giy Lys 505 Arg Phe Phe Pro Arg Cys Ser 510 WO 98/26082 PCT/EP97/07012 -148- Ala Val Leu 515 Asp Gin Ile Met Asn 520 Cys Glu Asp Leu Gin Leu Ala Cys Gly 530 Glu Asp Asp Thr Glu Lys Arg Leu Gin Lys Lys Gin 540 Phe Ser Glu Asp Arg Met Glu Ile Gin Glu 550 Thr Leu Lys Lys Ala 555 Asn 560 Leu Glu Leu Gly Leu Ser Leu Thr Ser Thr Ser Ser Thr Ser 575 Lys Ser Thr Gly 580 Gly Lys Arg Ser Asn 585 Arg Lys Leu Ser His Arg Arg 590 Arg INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 1597 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1410 OTHER INFORMATION: /product= "Altered form of NIM1" /note= "N-terminal deletion compared to wild-type NIM1 sequence." WO 98/26082 PCT/EP97/07012 -149- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: ATG GAT TCG GTT GTG ACT GTT TTG GCT TAT GTT TAC AGC AGC AGA GTG Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val 1 5 10 AGA CCG CCG CCT Arg Pro Pro Pro AAA GGA GTT TCT GAA Lys Gly Val Ser Glu 25 TGC GCA GAC GAG AAT TGC TGC Cys Ala Asp Glu Asn Cys Cys CAC GTG GCT His Val Ala TGC CGG CCG GCG GTG GAT TTC ATG TTG GAG GTT CTC TAT Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr 40 TTG GCT Leu Ala TTC ATC TTC AAG ATC Phe Ile Phe Lys Ile 55 CCT GAA TTA ATT ACT CTC TAT CAG AGG Pro Glu Leu Ile Thr Leu Tyr Gln Arg

CAC

His TTA TTG GAC GTT GTA Leu Leu Asp Val Val 70 GAC AAA GTT GTT ATA Asp Lys Val Val Ile 75 ATA TGT GGT AAA GCT Ile Cys Gly Lys Ala 90 GAG GAC ACA TTG GTT Glu Asp Thr Leu Val TGT ATG AAG CTA TTG Cys Met Lys Leu Leu 240 ATA CTC AAG CTT GCT Ile Leu Lys Leu Ala GAT AGA TGT AAA GAG ATT ATT GTC AAG Asp Arg Cys Lys Glu Ile Ile Val Lys 100 105 CTT GAA AAG TCA TTG CCG GAA GAG CTT Leu Glu Lys Ser Leu Pro Glu Glu Leu 115 120 TCT AAT GTA GAT ATG GTT AGT Ser Asn Val Asp Met Val Ser 110 GTT AAA GAG ATA ATT GAT AGA Val Lys Glu Ile Ile Asp Arg 125 288 336 384 CGT AAA GAG CTT GGT TTG GAG GTA CCT AAA GTA AAG AAA CAT GTC TCG WO 98/26082 PCT/EP97/07012 150- Arg Lys 130 Glu Leu Gly Leu Glu Val Pro Lys Val 135 Lys 140 Lys His Val Ser

AAT

Asn 145 GTA CAT AAG GCA CTT Val His Lys Ala Leu 150 GAC TCG GAT GAT ATT Asp Ser Asp Asp Ile 155 GAG TTA GTC Glu Leu Val AAG TTG Lys Leu 160 CTT TTG AAA GAG GAT Leu Leu Lys Glu Asp 165 TTC GCT GTT GCA TAT Phe Ala Val Ala Tyr 180 CAC ACC AAT CTA GAT GAT His Thr Asn Leu Asp Asp 170 GCG TGT GCT CTT CAT Ala Cys Ala Leu His 175 528 576 TGC AAT GTG AAG Cys Asn Val Lys 185 ACC GCA ACA GAT CTT TTA AAA Thr Ala Thr Asp Leu Leu Lys 190 CTT GAT CTT Leu Asp Leu 195 GCC GAT GTC AAC CAT Ala Asp Val Asn His 200 AGG AAT CCG AGG GGA TAT ACG GTG Arg Asn Pro Arg Gly Tyr Thr Val 205 GAG CCA CAA TTG ATA CTA TCT CTA Glu Pro Gin Leu Ile Leu Ser Leu 220 CTT CAT Leu His 210 GTT GCT GCG ATG CGG AAG Val Ala Ala Met Arg Lys 215

TTG

Leu 225 GAA AAA GGT GCA AGT Glu Lys Gly Ala Ser 230 GCA TCA GAA GCA ACT Ala Ser Glu Ala Thr 235 TTG GAA GGT AGA ACC Leu Glu Gly Arg Thr 240 624 672 720 768 816 GCA CTC ATG ATC Ala Leu Met Ile ATC CCG GAG CAA Ile Pro Glu Gin 260

GCA

Ala 245 CAA GCC ACT Gin Ala Thr ATG GCG GTT GAA TGT AAT AAT Met Ala Val Glu Cys Asn Asn 250 255 AAA GGC CGA CTA TGT GTA GAA Lys Gly Arg Leu Cys Val Glu 270 TGC AAG CAT TCT CTC Cys Lys His Ser Leu 265 ATA CTA GAG CAA GAA GAC AAA CGA GAA CAA ATT CCT AGA GAT GTT CCT WO 98/26082 WO 9826082PCT/EP97/07012 151 Ile Leu GlU 275 Gin Glu Asp Lys Arg 280 Glu Gin Ile Pro Arg 285 Asp Val Pro CCC TCT Pro Ser 290 TTT GCA GTG GCG GCC Phe Ala Val Ala Ala 295 GAT GAA TTG AAG ATG ACG CTG CTC GAT A sp Giu Leu Lys Met Thr Leu Leu Asp 300

CTT

Leu 305 GAA AAT AGA GTT GCA Giu Asn Arg Val Ala 310 CTT GCT CAA CGT CTT Leu Ala Gin Arg Leu 315 GCC GAA ATG AAG CGA Ala Glu Met Lys Gly 330 TTT CCA ACG GAA GCA Phe Pro Thr Glu Aia 320 ACA TGT GAG TTC ATA Thr Cys Giu Phe Ile 335 960 1008 CAA GCT GCA ATC GAG Gin Ala Ala Met Giu 325 GTG ACT AGC CTC Val Thr Ser Leu 340 GAG CCT GAC CGT CTC ACT GGT ACG AAG AGA ACA TCA Giu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser 345 350 1056 CCG GGT Pro Gly AGA CTA Arg Leu 370 GTA AAG ATA GCA CCT Val Lys Ile Ala Pro 355 AAA C CTT TCT AAA Lys Ala Leu Ser Lys 375 TTC AGA ATC CTA GAA GAG CAT CAA AGT Phe Arg Ile Leu Ciu Giu His Gin Ser 360 365 ACC GTG GAA CTC CCC AAA CGA TTC TTC Thr Val Giu Leu Gly Lys Arg Phe Phe 380 1104 1152

CCC

Pro 385 CCC TGT TCG GCA GTG Arg Cys Ser Ala Val 390 CTC GAC CAG ATT ATG Leu Asp Gin Ile Met 395 AAC TCT Asn Cys GAG GAC TTG Giu Asp Leu 400 1200 ACT CAA CTG GCT TGC CCA GAA Thr Gin Leu Ala Cys Gly Glu 405 CAC GAC ACT GCT Asp Asp Thr Ala 410 GAG AAA CGA CTA CAA Glu Lys Arg Leu Gin 415 1248 AAG AAG CAA AGG TAC ATG GAA ATA CAA GAG ACA CTA AAC AAG CCC TTT 1296 WO 98/26082 152 PCTIEP97O7012 Lys Lys Gin AGT GAG GAC Ser Glu Asp 435 TCT TCC ACA Ser Ser Thr 450 Arg 420

AAT

Asn Tyr Met Glu Ile TTG GAA TTA OGA Leu Glu Leu Gly 440 Gin Giu Thr Leu 425 AAT TTG TCC CTG Asn Leu Ser Leu Lys Lys Ala Phe 430 ACA GAT TCG ACT Thr Asp Ser Thr 445 TCG AAA TCA ACC GGT GGA AAG AGG TCT AAC CGT Ser Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg 455 460 kAA CTC ys Leu TCT CAT CGT CGT CGG TGA GACTCTTGCC TCTTAGTGTA ATTTTTGCTG Ser His Arg Arg Arg 465 470 TACCATATAA TTCTGTTTTC ATGATGACTG TAACTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT ATTATTGCTG CAGGTGTGCT TCAA~ACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG ATTTGTA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 470 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val 1 5 10 1344 1392 1440 1500 1560 1597 WO 98/26082 WO 9826082PCTIEP97/07012 153- Arg Pro Pro His Val Ala 35 Pro Lys Gly Val Ser Glu Cys 25 Ala Asp Giu Asn Cys Cys Val Leu Tyr Cys Arg Pro Ala Vai 40 Asp Phe Met Leu Giu Leu Ala Phe Ile Phe Lys Ile Pro Giu Leu Ile Leu Tyr Gin Arg His Leu Leu Asp Vai Asp Lys Val Val Ile Giu Asp Thr Leu Ile Leu Lys Leu Asn Ile Cys Gly Aia Cys Met Lys Leu Leu Asp Arg Cys Leu Giu Lys 115 Lys 100 Giu Ile Ile Val Lys 105 Ser Asn Val Asp Met Val Ser 110 Ile Asp Arg Ser Leu Pro Giu Giu 120 Leu Vali Lys Giu Ile 125 Arg Lys 130 Giu Leu Giy Leu Val Pro Lys Vai Lys His Vai Ser Asn 145 Vai His Lys Aia Leu 150 Asp Ser Asp ASP Giu Leu Val Lys Leu 160 Leu Leu Lys Giu His Thr Asn Leu Asp 170 ASP Ala Cys Ala Leu His 175 130 Phe Ala Val Ala 180 Tyr Cys Asn Vai Lys 185.

Thr Ala Thr Asp Leu Leu Lys 190 Tyr Thr Val Leu Asp Leu 195 Ala Asp Val Asn His Arg Asn Pro Arg 200 Gly 205 WO 98/26082 WO 9826082PCTEP97/O7012 154 Leu His 210 Vai Ala Ala Met Arg 215 Ala Leu 225 Ala Glu Lys Gly Ala Lys Giu Pro Gin Leu Ile 220 Ser Giu Ala Thr Leu Giu 235 Ala Thr Met Ala Val Giu 250 Ser Leu Lys Gly Arg Leu Leu Met Ile Ala 245 Cys Gin Leu Ser Leu Gly Arg Thr 240 Cys Asn Asn 255 Cys Val Giu 270 Asp Val Pro Leu Leu Asp Ile Pro Giu Gin 260 Gin Lys His Ile Leu Giu 275 Pro Ser Phe 290 Giu Asp Lys Ala Val Ala Ala 295 Leu Arg Giu 280 Asp Giu Aia Gin Gin Ile Pro Leu Lys Met 300 Arg Leu Phe 315 Lys Gly Thr Arg 285 Thr Leu 305 Gin Glu Asn Arg Val Ala 310 Ile Pro Thr Giu Ala 320 Ala Ala Met Giu 325 Giu Ala Giu Met Cys Giu Phe Ile 335 Val Thr Ser Leu 340 Lys Pro Asp Arg Leu 345 Arg Gly Thr Lys Pro Gly Val 355 Arg Leu Lys Ile Ala Pro Phe 360 Ile Leu Giu Giu 365 Lys Arg Thr Ser 350 His Gin Ser Arg Phe Phe Ala Leu Ser 370 Pro Arg Lys 375 Thr Val Glu Leu Gly 380 Met Asn 395 Cys Ser Ala 385 Val 390 Leu Asp Gin Ile CYS Giu ASP WO 98/26082 PCT/EP97/07012 -155- Thr Gin Leu Ala Cys 405 Gly Glu Asp Asp Ala Glu Lys Arg Leu Gin 415 Lys Lys Gin Tyr Met Glu Ile Gin 425 Glu Thr Leu Lys Lys Ala Phe 430 Asp Ser Thr Ser Glu Asp 435 Asn Leu Glu Leu Gly 440 Asn Leu Ser Leu Thr 445 Ser Ser 450 Thr Ser Lys Ser Thr 455 Gly Gly Lys Arg Ser 460 Asn Arg Lys Leu His Arg Arg Arg INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 1608 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 30 (ix) FEATURE: NAME/KEY: CDS LOCATION: 43..1608 OTHER INFORMATION: /product= "Altered 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 WO 98/26082 WO 9826082PCT/EP97/07012 -156- Met Asp Thr Thr 1 I ATT GAT GGA TTC GCC GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC Thr Ser Phe Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser GCT ACC GAT AAC ACC Ala Thr Asp Asn Thr GTA CTC ACC GGA CCT Val Leu Thr Gly Pro TTC GAA TCC GTC TTT Phe Giu Ser Val Phe GAC TCC TCT ATT GTT Asp Ser Ser Ile Val 30 GAT GTA TCT GCT CTG Asp Val Ser Ala Leu 45 TAT CTG GCC GCC GAA CAA Tyr Leu Ala Ala Giu Gin CAA TTG CTC TCC AAC AGC Gin Leu Leu Ser Asn Ser GAC TCG CCG Asp Ser Pro 60 GAT GAT TTC TAC AGC GAC GCT AAG Asp Asp Phe Tyr Ser Asp Ala Lys CTT GTT Leu Val CTC TCC GAC GGC CGG Leu Ser Asp Gly Arg 75 GAA GTT TCT TTC CAC CGG TGC GTT TTG Glu Val Ser Phe His Arg Cys Val Leu 294

TCA

Ser GCG AGA AGC TCT TTC Ala Arg Ser Ser Phe 90 TTC AAG AGC GCT TTA Phe Lys Ser Ala Leu 95 GCC GCC GCT AAG AAG Ala Ala Ala Lys Lys 100 342 GAG AAA GAC TCC AAC AAC ACC GCC GCC GTG Glu Lys Asp Ser Asn Asn Thr Ala Ala Val 105 110 AAG CTC GAG CTT AAG GAG Lys Leu Glu Leu Lys Glu 115 390 ATT GCC Ile Ala AAG GAT Lys Asp 120 TAC GAA GTC GGT TTC Tyr Glu Val Gly Phe 125 GAT TCG GTT GTG ACT GTT TTG Asp Ser Val Val Thr Val Leu 130 GCT TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT WO 98/26082 WO 9826082PCT/EP97/07O12 -157 Ala Tyr Val 135 Tyr Ser Ser Arg Val 140 Arg Pro Pro Pro Lys 145 Gly Val Ser GAA TGC Glu Cys 150 GCA GAC GAG AAT TGC Ala Asp Glu Asn Cys 155 TGC CAC GTG GCT TGC COG CCG GCG GTG Cys His Vai Ala Cys Arg Pro Ala Val 160

GAT

Asp 165 TTC ATG TTG GAG GTT Phe Met Leu Giu Val 170 CTC TAT TTG GCT TTC Leu Tyr Leu Ala Phe 175 CAG AGO CAC TTA TTG Gin Arg His Leu Leu 190 ATC TTC AAG ATC CCT Ile Phe Lys Ile Pro 180 GAC GTT GTA GAC AAA Asp Val Val Asp Lys 195 582 630 OAA TTA ATT ACT CTC Glu Leu Ile Thr Leu 185 OTT OTT ATA GAG GAC ACA TTG OTT Val Val Ile Glu Asp Thr Leu Val 200

ATA

Ile 205 CTC AAO CTT OCT AAT ATA TOT Leu Lys Leu Ala Asn Ile Cys 210 678

GOT

Gly AAA OCT TGT ATG Lys Aia Cys Met 215 AAG CTA TTG Lys Leu Leu 220 OAT AGA TOT AAA GAO ATT ATT OTC Asp Arg Cys Lys iu Ile Ile Val 225 726 774 AAG TCT Lys Ser 230 AAT OTA GAT ATG OTT Asn Val Asp Met Val 235 ACT CTT GAA AAG TCA TTO CCO OAA GAG Ser Leu Giu Lys Ser Leu Pro Giu Giu 240

CTT

Leu 245 OTT AAA GAG ATA ATT Val Lys Gu Ile Ile 250 OAT AGA COT AAA GAO Asp Arg Arg Lys Giu 255 CTT GOT TTG GAG OTA Leu Oly Leu Oiu Val 260 CCT AAA OTA AAG AAA Pro Lys Val Lys Lys 265 CAT GTC His Val TCG AAT Ser Asn

GTA

Val 270 CAT AAG OCA CTT GAC TCG His Lys Ala Leu Asp Ser 275 OAT OAT ATT GAG TTA GTC AAG TTO CTT TTG AAA GAG OAT CAC ACC A6AT 918 WO 98/26082 -158- Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp 280 285 PCT/EP97/07012 His Thr Asn 290 CTA GAT Leu Asp

GAT

Asp 295 GCG TGT GCT CTT CAT Ala Cys Ala Leu His 300 TTC GCT GTT GCA TAT TGC AAT GTG Phe Ala Val Ala Tyr Cys Asn Val 305 AAG ACC Lys Thr 310 GCA ACA GAT CTT TTA Ala Thr Asp Leu Leu 315 AAA CTT GAT CTT GCC GAT GTC AAC CAT Lys Leu Asp Leu Ala Asp Val Asn His 320 1014 AGG AAT CCG AGG GGA Arg Asn Pro Arg Gly 325 GAG CCA CAA TTG ATA Glu Pro Gin Leu Ile 345 TAT ACG GTG CTT CAT Tyr Thr Val Leu His 330 CTA TCT CTA TTG GAA Leu Ser Leu Leu Glu 350 GTT GCT GCG ATG CGG AAG Val Ala Ala Met Arg Lys 335 340 AAA GGT GCA AGT GCA TCA Lys Gly Ala Ser Ala Ser 355 1062 1110 GAA GCA ACT TTG GAA GGT AGA ACC GCA Glu Ala Thr Leu Glu Gly Arg Thr Ala 360 365 CTC ATG ATC GCA AAA CAA GCC Leu Met Ile Ala Lys Gin Ala 370 1158 ACT ATG GCG Thr Met Ala 375 GTT GAA TGT AAT AAT Val Glu Cys Asn Asn 380 ATC CCG GAG CAA TGC AAG CAT TCT Ile Pro Glu Gin Cys Lys His Ser 385 1206 CTC AAA Leu Lys 390 GGC CGA CTA TGT GTA Gly Arg Leu Cys Val 395 GAA ATA CTA GAG CAA GAA GAC AAA CGA Glu Ile Leu Glu Gin Glu Asp Lys Arg 400 1254 30

GAA

Glu 405 CAA ATT CCT AGA GAT Gin Ile Pro Arg Asp 410 GTT CCT CCC TCT TTT Val Pro Pro Ser Phe 415 GCA GTG GCG GCC GAT Ala Val Ala Ala Asp 420 1302 GAA TTG AAG ATG ACG CTG CTC GAT CTT GAA AAT AGA GTT GCA CTT GCT 1350 WO 98/26082 PCT/EP97/07012 -159- Glu Leu Lys CAA CGT CTT Gin Arg Leu Met Thr 425 Leu Leu Asp Leu Glu 430 Asn Arg Val Ala Leu Ala 435 TTT CCA ACG GAA GCA CAA Phe Pro Thr Glu Ala Gin 440 445 ACA TGT GAG TTC ATA GTG Thr Cys Glu Phe Ile Val 460 GCT GCA ATG GAG ATC GCC GAA Ala Ala Met Glu Ile Ala Glu 450 ACT AGC CTC GAG CCT GAC CGT Thr Ser Leu Glu Pro Asp Arg 465 1398 1446 ATG AAG GGA Met Lys Gly 455 CTC ACT Leu Thr 470 GGT ACG AAG AGA ACA Gly Thr Lys Arg Thr 475 TCA CCG GGT GTA AAG ATA GCA CCT TTC Ser Pro Gly Val Lys Ile Ala Pro Phe 480 1494

AGA

Arg 485 ATC CTA GAA GAG CAT Ile Leu Glu Glu His 490 CAA AGT AGA CTA AAA Gin Ser Arg Leu Lys 495 TTC TTC CCG CGC TGT Phe Phe Pro Arg Cys 510 GCG CTT TCT AAA ACC Ala Leu Ser Lys Thr 500 TCG GCA GTG CTC GAC Ser Ala Val Leu Asp 515 1542 1590 GTG GAA CTC GGG AAA Val Glu Leu Gly Lys 505 CAG ATT ATG AAC TGT TGA Gin Ile Met Asn Cys 520 INFORMATION FOR SEQ ID NO:27: 1608 SEQUENCE CHARACTERISTICS: LENGTH: 522 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 98/26082-16- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: PCT/EP97/07012 Met 1 Thr Asp Thr Thr Ser Phe Val Ala Glu Gin Asp Gly Phe Ala Thr Asp Asn Thr 25 Pro Asp Ser Tyr 10 Asp Ser Ser Asp Val Ser Glu Ile Ser Ser Ile Ala Val Leu Thr Leu Ser Asn Giy 40 Val Ala Val Tyr Leu Leu Gin Leu Asp Phe Tyr Ser Phe Glu Ser Leu Phe Asp Ser Pro Asp Ser Ala Lys Leu Ser Asp Gly Arg 75 Phe Glu Lys Arg Cys Vai Leu Ser Glu Arg Ser Ser Vai Ser Phe His Ser Ala Leu Ala Ala Val Lys Leu Ala Ala Lys Glu Leu Lys 115 Vai Thr Val Lys 100 Glu Lys Asp Ser Asn 105 Tyr Thr Ala 110 Asp Ser Val Ile Ala Lys Asp 120 Tyr Giu Val Gly Phe 125 Arg Leu Ala Tyr 130 Gly Val 135 Ala Ser Ser Arg Pro Pro Pro Lys 145 Val Ser Glu Cys 150 Asp Glu Asn Cys 155 His Val Aia Cys 160 Arg Pro Ala Val Asp 165 Phe Met Leu Glu Vai 170 Leu Tyr Leu Ala Phe Ile 175 WO 98/26082 PCT/EP97/07012 -161 Giu Leu Ile Thr Leu Tyr Gin Arg His 185 Phe Lys Ile Leu Leu Asp 190 Leu Lys Leu Vai Vai Asp 195 Lys Vai Vai Ile Giu 200 Asp Thr Leu Val Ile 205 Ala Asn 210 Ile Cys Giy Lys Aia 215 Cys Met Lys Leu Leu 220 Asp Arg Cys Lys Giu 225 Ile Ile Val Lys Ser Asn Vai Asp Met 230 Ser Leu Giu Lys Ser 240 Leu Pro Giu Giu Leu 245 Vai Lys Giu Ile Ile 250 Asp Arg Arg Lys Giu Leu 255 Giy Leu Giu Ala Leu Asp 275 Val 260 Pro Lys Vai Lys His Val Ser Asn Vai His Lys 270 Leu LysGiu Ser Asp Asp Ile Giu 280 Leu Val Lys Leu Leu 285 Asp His 290 Thr Asn Leu Asp Ala Cys Aia Leu Phe Ala Val Ala Tyr 305 Cys Asn Val Lys Thr 310 Ala Thr Asp Leu Leu 315 Lys Leu Asp Leu Asp Val Asn His Arg 325 Asn Pro Arg Gly Tyr 330 Thr Val Leu His Val Ala 335 Ala Met Arg Giu Pro Gin Leu Ile 345 Leu Ser Leu Leu Giu Lys Gly 350 Ala Ser Ala 355 Ser Giu Ala Thr Leu Giu Gly Arg Thr Ala Leu Met Ile 360 365 WO 98/26082 WO 9826082PCT/EP97/07012 162 Ala Lys 370 Cys Lys Gin Ala Thr Met His Ser Leu Lys 390 Lys Arg Glu Gin Ala 375 Gly Val Giu Cys Arg Leu Cys Asn Asn Ile Pro Glu Gin 380 Val Glu Ile Leu Glu Gin 385 Giu Asp Ile Pro Arg 405 Glu Vai Ala Ala Asp 420 Ala Leu Lys Met Thr Leu 425 Pro Thr Val Ala Leu 435 Giu Ile Ala Gin Arg Leu Phe 440 Thr Val Pro Pro Ser Phe Ala 415 Leu Asp Leu Giu Asn Arg 430 Giu Ala Gin Ala Ala Met 445 Phe Ile Val Thr Ser Leu 460 Thr Ser Pro Gly Val Lys Glu Met Lys 450 Gly 455 Gly Cys Giu Giu 465 Ile Pro Asp Arg Leu Thr Lys Arg Ala Pro Phe Arg 485 Val Leu Giu GiU 475 His Gin 490 Arg Phe 480 Ser Arg Leu Lys Ala 495 Phe Pro Arg Cys Ser 510 Leu Ser Lys Thr 500 Giu Leu Gly Lys Ala Val Leu Asp Gin Ile 515 INFORMATION FOR SEQ 5 Met Asn C 520 ID NO:28: 05 ,ys SEQUENCE CHARACTERISTICS: LENGTH: 1194 base pairs TYPE: nucleic acid STRANDEDNESS: single WO 98/26082 PCT/EP97/07012 -163- TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1194 OTHER INFORMATION: /product= "Altered form of NIM1" /note= "N-terminal/C-terminal chimera." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

ATG

Met 1 GAT TCG Asp Ser GTT GTG ACT GTT TTG GCT TAT Val Val Thr Val Leu Ala Tyr 5 10 GTT TAC AGC AGC Val Tyr Ser Ser AGA GTG Arg Val AGA CCG CCG Arg Pro Pro AAA GGA GTT TCT GAA Lys Gly Val Ser Glu TGC GCA GAC GAG AAT TGC TGC Cys Ala Asp Glu Asn Cys Cys CAC GTG GCT TGC CGG CCG GCG His Val Ala Cys Arg Pro Ala GTG GAT TTC ATG TTG GAG GTT CTC TAT Val Asp Phe Met Leu Glu Val Leu Tyr 40 CCT GAA TTA ATT ACT CTC TAT CAG AGG Pro Glu Leu Ile Thr Leu Tyr Gin Arg 144 192

TTG

Leu GCT TTC Ala Phe TTA TTG Leu Leu ATC TTC AAG ATC Ile Phe Lys Ile 55

CAC

His

ATA

Ile GAC GTT GTA Asp Val Val 70 GAC AAA GTT GTT ATA Asp Lys Val Val Ile 75 GAG GAC ACA TTG GTT Glu Asp Thr Leu Val 240 CTC AAG Leu Lys CTT GCT AAT ATA TGT GGT AAA GCT TGT ATG AAG CTA TTG Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu WO 98/26082 WO 9826082PCT/EP97/07012 -164 GAT AGA TGT Asp Arg Cys

AAA

Lys 100 GAG ATT ATT GTC AAG Glu Ile Ile Val Lys 105 TCT AAT GTA OAT Ser Asn Val Asp ATG GTT AGT Met Val Ser 110 336 CTT GAA AAG TCA TTG CCG GAA GAG Leu Glu Lys Ser Leu Pro Olu Giu 115 120 CTT OTT AAA GAG ATA ATT OAT AGA Leu Val Lys Ou Ile Ile Asp Arg 125 CCT AAA GTA AAG AAA CAT GTC TCG Pro Lys Val Lys Lys His Val Ser 140 COT AAA Arg Lys 130 GAG CTT GGT TTG GAG Oiu Leu Oly Leu Oiu 135

AAT

Asn 145 GTA CAT AAO GCA CTT Val His Lys Ala Leu 150 GAC TCG GAT GAT ATT Asp Ser Asp Asp Ile 155 ACC AAT CTA OAT OAT Thr Asn Leu Asp Asp 170 GAG TTA OTC AAO TTG Glu Leu Val Lys Leu 160 000 TOT OCT CTT CAT Ala Cys Ala Leu His 175 CTT TTO AAA GAG OAT Leu Leu Lys Olu Asp 165 480 528 576 624 TTC OCT OTT Phe Ala Val CTT OAT OTT Leu Asp Leu 195 OCA TAT TOO AAT OTO AAO Ala Tyr Cys Asn Val Lys 180 185 0CC OAT OTC AAC CAT AGO Ala Asp Val Asn His Arg 200 ACC GCA ACA OAT CTT TTA AAA Thr Ala Thr Asp Leu Leu LYS 190 AAT CO AGO OGA TAT ACO GTG Asn Pro Arg Gly Tyr Thr Val 205 CTT CAT Leu His 210 GTT OCT 000 ATG CG Val Ala Ala Met Arg 215 AAO GAG CCA CAA TTG ATA CTA TCT CTA Lys Olu Pro Gin Leu Ile Leu Ser Leu 220 672 TTG GAA AAA GOT OCA AOT OCA TCA GAA OCA ACT TTG GAA GGT AGA ACC Leu Glu Lys Oly Ala Ser Ala Ser Giu Ala Thr Leu Olu Gly Arg Thr 720 WO 98/26082 WO 9826082PCTIEP97/07012 165- 240 GCA CTC ATG ATC GCA Ala Leu Met Ile Ala 245 AAA CAA GCC ACT ATb Lys Gin Ala Thr Met 250 GCG GTT GAA Ala Val Giu TGT AAT AAT Cys Asn Asn 255 768 ATC CCG GAG CAA TGC AAG CAT TCT CTC Ile Pro Giu Gin Cys Lys His Ser Leu 260 265 ATA CTA GAG CAA GAA GAG AAA CGA GAA Ile Leu Glu Gin Glu Asp Lys Arg Giu 275 280 AAA GGC CGA CTA TGT GTA GAA Lys Gly Arg Leu Cys Val Giu 270 CAA ATT CCT AGA GAT GTT CCT Gin Ile Pro Arg Asp Val Pro 285 816 864 CCC TCT Pro Ser 290 TTT GCA GTG GCG GCC Phe Ala Val Ala Ala 295 GAT GAA TTG AAG ATG ACG CTG CTC GAT Asp Glu Leu Lys Met Thr Leu Leu Asp 300

CTT

Leu 305 GAA AAT AGA GTT GCA Giu Asn Arg Val Aia 310 CTT GCT CAA CGT CTT Leu Ala Gin Arg Leu 315 TTT CCA ACG GAA GCA Phe Pro Thr Giu Ala 320 CAA GCT GCA ATG GAG Gin Ala Ala Met Giu 325 ATC GCC GAA ATG AAG Ile Ala Giu Met Lys 330 CCT GAG CGT CTC ACT Pro Asp Arg Leu Thr 345 GGA ACA TGT GAG TTC Gly Thr Cys Giu Phe 335

ATA

Ile 960 1008 1056 1104 GTG ACT AGO Val Thr Ser CCG GGT GTA Pro Gly Val 355 AGA CTA AAA Arg Leu Lys GGT ACG AAG AGA ACA TCA Gly Thr Lys Arg Thr Ser 350 AAG ATA GCA OCT TTC LYS Ile Ala Pro Phe 360 AGA ATO CTA GAA GAG CAT CAA AGT Arg Ile Leu Glu Glu His Gin Ser 365 GOG OTT TCT AAA AGO GTG GAA CTC GGG AAA CGA TTC TTC Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe 1152 WO 98/26082-16- 370 375 380 CCG CGC TGT TCG GCA GTG CTC GAC CAG ATT ATG AAC TGT TGA Pro Arg Cys Ser Ala Val Leu Asp Gin Ile Met Asn Cys* 385 390 395 INFORMATION FOR SEQ ID NO:29: 0 SEQUENCE CHARACTERISTICS: LENGTH: 398 amino acids TYPE: amino acid TOPOLOGY: linear PCT/EP97/07012 1194 Met i Arg (i i) (Xi) Asp Sex MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID NO:29: *Val Val 5 Pro Lys Thr Val Leu Ala 10 Cys Vai Tyr Ser Ser Arg Val Pro Pro Gly Val Ser Ala ASP Glu Asn Cys Cys Vai Leu Tyr His Val Ala Leu Aia Phe Cys Arg Pro Ala Val 40 Pro Asp Phe Met Leu Glu Leu Ile Phe Lys Giu Leu Ile Thr Giu Tyr Gin Arg His Leu Leu Asp Vai Val 70 Asp Lys Val Val Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Giy Lys Ala Cys Met Lys Leu Leu 90 WO 98/26082 16- Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val 100 105 PCT/EP97/07012 Asp Met Val. Ser 110 Leu Glu Lys 115 Ser Leu Pro Giu Glu 120 Leu Val Lys Glu Ile 125 Ile Asp Arg Arg Lys 130 Giu Leu Gly Leu Glu Val. Pro Lys Val 135 Lys 140 Lys His Val Ser Asn 145 Val His LYS Ala Asp Ser Asp Asp Glu Leu Val Lys Leu Leu Lys Glu Asp 165 His Thr Asn Leu Asp 170 Asp Ala Cys Ala Leu His 175 Phe Ala Val Leu Asp Leu 195 Ala 180 Tyr Cys Asn Val Lys 185 Thr Ala Thr Asp Leu Leu Lys 190 Tyr Thr Val Ala Asp Val Asn Arg Asn Pro Arg Gly 205 Leu His Val Ala Ala Met 210 Arg 215 Lys Glu Pro Gin Leu 220 Ile Leu Ser Leu Leu 225 Glu Lys Gly Ala Ala Ser Giu Ala Leu Glu Gly Arg Ala Leu Met Ile Ala 245 Lys Gin Ala Thr Met 250 Ala Val Glu Cys Asn Asn 255 Ile Pro Glu Gin Cys Lys His Ser 260 Leu 265 LYS Gly Arg Leu -Cys Val GlU 270 Asp Val Pro Ile Leu Giu 275 Gin Glu Asp Lys Arg 280 Glu Gin Ile Pro Arg 285 WO 98/26082 PCT/EP97/07012 -168- Pro Ser Phe 290 Ala Val Ala Ala 295 Asp Glu Leu Lys Met 300 Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala Gin Arg 305 310 Phe Pro Thr Glu Ala 320 Gin Ala Ala Met Glu 325 Ile Ala Glu Met Lys 330 Gly Thr Cys Glu Phe Ile 335 Val Thr Ser Pro Gly Val 355 Leu 340 Glu Pro Asp Arg Leu 345 Thr Gly Thr Lys Arg Thr Ser 350 His Gin Ser Lys Ile Ala Pro Phe 360 Arg Ile Leu Glu Glu 365 Arg Leu 370 Lys Ala Leu Ser Lys 375 Thr Val Glu Leu Gly 380 Lys Arg Phe Phe Pro 385 Arg Cys Ser Ala Leu Asp Gin Ile Met Asn Cys 395 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 786 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..786 OTHER INFORMATION: /product= "Altered form of NIM1" WO 98/26082-16- /note= "Ankyrin domains of NIMi." (xi) SEQUENCE DESCRIPTION: SEQ ID PCTIEP97/07012

ATG

Met 1 GAC TCC AAC AAC ACC GCC GCC GTG Asp Ser Asn Asn Thr Ala Ala Val 5 AAG CTC GAG CTT AAG GAG ATT Lys Leu Glu Leu Lys 10 Giu Ile GCC AAG GAT TAC Ala Lys Asp Tyr GAA GTC GGT TTC GAT Glu Val Oly PheAsp 25 TCG OTT GTG ACT GTT TTG GCT Ser Val Val Thr Val Leu Ala TAT GTT TAC Tyr Val Tyr AGC AGC AGA OTO AGA CCG CCG CCT AAA GGA GTT TCT GAA Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Olu 40 TGC OCA Cys Ala GAC GAG AAT TGC TGC Asp Glu Asn Cys Cys 55 CAC GTG OCT TOO CGG CCG GCG OTO GAT His Val Ala Cys Arg Pro Ala Val Asp

TTC

Phe ATO TTG GAG GTT CTC Met Leu Glu Val Leu 70 TAT TTG GCT TTC ATC Tyr Leu Ala Phe Ile 75 AOG CAC TTA TTG GAO Arg His Leu Leu Asp 90 TTC AAG ATC CCT GAA Phe Lys Ile Pro Glu GTT GTA GAC AAA GTT Val Val Asp Lys Val 240 288 TTA ATT ACT CTC TAT Leu Ile Thr Leu Tyr GTT ATA GAG GAC ACA TTG GTT ATA CTO Val Ile Olu Asp Thr Leu Val Ile Leu 100 105 AAG CTT GCT AAT ATA TOT GOT Lys Leu Ala Asn Ile Cys Gly 110 AAA GCT Lys Ala TGT ATO Cys Met 115

AAG

Lys CTA TTG GAT AGA TGT Leu Leu Asp Arg Cys AAA GAG Lys Giu

ATT

Ile 125 ATT GTC AAG Ile Val Lys WO 98/26082 PC :T/EP97/07012 -170- TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA GAG CTT 432 Ser

GTT

Val 145 Asn Val Asp Met Val 130 AAA GAG ATA ATT GAT Lys Glu Ile Ile Asp 150 Ser 135 Leu Glu Lys Ser Leu 140 Pro Glu Glu Leu AGA CGT AAA GAG CTT Arg Arg Lys Glu Leu 155 GGT TTG GAG GTA CCT Gly Leu Glu Val Pro 160

AAA

Lys GTA AAG AAA CAT GTC TCG AAT Val Lys Lys His Val Ser Asn 165 GTA CAT Val His 170 AAG GCA CTT Lys Ala Leu GAC TCG GAT Asp Ser Asp 175 GAT ATT GAG TTA Asp Ile Glu Leu 180 GTC AAG TTG CTT TTG AAA GAG GAT CAC ACC AAT CTA Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Leu 185 190 GCT CTT CAT TTC GCT GTT GCA TAT TGC AAT GTG AAG Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val Lys 200 205 GAT GAT GCG Asp Asp Ala 195 ACC GCA Thr Ala 210 ACA GAT CTT TTA AAA Thr Asp Leu Leu Lys 215 CTT GAT CTT GCC GAT GTC AAC CAT AGG Leu Asp Leu Ala Asp Val Asn His Arg 220 672

AAT

Asn 225 CCG AGG GGA TAT ACG Pro Arg Gly Tyr Thr 230 GTG CTT CAT GTT GCT Val Leu His Val Ala 235 GCG ATG CGG AAG GAG Ala Met Arg Lys Glu 240 720

CCA

Pro CAA TTG ATA CTA Gin Leu Ile Leu 245 TCT CTA TTG GAA AAA Ser Leu Leu Glu Lys 250 GGT GCA AGT GCA TCA GAA Gly Ala Ser Ala Ser Glu 255 GCA ACT Ala Thr TTG GAA GGT TGA Leu Glu Gly 260 WO 98/26082 171 -PCTIEP97/07012 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 262 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: Met Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Giu 1 5 10 Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Leu Lys Glu Ile Thr Val Leu Ala Ser Tyr Val Tyr Cys Ala Asp Ser Arg Val 25 Arg Pro 40 His Val Val Ser Glu Glu Asn Cys Cys Tyr Pro Pro Lys Ala Cys Arg Phe Ile Phe Gly Pro Ala Val Asp Phe Leu Leu Glu Val Leu Ala Lys Ile Pro Glu Ile Thr Leu Tyr 85 Ile Glu Asp Thr 100 Arg His Leu Leu 90 Val Val Asp Lys Val Val Leu Val Ile Leu 105 Lys Leu Ala Asn Ile Cys Gly 110 Ile Val Lys LYS Ala Cys Met LYS Leu Leu 115 Asp Arg 120 Cys Lys Glu Ile 125 WO 98/26082 WO 9826082PCT/EP97O7O12 172 Ser Asn 130 Val Asp Met Val Ser 135 Leu Glu Lys Ser Leu 140 Pro Glu Glu Leu Val 145 Lys Glu Ile Ile Asp 150 Arg Arg Lys Glu Leu 1.55 Gly Leu Glu Val Pro 160 Lys Val Lys Lys His 165 Val Ser Asn Val Lys Ala Leu Asp Ser Asp 175 Asp Ile Glu Asp Asp Ala 195 Val Lys Leu Leu Leu 185 Lys Glu Asp His Thr Asn Leu 190 Asn Val Lys Cys Ala Leu His Phe 200 Ala Val Ala Tyr Thr Ala 210 Thr Asp Leu Leu Leu Asp Leu Ala Asp 220 Val Asn His Arg ASn 225 Pro Arg Gly Tyr Thr 230 Val Leu His Val Ala 235 Ala Met Arg Lys Glu 240 Pro Gin Leu Ile Leu 245 Ser Leu Leu Glu Gly Ala Ser Ala Ser Glu 255 Ala Thr Leu Glu Gly 260 INFORMATION FOR SEQ ID NO:32: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single CD) TOPOLOGY: linear WO 98/26082 PCT/EP97/n712 -173- (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: CAACAGCTTC GAAGCCGTCT TTGACGCGCC GGATG INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: CATCCGGCGC GTCAAAGACG GCTTCGAAGC TGTTG INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid

B

WO 98/26082 PCT/IEP.7/n701f -174- DESCRIPTION: /desc "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: GGAATTCAAT GGATTCGGTT GTGACTGTTT TG 32 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID GGAATTCTAC AAATCTGTAT ACCATTGG 28 INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid WO 98/26082 PCT/EPO7/n7n -175- DESCRIPTION: /desc "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: CGGAATTCGA TCTCTTTAAT TTGTGAATTT C 31 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: GGAATTCTCA ACAGTTCATA ATCTGGTCG 29 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid WO 98/26082 PCT/EP97/07012 176 DESCRIPTION: /desc "oligonucleotide' (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: GGAATTCAAT GGACTCCAAC AACACCGCCG C 31 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: GGAATTCTCA ACCTTCCAAA GTTGCTTCTG ATG

Claims (30)

1. A recombinant DNA molecule that encodes an altered from of a NIM1 protein acting as a dominant-negative regulator of the SAR signal transduction pathway wherein said 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; 520mM NaPO 4 pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55 0 C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55 0 C.

2. The DNA molecule according to claim 1, wherein said altered form of the NIM1 protein has alanines instead of serines in amino acid positions corresponding to positions 55 and 59 of SEQ ID NO: 3.

3. The DNA molecule according to claim 2, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO: 23.

4. The DNA molecule according to claim 3, wherein said DNA molecule comprises 20 the nucleotide sequence shown inn SEQ ID NO: 22 and all DNA. The DNA molecule according to claim 1, wherein the altered form of the NIM1 protein is a truncated version of the NIM1 gene product.

6. The DNA molecule according to claim 1, wherein said altered form of the NIM1 protein has an N-terminal truncation of amino acids corresponding approximately to amino acid positions 1-125 of SEQ ID NO: 3.

7. The DNA molecule according to claim 6, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO:

8. The DNA molecule according to claim 7, wherein said DNA molecule comprises AL/ the nucleotide sequence shown in SEQ ID NO: 24. P\OPER\MKR\SPEC"663-98 spe.d26/07 -178-

9. The DNA molecule according to claim 1, wherein said altered form the NIM1 protein has a C-terminal truncation of amino acids corresponding approximately to amino acid positions 522-593 of SEQ ID NO: 3.

10. The DNA molecule according to claim 9, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO: 27.

11. The DNA molecule according to claim 10, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO: 26.

12. The DNA molecule according to claim 1, wherein said altered form of the NIM1 protein has an N-terminal truncation of amino acids corresponding approximately to amino acid positions 1-125 of SEQ ID NO: 2 and a C-terminal truncation of amino acids corresponding approximately to amino acid positions 522-593 of SEQ 15 ID NO: 3.

13. The DNA molecule according to claim 12, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO: 29. 4 20 14. The DNA molecule according to claim 13, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO: 28. The DNA molecule according to claim 1, wherein said altered form of the NIM1 protein consists essentially of ankyrin motifs corresponding approximately to amino acid positions 103-362 of SEQ ID NO: 3.

16. The DNA molecule according to claim 15, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO: 31.

17. The DNA molecule according to claim 16, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:

18. A chimeric gene comprising a promoter active in plants operatively linked to the DNA molecule according to any one of claims 1 to 17. P:OPERtMMR'SPEC663-9&266doc-29M90 -179-

19. A recombinant vector comprising the chimeric gene of claim 18, wherein said vector is capable of being stably transformed into a host cell.

20. A method of activating SAR in a plant, comprising transforming the plant with the recombinant vector of claim 19, wherein said altered form of the NIM1 protein is expressed in said transformed plant and activates SAR in said plant.

21. A method of conferring broad spectrum disease resistance to a plant, comprising transforming the plant with the recombinant vector of claim 19, wherein said altered form of the NIM1 protein is expressed in said transformed plant and confers broad spectrum disease resistance to said plant.

22. A method of conferring a CIM phenotype to a plant, comprising transforming the plant with the recombinant vector of claim 19, wherein said altered form of the NIM1 protein is expressed in said transformed plant and confers a CIM phenotype to said plant. O:oe. o

23. A host cell stably transformed with the vector of claim 19.

24. The host cell of claim 23, which is a plant cell. A plant, plant cells and the descendants thereof comprising the chimeric gene of claim 18 which have a broad spectrum of disease resistance.

26. A plant, plant cells and the descendants thereof, wherein a NIM1 protein as encoded by DNA according to claim 1 and involved in the signal transduction cascade leading to systemic acquired resistance in plants is expressed in said transformed plant at higher levels than in a wild type plant.

27. A plant, plant cells and the descendants thereof of claim 25 or 26, wherein said plant is selected from the group consisting of gymnosperms, monocots, and dicots. P:\OPERMKVRSPEC 5663I-98-266.dcO29/0900 -180-

28. A plant, plant cells and the descendants thereof of claim 25 or 26, wherein said plant is a crop plant.

29. A plant, plant cells and the descendants thereof of claim 25 or 26, wherein said plant is selected from the group consisting of rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, 10 pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane. A method of conferring a CIM phenotype to a plant cell, a plant and the descendants thereof, comprising transforming the plant with the recombinant 15 vector comprising the chimeric gene comprising a promoter active in plants operatively linked to the DNA molecule of claim 1, wherein said vector is capable of being stably transformed into a host wherein said NIM1 protein is expressed in said transformed plant at higher levels than in a wild type plant. *e

31. A method of activating systemic acquired resistance in a plant cell, a plant and the descendants thereof, comprising transforming the plant with the recombinant vector comprising the chimeric gene comprising a promoter active in plants operatively linked to the DNA molecule of claim 1, wherein said vector is capable of being stably transformed into a host, wherein said NIM1 protein is expressed in said transformed plant at higher levels than in a wild type plant.

32. A method of conferring broad spectrum disease resistance to a plant cell, a plant and the descendants thereof, comprising transforming the plant with the recombinant vector comprising the chimeric gene comprising a promoter active in plants operatively linked to the DNA molecule of claim 1, wherein said vector is capable of being stably transformed into a host, wherein said NIM1 protein is expressed in said transformed plant at higher levels than in a wild type plant. OP:UER\MKRSPECS4S63I-98-266,d 29/909 -181

33. Use of a transgenic plant or the descendants thereof comprising a chimeric gene according to claim 18 in an agricultural method.

34. A commercial bag comprising seed of a transgenic plant comprising at least one altered form of a NIM1 protein or a NIM1 protein that is expressed in said transformed plant at higher levels than in a wild type plant together with a suitable carrier in an amount sufficient to act as a dominant-negative regulator of the SAR signal transduction pathway, together with lable instructions for the use thereof for conferring broad spectrum disease resistance to plants.

35. A recombinant DNA molecule according to claim 1, substantially as hereinbefore described with reference to the Examples. 15 DATED this 29th day of September, 2000 NOVARTIS AG by its Patent Attorneys DAVIES COLLISON CAVE