EP0944728A1

EP0944728A1 - METHODS OF USING THE $i(NIM1) GENE TO CONFER DISEASE RESISTANCE IN PLANTS

Info

Publication number: EP0944728A1
Application number: EP97952940A
Authority: EP
Inventors: John Andrew Ryals; Kay Ann Lawton; Scott Joseph Uknes; Henry-York Steiner; Michelle Denise Hunt; Leslie Bethards Friedrich
Original assignee: Novartis AG
Current assignee: Syngenta Participations AG
Priority date: 1996-12-13
Filing date: 1997-12-12
Publication date: 1999-09-29
Also published as: NL1007779A1; JP2001505774A; FR2757875A1; BR9714398A; AU727179B2; WO1998026082A1; AU5663198A; IT1298472B1; NL1007779C2; ITMI972741A1; CA2273189A1

Abstract

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

Description

METHODS OF USING THE 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, 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.Λ , 5-12 (1997), incorporated by reference herein in its entirety; Bi et al., Plant J. 8, 235-245 (1995), incorporated by reference herein in its entirety; Mauch-Mani and Slusarenko, Plant Cell8, 203-212 (1996), incorporated by reference herein in its entirety). These observations indicate that the SAR signal transduction pathway is critical for maintaining plant health.

Conceptually, the SAR response can be divided into two phases. In the initiation phase, a pathogen infection is recognized, and a signal is released that travels through the 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 ai., 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., 1995P/aπt Ce//7, 1691-1701 (1995), incorporated by reference herein in its entirety; Vernooij et al., Plant Cell 6, 959-965 (1994), incorporated by reference herein in its entirety).

Recently, Arabidopsis has emerged as a model system to study SAR (Uknes et al., Plant Cell 4, 645-656 (1992), incorporated by reference herein in its entirety; Uknes et al., Mol. Plant-Microbe Interact. 6, 692-698 (1993), incorporated by reference herein in its entirety; Cameron et al., Plant J. 5, 715-725 (1994), incorporated by reference herein in its entirety; Mauch-Mani and Slusarenko, Mol. Plant-Microbe Interact. 7, 378-383 (1994), incorporated by reference herein in its entirety; Dempsey and Klessig, Bulletin de L'lnstitut Pasteur93, 167-186 (1995), incorporated by reference herein in its entirety). It has been demonstrated that SAR can be activated in Arabidopsis by both pathogens and chemicals, such as SA, 2,6-dichloroisonicotinic acid (INA) and benzo(1 ,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) (Uknes et al., 1992; Vernooij et al., Mol. Plant-Microbe Interact. 8, 228-234 (1995), incorporated by reference herein in its entirety; Lawton et al., Plant J. 10, 71-82 (1996), incorporated by reference herein in its entirety). Following treatment with either INA or BTH or pathogen infection, at least three pathogenesis-related (PR) protein genes, namely, PR-1, PR-2, and PR-5 are coordinately induced concomitant with the onset of resistance (Uknes et al., 1992, 1993). In tobacco, the best characterized species, treatment with a pathogen or an immunization compound induces the expression of at least nine sets of genes (Ward et al., Plant CellZ, 1085-1094 (1991), incorporated by reference herein in its entirety). Transgenic disease-resistant plants have been created by transforming plants with various SAR genes (U.S. 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 /s (lesions simulating disease) mutants and acd2 (accelerated cell death) (Dietrich et al., CellH, 551- 563 (1994), incorporated by reference herein in its entirety; Greenberg et al., Cell 77, 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 /sd 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 /sd mutants and acd2, cim mutants have elevated SA and SAR gene expression and resistance, but in contrast to /sd or acd2, do not display detectable lesions on their leaves, cprl (constitutive expresser of P_R genes) may be a type of dm 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 Cell 6, 1845-1857 (1994), incorporated by reference herein in its entirety).

Mutants have also been isolated that are blocked in SAR signaling, ndrl (non-race- specific disease resistance) is a mutant that allows growth of both Pseudomonas syringae containing various avirulence genes and also normally avirulent isolates of Peronospora parasitica (Century et al., Proc. Natl. Acad.Sci. USA 92, 6597-6601 (1995), incorporated by reference herein in its entirety). Apparently this mutant is blocked early in SAR signaling. nprl (nonexpresser of PR genes) is a mutant that cannot induce expression of the SAR signaling pathway following INA treatment (Cao et al., Plant Cell 6, 1583-1592 (1994), incorporated by reference herein in its entirety), eds (enhanced disease susceptibility) mutants have been isolated based on their ability to support bacterial infection following inoculation of a low bacterial concentration (Glazebrook et al., Genetics 143, 973-982 (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). nim1 (noninducible immunity) is a mutant that supports P. parasitica (i.e., causal agent of downy mildew disease) growth following INA treatment (Delaney et al., 1995; International PCT Application WO 94/16077). Although nim1 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. nim1 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 nim1 and nprl phenotypes, respectively (Ryals et al., Plant Cell 9, 425-439 (1997), incorporated by reference herein in its entirety; Cao et al., Cell 88, 57-63 (1997), incorporated by reference herein in its entirety). The wild-type NIM1 gene product is involved in the signal transduction cascade leading to both SAR and gene- for-gene disease resistance in Arabidopsis (Ryals et al., 1997). Ryals et al., 1997 also report the isolation of five additional alleles of nim1 that show a range of phenotypes from weakly impaired in chemically induced PR-1 gene expression and fungal resistance to very strongly blocked. Transformation of the wild-type NPR1 gene into nprl mutants not only complemented the mutations, restoring the responsiveness of SAR induction with respect to PR-gene expression and disease resistance, but also rendered the transgenic plants more resistant to infection by P. syringae in the absence of SAR induction (Cao et al., 1997).

NF-κB/lκB Signal Transduction Pathways

NF-κB/lκB signaling pathways have been implicated in disease resistance responses in a range of organisms from Drosophila to mammals. In mammals, NF-κB/lκB signal transduction can be induced by a number of different stimuli including exposure of cells to lipopolysaccharide, tumor necrosis factor, interleukin 1 (IL-1), or virus infection (Baeuerle and Baltimore, Ce//87, 13-20 (1996); Baldwin, Annu. Rev. Immunol. 14, 649-681 (1996)). The activated pathway leads to the synthesis of a number of factors involved in inflammation and immune responses, such as IL-2, IL-6, IL-8 and granulocyte/macrophage- colony stimulating factor (deMartin et al., Gene 152, 253-255 (1995)). In transgenic mouse studies, the knock out of NF-κB/lκB signal transduction leads to a defective immune response including enhanced susceptibility to bacterial and viral pathogens (Beg and Baltimore, Science 27 , 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-κB activation in mammalian cells (Kopp and Ghosh, Science 265, 956-959 (1994)), while SA activates signal transduction in

Arabidopsis. Bacterial infection of Drosophila activates 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., Ce//75, 753-763 (1993); Lemaitre et al., Ce//86, 973- 983 (1996)). This induction is dependent on the gene product of dorsal and dif, two NF-κB homologs, and is repressed by cactus, an lκB 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 by this definition is not intended to be exclusive of any other type of plant tissue. Protoplast: a plant cell without a cell wall.

Descendant plant: a sexually or asexually derived future generation plant which includes, but is not limited to, progeny plants. Transgenic plant: a plant having stably incorporated recombinant DNA in its genome.

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

Recombinant DNA technology: Technology which produces recombinant DNA in vitro and transfers the recombinant DNA into cells where it can be expressed or propagated (See, Concise Dictionary of Biomedicine and Molecular Biology, Ed. Juo, CRC Press, Boca Raton (1996)), for example, transfer of DNA into a protoplast(s) or cell(s) in various forms, including, for example, (1) naked DNA in circular, linear or supercoiled forms, (2) DNA contained in nucleosomes or chromosomes or nuclei or parts thereof, (3) DNA complexed or associated with other molecules, (4) DNA enclosed in liposomes, spheroplasts, cells or protoplasts or (5) DNA transferred from organisms other than the host organism (ex. Agrobacterium tumefiaciens). These and other various methods of introducing the recombinant DNA into cells are known in the art and can be used to produce the transgenic cells or transgenic plants of the present invention. Recombinant DNA technology also includes the homologous recombination methods described in Treco et al., WO 94/12650 and Treco et al., WO 95/31560 which can be applied to increasing peroxidase activity in a monocot. Specifically, regulatory regions (ex. promoters) can be introduced into the plant genome to increase the expression of the endogenous peroxidase. Also included as recombinant DNA technology is the insertion of a peroxidase coding sequence lacking selected expression signals into a monocot and assaying the transgenic monocot plant for increased expression of peroxidase due to endogenous control sequences in the monocot. This would result in an increase in copy number of peroxidase coding sequences within the plant. The initial insertion of the recombinant DNA into the genome of the R° plant is not defined as being accomplished by traditional plant breeding methods but rather by technical methods as described herein. Following the initial insertion, transgenic descendants can be propagated using essentially traditional breeding methods.

Chimeric gene: A DNA molecule containing at least two heterologous parts, 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 DNA technology.

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

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

Within the scope of the present invention a DNA molecule is described that encodes the NIM1 protein hybridizing under the following conditions to clone BAC-04, ATCC Deposit No. 97543: hybridization in 1%BSA; 520mM NaPO₄, pH7.2; 7% lauryl sulfate, sodium salt; 1 mM 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 an especially preferred embodiment, the NIM1 gene is comprised within clone BAC-04, ATCC Deposit No. 97543.

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 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 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; 1 mM 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 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.

In an especially preferred embodiment, the NIM1 protein is expressed in a transformed plant at higher levels than in a wild type plant. 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 a NIM1 gene coding sequence, wherein the encoded NIM1 protein is expressed in the transformed plant at higher levels than in a wild type 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 a NIM1 gene coding sequence, wherein the encoded NIM1 protein is expressed in the transformed plant at higher levels than in a wild type 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 a NIM1 gene coding sequence, wherein the encoded NIM1 protein is expressed in the transformed plant at higher levels than in a wild type plant. Another aspect of the present invention exploits both the recognition that the SAR pathway in plants shows functional parallels to the NF-κB/lκB 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 lκB subclass α. Mutations of lκB have been described that act as super-repressors or dominant-negatives of the NF-κB/lκB 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 pathway. These altered forms of NIM1 confer the opposite phenotype in plants transformed therewith as the nim1 mutant; plants i.e., 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 T_m 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 T_m and preferably in the range of about 12-15°C below the calculated melting temperature T_m and in the case of oligonucleotides in the range of about 5-10°C below the melting temperature T_m.

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 NaPθ4, pH7.2; 7% lauryl sulfate, sodium salt; 1 mM 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: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 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 NaPθ4, 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 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.

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 NaPθ4, pH7.2; 7% lauryl sulfate, sodium salt; 1 mM 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: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. 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 N0:28. An exemplary dominant-negative form of the NIM1 protein with a C- terminal 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 NaPθ4, pH7.2; 7% lauryl sulfate, sodium salt; 1 mM 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. 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 NO:30. 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 NO:30; especially preferred are the following conditions: hybridization in 1%BSA; 520mM NaP04, pH7.2; 7% lauryl sulfate, sodium salt; 1 mM 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 wild- type 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 of being stably transformed into a host cell, as well as a host cell stably transformed with such a vector. Preferably, the host cell is a plant, plant cells 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 dominant- negative 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 N0:2: hybridization in 1 %BSA; 520mM NaPO₄, pH7.2; 7% lauryl sulfate, sodium salt; 1 mM 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.

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 55 and 59 of SEQ ID N0:3, wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID N0:22: hybridization in 1%BSA; 520mM NaPθ4, pH7.2; 7% lauryl sulfate, sodium salt; 1 mM 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.

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 NaP04, pH7.2; 7% lauryl sulfate, sodium salt; 1 mM 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.

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 N0:3, wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID N0:26: hybridization in 1%BSA; 520mM NaPθ4, 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.

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 NaPθ4, pH7.2; 7% lauryl sulfate, sodium salt; 1 mM 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.

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 NaPθ4, 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. 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

(a) 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;

(b) 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 (a); and (c) isolating and multiplying a DNA molecule comprising a DNA portion containing a

DNA portion encoding an altered form of a NIM1 protein. A method of isolating a DNA molecule comprising a DNA portion containing an altered form of a NIM1 sequence comprising

(a) 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;

(b) 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 (a); and

(c) 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 descendant plants with known chemical inducers (e.g. 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 (i.e. non- mutant) 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 (e.g. crop plant species) facilitates efficacious fungicide screening procedures for compounds against important pathogens of crop plants.

The Arabidopsis thaliana nim1 Mutant

An Arabidopsis thaliana mutant called nim1 (noninducible immunity) that supports P. parasitica (i.e., causal agent of downy mildew disease) growth following INA treatment is described in Delaney et al., 1995. Although nim1 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. nim1 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 nim1 mutant (herein designated nim1-1) 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 nim1-2, nim1-3, nim1-4, nim1-5, and nim1-6) were isolated from 280,000 M₂ 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-i plants were then scored for their ability to support fungal growth following INA treatment. As shown in Table 3 of the Examples, all nim1-1, nim1-2, nim1-3, nim1-4, and nim1-6 F^ plants were phenotypically wild type, indicating a recessive mutation in each line. nim1-5 showed the nim phenotype in all 35 F-i plants, indicating that this particular mutant is dominant. For verification, the reciprocal cross was carried out using nim1 -5 as the pollen donor to fertilize Ws-0 plants. In this case, all 18 F, plants were phenotypically nim, confirming the dominance of the nim1-5 mutation.

To determine whether the nim 1-2 through nim1-6 mutations were allelic to the previously characterized nim1- 1 mutation, pollen from nim1-1 was used to fertilize nim1-2 through nim1-6. Because nim1-1 carried resistance to kanamycin, F-i descendants were identified by antibiotic resistance. In all cases, the kanamycin-resistant F^ plants were nim, indicating they were all allelic to the nim1-1 mutant. Because the nim1-5 mutant is dominant and apparently homozygous for the mutation, it was necessary to analyze nim1-1 complementation in the F₂ generation. If nim1-1 and nim1-5 were allelic, then the expectation would be that all F₂ plants have a nim phenotype. If not, then 13 of 16 F₂ 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 nim 1 -5 carries a point mutation in the NIM1 gene (infra), it is considered to be a nim1 allele. To determine the relative strength of the different nim1 alleles, each mutant was analyzed for the growth of P. parasitica under normal growth conditions and following pretreatment with either SA, INA, or BTH. As shown in Table 1 , during normal growth, nim1-1, nim1-2, nim1-3, nim1-4, and nim1-6 a\\ supported approximately the same rate of fungal growth, which was somewhat faster than the Ws-0 control. The exception was the n/m7-5 plants, in which fungal growth was delayed by several days relative to both the other nim1 mutants and the Ws-0 control, but eventually all of the nim1-5 plants succumbed to the fungus. Following SA treatment, the mutants could be grouped into three classes: nim1-4 and nim1-6 showed a relatively rapid fungal growth; nim1-1, nim1-2, nim1-3 p\an\s exhibited a somewhat slower rate of fungal growth; and fungal growth in /wn 7 -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 nim1-4 was the most severely compromised in its ability to restrict fungal growth following chemical treatment; nim1-1, nim1-2, nim1-3, and nim1-6were 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 nim1-4 being the most severely compromised, nim1-1, nim1-2, nim1-3 and nim 1-6 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 nim1 alleles. RNA was extracted from plants 3 days after either water or chemical treatment, or 14 days after inoculation with a compatible fungus (P. 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 nim1-1, nim1-2, and nim 1-3 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 nim1-4 and nim 1-6 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 nim1 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 (nim1-4 and nim1-6); moderately compromised alleles (nim1-1, nim1-2, and nim1-3); and a weakly compromised allele (nim1-5). Fine Structure Mapping of the nim1 Mutation

To determine a rough map position for NIM1, 74 F₂ nim phenotype plants from a cross between nim 1-1 (Ws-0) and Landsberg erecta (Let) 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), nim1 was found to lie about 8.2 centimorgans (cM) from nga128 and 8.2 cM from ngal 11 on the lower arm of chromosome 1. In subsequent analysis, nim1-1 was found to lie between ngal 1 1 and about 4 cM from the SSLP marker ATHGENEA. For fine structure mapping, 1138 nim plants from an F₂ population derived from a cross between nim1-1 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 n/tτ77, giving a genetic distance of approximately 4.1 cM (93 of 2276), and 239 recombinant chromosomes were identified between nga111 and nim1, 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 nim1) and W85.1 (0.6 cM from nim1) 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 recombinant. Because these clones overlap to form a large contig (>100 kb), and include AFLP markers that flank nim1, 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 nim1 was located between L84.Y1 and L84.8, representing a gap of about 0.09 cM.

A cosmid library was constructed in the Agrobacterium-compa ib\e 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 nim1-1, and transformants were evaluated for their ability to complement the mutant phenotype. Cosmids D5, E1 , and D7 were all found to complement nim1-1, 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 nim1 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 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 nimi 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 nim 1-6 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 nim 1 -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 nim1-2, there is a cytidine-to-thymidine transition at position 3763 that changes a histidine to a tyrosine. In nim1-3, 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 nim1-4 and nim 1-5 have a guanosine-to-adenosine transition at position 4160 changing an arginine to a lysine, and in nim1-6, 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 t7/^'t777-5 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, 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. 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 NaP0₄, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride) at 55°C for 18-24h] with ³ P-radiolabelled >4rab/dops s t/7a//aπa NIM1 cDNA. Following hybridization the blots were washed under low stringency conditions [6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C; 1XSSC is 0.15M NaCI, 15mM Na-citrate (pH7.0)] and exposed to X-ray film to visualize bands that correspond to NIML

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 (e.g., 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. 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 lκBα

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 nimi gene, which gives the host plants extreme sensitivity to a wide variety of pathogens and renders them unable to respond to pathogens and chemical inducers of SAR. The wildtype NIM1 gene of Arapidopsis has been mapped and sequenced (SEQ ID NO: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 disease- resistance 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-κB and lκB (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 IKBCC. Despite the failings of the computer programs, pair-wise visual inspections between the NIM1 protein (SEQ ID NO:3) and 70 known ankyrin-containing proteins were carried out, and striking similarities were found to members of the lκBα 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 lκBα proteins from mouse, rat, and pig (SEQ ID NOs: 18, 19, and 20, respectively). NIM1 contains several important structural domains of lκBα throughout the entire length of the protein, including ankyrin domains (indicated by the dashed underscoring in Figure 9), 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 lκBα share identity at 30% of the residues and conservative replacements at 50% of the residues. Thus, there is homology between lκBα and NIM1 throughout the proteins, with an overall similarity of 80%.

One way in which lκBα protein functions in signal transduction is by binding to the cytosolic transcription factor NF-κB and preventing it from entering the nucleus and altering transcription of target genes (Baeuerle and Baltimore, 1996; Baldwin, 1996). The target genes of NF-κB 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, lκBα is phosphorylated at two serine residues (amino acids 32 and 36 of Mouse IKBOC). This programs ubiquitination at a double lysine (amino acids 21 and 22 of Mouse IKBCC). Following ubiquitination, the NF-κB/lκB complex is routed through the proteosome where lκBα is degraded and NF-κB is released to the nucleus. The phosphorylated serine residues important in lκBα function are conserved in NIM1 within a large contiguous block of conserved sequence from amino acids 35 to 84 (Figure 9). In contrast to lκBα, 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 lκBα 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 IKBCC function, NIM1 is expected to function like the IKBCC, 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-κB homolog is kept out of the nucleus, and SAR gene expression and resistance responses are allowed to occur. In the nimi mutant plants a non-functional NIM1 gene product is present. Therefore, in accordance with this model, the NF-κB 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-κB 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., EMB0 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 2.7 A: 784-787 (1996)). These mutant forms of IKB bind to NF-κB but are not phosphorylated or ubiquitinated and therefore are not degraded. NF-κB 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 nimi 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. Phosphorylation of serine residues in human lκBα is required for stimulus activated degradation of lκBα thereby activating NF-κB. Mutagenesis of the serine residues (S32 and S36) in human lκBα to alanine residues inhibits stimulus-induced phosphorylation, thus blocking lκBα proteosome-mediated degradation (Traenckner et al., 1995; Brown et al., 1996; Brockman et al., 1995; Wang et al., 1996). This altered form of lκBα can function as a dominant-negative form by retaining NF-κB 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 lκBα. 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 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 261 -317 of human IkBa may result in enhanced intrinsic stability by blocking constitutive phosphorylation of serine and threonine residues in the C- terminus. This altered form of lκBα 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 NIML 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 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. 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 tabacr^', insects such as aphids, e.g. Myzus persicae; and lepidoptera, e.g., Heliothus spp.; and nematodes, e.g., 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. 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 (i.e., 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 λgtH , λgtIO 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 expression 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, i.e., 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. 2£7): 1099-1104 (1983)), and the EPSPS gene, which confers resistance to glyphosate (U.S. 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 Accl fragment from pUC4K carrying an NPTII (Messing & Vierra, Gene 19: 259-268 (1982): Bevan et al., Nature 304: 184-187 (1983): McBride et al., Plant Molecular Biology 14: 266-276 (1990)). Xhol linkers are ligated to the Ecσ/Wfragment 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 Sa/7-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, Bell, 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 >4gro.3acter/^'/vt77-mediated transformation, the RK2-derived trfA function for mobilization between E. coli and other hosts, and the Or/Tand Or/Vfunctions 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. coli and 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 (e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. a. pCIB3064: pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques in combination with selection by the herbicide basta (or phosphinothricin). The plasmid pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator and is described in the PCT published application WO 93/07278. The 35S promoter of this vector contains two ATG sequences 5' of the start site. These sites are mutated using standard PCR techniques in such a way as to remove the ATGs and generate the restriction sites Sspl and Pvull. The new restriction sites are 96 and 37 bp away from the unique Sail site and 101 and 42 bp away from the actual start site. The resultant derivative of pCIB246 is designated pCIB3025. The GUS gene is then excised from pCIB3025 by digestion with Sail and 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 Smal fragment containing the bat-gene from Streptomyces viridochromogenes is excised and inserted into the Hpal site of pCIB3060 (Thompson et al. EMBO J 6: 2519-2523 (1987)). This generated pCIB3064, which comprises the bargene under the control of the CaMV 35S promoter and terminator for herbicide selection, a gene for ampicillin resistance (for selection in E. coli) and a polylinker with the unique sites Sphl, Pstl, Hindlll, and BamHI. This vector is suitable for the cloning of plant expression cassettes containing their own regulatory signals.

b. pSOG19 and pSOG35: pSOG35 is a transformation vector which utilizes the E. coli 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 Adh1 gene (-550 bp) and 18 bp of the GUS untranslated leader sequence from pSOGIO. A 250-bp fragment encoding the E. coli dihydrofolate reductase type II gene is also amplified by PCR and these two PCR fragments are assembled with a Sacl-Pstl fragment from pB1221 (Clontech) which comprises the pUC19 vector backbone and the nopaline synthase terminator. Assembly of these fragments generates pSOG19 which contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene and the nopaline synthase terminator. Replacement of the GUS leader in pSOG19 with the leader sequence from Maize Chlorotic Mottle Virus (MCMV) generates the vector pSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistance and have Hindlll, Sphl, Pstl and EcoRI sites available for the cloning of foreign substances. 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 tm/transcriptional 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 Xhol sites in addition to the existing EcoRI site. This derivative is designated pCGN1761 ENX. pCGN1761 ENX is useful for the cloning of cDNA sequences or gene sequences (including microbial ORF sequences) within its polylinker for the purpose of their expression under the control of the 35S promoter in transgenic plants. The entire 35S promoter-gene sequence-tm/ terminator cassette of such a construction can be excised by Hindlll, Sphl, Sail, and Xbal sites 5' to the promoter and Xbal, BamHI and 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 pCGN1761 ENX 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. pCGN1761 ENX 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 pCGN1761 ENX/Sph-. pCGN1761 ENX/Sph- is cleaved with EcoRI, and ligated to an annealed molecular adaptor of the sequence 5'-AATTCTAAAGCATGCCGATCGG-375'- AATTCCGATCGGCATGCTTTA-3' (SEQ ID NO's: 12 and 13). This generates the vector pCGNSENX, which incorporates the uasAoptimized 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 Xhol sites are retained. An alternative vector is constructed which utilizes an Ncol site at the initiating ATG.

This vector, designated pCGN1761 NENX 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 pCGN1761 ENX EcoRI site. Thus the vector includes the qivasAoptimized sequence TAAACC adjacent to the initiating ATG which is within the Ncol site. Downstream sites are EcoRI, Notl, and Xhol. Prior to this manipulation, however, the two Ncol sites in the pCGN1761 ENX 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 pCGN1761 ENX 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 35S 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-1a 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 pCGN1761 ENX (Uknes et al. 1992). pCIB1004 is cleaved with Ncol and the resultant 3' overhang of the linearized fragment is rendered blunt by treatment with T4 DNA polymerase. The fragment is then cleaved with Hindlll and the resultant PR-la-promoter- containing fragment is gel purified and cloned into pCGN1761 ENX from which the double 35S promoter has been removed. This is done by cleavage with Xhol and 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 pCGN1761 ENX derivative with the PR-1a promoter and the tml terminator and an intervening polylinker with unique EcoRI and Notl sites. Selected NIM1 genes can be inserted into this vector, and the fusion products (i.e. 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 ,2,3- thiadiazolecarboxylic acid, benzo-1 ,2,3-thiadiazolethiocarboxylic acid, cyanobenzo-1 ,2,3- thiadiazole, 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-7- thiocarboxylic 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-7- carboxylate, n-propyl benzo-1 ,2,3-thiadiazole-7-carboxyiate, 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 (SA), 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 haioisonicotinic 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. 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 >Act/gene 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 Actl-^'mtron 1 , Adhlδ' flanking sequence and >4d 7/-intron 1 (from the maize alcohol dehydrogenase gene) and sequence from the CaMV 35S promoter. Vectors showing highest expression were fusions of 35S and Actl intron or the Actl 5' flanking sequence and the Actl intron. Optimization of sequences around the initiating ATG (of the GUS reporter gene) also enhanced expression. The promoter expression cassettes described by McElroy et al. (Mol. Gen. Genet. 23J.: 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 (e.g. sunflower - Binet et al. Plant Science 79: 87-94 (1991) and maize - Christensen et al. Plant Molec. Biol. 12: 619-632 (1989)). The maize ubiquitin promoter has been developed in transgenic 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 (pAHC25) 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 pAHC25 or any of the transformation vectors described in this application, modified by the introduction of the appropriate ubiquitin promoter and/or intron sequences. f. 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 pCGN1761 ENX 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-lnducible Promoters:

Wound-inducible promoters may also be suitable for expression of NIM1 genes of the invention. Numerous such promoters have been described (e.g. Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22: 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 wunl gene. Xu et al. show that a wound-inducible promoter from the dicotyledon potato (pin2) is active in the monocotyledon rice. Further, Rohrmeier & Lehle describe the cloning of the maize Wipl cDNA which is wound induced and which can be used to isolate the cognate promoter using standard techniques. Similar, Firek et al. and Warner etal. 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 35S 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. i. Leaf-Specific Expression:

A maize gene encoding phosphoenol carboxylase (PEPC) has been described by Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)). Using standard molecular 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 (J. 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 Tsp509l 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-Sphl fragment 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 Tsp509l-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 (e.g. 35S, PR-1a, 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' Tsp509l 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. 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 pCGN1761 ENX/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 rbcSSA 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 Notl and Xhol sites of pCGN1761 ENX. 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 a/// site at the amplified ATG, which following restriction enzyme cleavage with the appropriate enzyme is ligated into Sp/V-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 pCGN1761 ENX with the BamHI-Sphl fragment 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 Pstl and 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 terminus blunt. The promoter-transit peptide fragment thus generated is cloned into the prepared pCGN1761 ENX vector, generating a construction comprising the rbcS-δA 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 Xhol cloning sites. This construction is designated pCGN1761 rbcS/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 tml terminator, 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 Adhl gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells. Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et 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 effective in enhancing expression (e.g. 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 (e.g. 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 (e.g. 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 cleavage site and the transgene ATG or, alternatively, replacement of some amino acids within the transgene sequence. Fusions constructed for chloroplast import can be tested for efficacy of chloroplast uptake by in vitro translation of in vitro transcribed constructions followed by in vitro chloroplast uptake using techniques described by Bartlett et al. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier pp 1081-1091 (1982) and Wasmann etal. 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. APPI. Genet. 75: 30-36; Klein et al., (1987) Nature 327: 70-73; Howell et al., (1980) Science 208:1265; Horsch et al., M 985) 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, J., Barnason, A., and Horsch, R.B. Transformation of Brassica napus with Agrobacterium tumefaciens based vectors. Pl.Cell Rep. 6:321-325, 1987; Block, M.d. Genotype independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens. Theor.appi. genet. 76:767-774, 1988; Deblock, M., Brouwer, D.D., 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, T.J., Skene, K.G.M., Cain, P.A., and Scott, N.S. Transgenic grapevines: regeneration of shoots expressing beta-glucuronidase. Pl.Cell Rep. 41 :1045-1049, 1990; Hinchee, M.A.W., Newell, C.A., ConnorWard, D.V., Armstrong, T.A., Deaton, W.R., Sato, S.S., and Rozman, R.J. Transformation and regeneration of non-solanaceous crop plants. Stadler.Genet.Symp. 203212.203-212, 1990; Barfield, D.G. and Pua, E.C. Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation. Pl.Cell Rep. 10:308-314, 1991 ; Cousins, Y.L., Lyon, B.R., and Llewellyn, D.J. Transformation of an Australian cotton cultivar: prospects for cotton improvement through genetic engineering. Aust. J. Plant Physiol. 18:481 -494. 1991 ; Chee, P.P. and Slightom, J.L. Transformation of Cucumber Tissues by Microprojectile Bombardment Identification of Plants Containing Functional and Nonfunctional Transferred Genes. GENE 118:255-260, 1992; Christou, P., Ford, T.L., and Kofron, M. The development of a variety-independent gene-transfer method for rice. Trends.Biotechnol. 10:239-246, 1992; D'Halluin, K., Bossut, M., Bonne, E., Mazur, B., Leemans, J., and Botterman, J. Transformation of sugarbeet (Beta vulgaris L.) and evaluation of herbicide resistance in transgenic plants. Bio/Technol. 0:309-314. 1992; Dhir, S.K., Dhir, S., Savka, M.A., Belanger, F., Kriz, A.L., Farrand, S.K., and Widholm, J.M. Regeneration of Transgenic Soybean (Glycine Max) Plants from Electroporated Protoplasts. Plant Physiol 99:81-88, 1992; Ha, S.B., Wu, F.S., and Thome, T. K. Transgenic turf-type tall fescue (Festuca arundinacea Schreb.) plants regenerated from protoplasts. PI. 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, A.M., Kononowicz, A.K., Zehr, U.B., Tomes, D.T., Axtell, J.D., Butler, L.G., Bressan, R.A., and Hasegawa, P.M. Transgenic Sorghum Plants via Microprojectile Bombardment. Proc Natacad Sci USA 90:11 '21 '2-11216, 1993; Christou, P. Philosophy and Practice of

Variety Independent Gene Transfer into Recalcitrant Crops. In Vitro Cell Dev Biol-Plant 2SP: 119-124, 1993; Damiani, F., Nenz, E., Paolocci, F., and Arcioni, S. Introduction of Hygromycin Resistance in Lotus spp Through Agrobacterium Rhizogenes Transformation. Transgenic Res 2:330-335, 1993; Davies, D.R., Hamilton, J., and Mullineaux, P. Transformation of Peas. Pl.Cell Rep. 12:180-183, 1993; Dong, J.Z. and Mchughen, A.

Transgenic Flax Plants from Agrobacterium Mediated Transformation Incidence of Chimeric Regenerants and Inheritance of Transgenic Plants. Plant Sci 91 :139-148, 1993; Fitch, M.M.M., Manshardt, R.M., Gonsalves, D., and Slightom, J.L. Transgenic Papaya Plants from Agrobacterium Mediated Transformation of Somatic Embryos. Pl.Cell Rep. 12:245- 249, 1993; Franklin, C.I. and Trieu, T.N. Transformation of the Forage Grass Caucasian Bluestem via Biolistic Bombardment Mediated DNA Transfer. Plant Physiol 102:167, 1993; Golovkin, M.V., Abraham, M., Morocz, S., Bottka, S., Feher, A., and Dudits, D. Production of Transgenic Maize Plants by Direct DNA Uptake into Embryogenic Protoplasts. Plant Sci 90:41-52, 1993; Guo, G.Q., Xu, Z.H., Wei, Z.M., and Chen, H.M. Transgenic Plants Obtained from Wheat Protoplasts Transformed by Peg Mediated Direct Gene Transfer. Chin Sci Bull 38:2072-2078. 1993; Asano, Y. and Ugaki, M. Transgenic plants of Agrostis alba obtained by electroporationmediated direct gene transfer into protoplasts. Pl.Cell Rep. 13, 1994; Ayres, N.M. and Park, W.D. Genetic Transformation of Rice. Crit Rev Plant Sci 13:219-239, 1994; Barcelo, P., Hagel, C, Becker, D., Martin, A., and Lorz, H. Transgenic Cereal (Tritordeum) Plants Obtained at High Efficiency by Microprojectile Bombardment of Inflorescence Tissue. PLANT J 5:583-592, 1994; Becker, D., Brettschneider, R., and Lorz, H. Fertile Transgenic Wheat from Microprojectile Bombardment of Scutellar Tissue. Plant J 5:299-307, 1994; Biswas, G.C.G., Iglesias, V.A., Datta, S.K., and Potrykus, I. Transgenic Indica Rice (Oryza Sativa L) Plants Obtained by Direct Gene Transfer to Protoplasts. J Biotechnol 32: 1-10, 1994; Borkowska, M., Kleczkowski, K., Klos, B., Jakubiec, J., and Wielgat, B. Transformation of Diploid Potato with an Agrobacterium Tumefaciens Binary Vector System .1. Methodological Approach. Ada Physiol Plant 16:225-230, 1994; Brar, G.S., Cohen, B.A., Vick, C.L., 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, M.C., Pautot, V., and Chupeau, Y. Recovery of Transgenic Trees After Electroporation of Poplar Protoplasts. Transgenic Res 3: 13-19, 1994; Eapen, S. and George, L. Agrobacterium Tumefaciens Mediated Gene Transfer in Peanut (Arachis Hypogaea L). Pl.Cell Rep. 13:582-586, 1994; Hartman, C.L., Lee, L., Day, P.R., and Turner, N.E. Herbicide Resistant Turfgrass (Agrostis Palustris Huds) by Biolistic Transformation. Bid-Technology 12:919923, 1994; Howe, G.T., Goldfarb, B., and Strauss, S.H. Agrobacterium Mediated Transformation of Hybrid Poplar Suspension Cultures and Regeneration of Transformed Plants. Plant Cell Tissue & Orgart Culture 36:59-71 , 1994; Konwar, B.K. Agrobacterium Tumefaciens Mediated Genetic Transformation of Sugar Beet (Beta Vulgaris L). J Plantbiochem Biotechnol 3:37 '-41 , 1994; Ritala, A., Aspegren, K., Kurten, U., Salmenkalliomarttila, M., Mannonen, L., Hannus, R., Kauppinen, V., Teeri, T.H., and Enari, T.M. Fertile Transgenic Barley by Particle Bombardment of Immature Embryos. Plant Mol Biol 24:317-325, 1994; Scorza, R., Cordts, J.M., Ramming, D.W., and Emershad, R.L. Transformation of Grape (Vitis Vinifera L) Somatic Embryos and Regeneration of Transgenic Plants. J Cell Biochem :102, 1994; Shimamoto, K. Gene Expression in Transgenic Monocots. Curr Opinbiotechnol 5:158-162, 1994; Spangenberg, G., Wang, Z.Y., Nagel, J., and Potrykus, I. Protoplast Culture and Generation of Transgenic Plants in Red Fescue (Festuca Rubra L). Plant Sci 97:83-94, 1994; Spangenberg, G., Wang, Z.Y., Nagel, J., and Potrykus, I. Gene Transfer and Regeneration of Transgenic Plants in Forage Grasses. J Cell Biochem :102, 1994; Wan, Y.C. and Lemaux, P.G. Generation of Large Numbers of Independently Transformed Fertile Barley Plants. Plant Physiol 104:3748, 1994; Weeks, J.T., Anderson, O.D., and BlechI, A.E. Stable Transformation of Wheat (Triticum Aestivum L) by Microprojectile Bombardment. J Cell Biochem :104, 1994; Ye, X.J., Brown, S.K., Scorza, R., Cordts, J., and Sanford, J.C. Genetic Transformation of Peach Tissues by Particle Bombardment. Jamer Sochortsci 119:367-373, 1994; Spangenberg, G., Wang, Z.Y., Nagel, J., and Potrykus, I. Protoplast Culture And Generation Of Transgenic Plants In Red Fescue (Festuca Rubra L). Plant

Science 1994 97:83-94, 1995. See also, U.S. Patent No. 5,639,949, 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 (e.g., 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.

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

Agrobacterium-medlated 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), US 4,795,855 (poplar)). Agrobacterium transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest (e.g. pCIB200 or pCIB2001) to an appropriate Agrobacterium strain which may depend of the complement of rgenes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)). The transfer of the recombinant binary vector to Agrobacterium is accomplished by a triparental mating procedure using E. coli carrying the recombinant binary vector, a helper E. coli strain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Hόfgen & 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 (e.g., 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 (i.e. co- transformation) and both these techniques are suitable for use with this invention. Co- transformation may have the advantage of avoiding complete vector construction and of generating transgenic plants with unlinked loci for the gene of interest and the selectable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable. However, a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al. Biotechnology 4: 1093-1096 (1986)). Patent Applications EP 0 292 435 ([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 H: 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-1 OOOHe 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 /nd/ca-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 10: 667-674 (1992)) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al. (Biotechnology 11: 1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102: 1077-1084 (1993)) using particle bombardment of immature embryos and immature embryo-derived callus. A preferred technique for wheat transformation, however, involves the transformation of wheat by particle bombardment of immature embryos and includes either a high sucrose or a high maltose step prior to gene delivery. Prior to bombardment, any number of embryos (0.75-1 mm in length) are plated onto MS medium with 3% sucrose (Murashiga & Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/l 2,4-D for induction of somatic embryos, which is allowed to proceed in the dark. On the chosen day of bombardment, embryos are removed from the induction medium and placed onto the osmoticum (i.e. induction medium with sucrose or maltose added at the desired concentration, typically 15%). 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 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.

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. (Ed.) American Society of Agronomy Madison, Wisconsin (1983); Mayo O., The Theory of Plant Breeding. Second Edition, Clarendon Press, Oxford (1987); Singh, D.P., Breeding for Resistance to Diseases and Insect Pests. Springer-Veriag, 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 and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc. Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical or biochemical means. Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines. Thus, the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines 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 seedbome 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 application- promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests. The protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods of application are also possible such as treatment directed at the buds or the fruit.

It is a further aspect of the present invention to provide new agricultural methods such as the methods examplified above which are characterized by the use of transgenic plants, transgenic plant material, or transgenic seed according to the present invention. The seeds may be provided in a bag, container or vessel comprised of a suitable packaging material, the bag or container capable of being closed to contain seeds. The bag, container or vessel may be designed for either short term or long term storage, or both, of the seed. Examples of a suitable packaging material include paper, such as kraft paper, rigid or pliable plastic or other polymeric material, glass or metal. Desirably the bag, container, or vessel is comprised of a plurality of layers of packaging materials, of the same or differing type. In one embodiment the bag, container or vessel is provided so as to exclude or limit water and moisture from contacting the seed. In one example, the bag, container or vessel is sealed, for example heat sealed, to prevent water or moisture from entering. In another embodiment water absorbent materials are placed between or adjacent to packaging material layers. In yet another embodiment the bag, container or vessel, or packaging material of which it is comprised is treated to limit, suppress or prevent disease, contamination or other adverse affects of storage or transport of the seed. An example of such treatment is sterilization, for example by chemical means or by exposure to radiation. Comprised by the present invention is a commercial bag comprising seed of a transgenic plant comprising 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 20- 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; 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 H₂0. Six individual plants are evaluated at each time point. Pseudomonas tabaci infected plants are rated on a 5 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. 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 nimi plants. Water, SA, INA, or BTH is applied to wild type (WT) and t7/^'m7 plants. After 3 days, RNA is prepared from these plants and examined for expression of PR-1 , PR-2, and PR-5.

FIGURE 2 depicts PR-1 gene expression in pathogen-infected Ws-O and nimi plants. Pathogen induction of PR-1 is diminished in nimi plants. Wild type (WT) and nimi 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 t7/^'tτ7 mutants and wild-type plants after pathogen infection or chemical treatment. Plants containing nimi alleles π/tn - 1, -2, -3, -4, -5, and -6 and Ws-0 (Ws) were treated with water (C), 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 etal., 1992).

FIGURE 4 shows the levels of SA accumulation in Ws-0 and nimi plants infected with P. syringae. nimi plants accumulate SNA following pathogen exposure. Leaves of wild type and nimi plantsare infiltrated with Pst DC3000(avrRpt2) or carrier medium (10 mM MgCI₂) alone. After 2 days, samples were collected from untreated, MgCI₂-treated, and DC3000(awflpf2)-treated plants. Bacteria-treated samples were separated into primary (infiltrated) and secondary (noninfiltrated) leaves. Free SA and total SA following hydrolysis with β-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. (A) Map position of NIM1 on chromosome 1. The total number of gametes scored is 2276.

(B) Yeast artificial chromosome (striped), bacterial artificial chromosome (BAC), and P1 clones used to clone NIML

(C) Cosmid clones that cover the NIM1 locus. The three cosmids that complement nim1-1 are shown as thicker lines. (D) 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 nimL 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 lκBα from mouse, rat, and pig. Vertical bars (I) above the sequences indicate amino acid identity between NIM1 and the lκBα sequences (matrix score equals 1.5); double dots (:) above the sequences indicate a similarity score >0.5; single dots (.) above the sequences indicate a similarity score <0.5 but >0.0; and a score <0.0 indicates no similarity and has no indicia above the sequences (see Examples). Locations of the mammalian IKBCC 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 lκBα proteins. The five ankyrin repeats in lκBα 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. 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 LISTING

SEQ ID NO: 1 - 9919-bp genomic sequence of NIM1 gene region 2 in Rgure 5D .

SEQ ID NO: 2 - 5655-bp genomic sequence in Rgure 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 eds 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 Rgure 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 Rgure 8. SEQ ID NO: 8 - Rice-3 AA sequence 33-155 from Rgure 8.

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

SEQ ID NO: 10 - Rioe-4 AA sequence 33-155 from Rgure 8.

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

SEQ ID NO: 12 - OligonucleotJde. SEQ ID NO: 13 - Oiigonucleotide.

SEQ ID NO: 14 - OligonucleotJde.

SEQ ID NO: 15 - OligonucleotJde.

SEQ ID NO: 16 - OligonucleotJde.

SEQ ID NO: 17 - Oiigonucleotide. SEQ ID NO: 18 is the mouse lκBα amino acid sequence from Figure 8.

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

SEQ ID NO: 20 is the pig IKBCC 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. 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 C- terminal 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 NIML SEQ ID NOs:32 through 39 are oiigonucleotide primers.

Definitions

acd. accelerated cell death mutant plant

AFLP: Amplified Fragment Length Polymorphism avrRpt2: avirulence gene Rpt2, isolated from Pseudomonas syringae

BAC: Bacterial Artificial Chromosome

BTH: benzo(1 ,2,3)thiadiazole-7-carbothioic acid S-methyl ester

CIM: Constitutive IMmunity phenotype (SAR is constitutively activated) cim: constitutive immunity mutant plant cM: centimorgans cpr7: constitutive expresser of PR genes mutant plant

Col-O: Arabidopsis ecotype Columbia

ECs: Enzyme combinations

Emwa: Peronospora parasitica isolate compatible in the Ws-0 ecotype of

Arabidopsis

EMS: ethyl methane sulfonate

INA: 2,6-dichloroisonicotinic acid

Ler: Arabidopsis ecotype Landsberg erecta

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

NahG: Arabidopsis line transformed with nahG gene ndr. non-race-specific disease resistance mutant plant nim: non-inducible immunity mutant plant

NIM1: the wild type gene, involved in the SAR signal transduction cascade

NIM1 : Protein encoded by the wild type NIM1 gene nimi: mutant allele of NIM1, conferring disease susceptibility to the plant; also refers to mutant Arabidopsis thaliana plants having the nimi mutant allele of

NIM1

Noco: Peronospora parasitica isolate compatible in the Col-0 ecotype of

Arabidopsis

ORF: open reading frame PCs: Primer combinations

PR: Pathogenesis Related

SA: salicylic acid

SAR: Systemic Acquired Resistance

SSLP: Simple Sequence Length Polymorphism UDS: Universal Disease Susceptible phenotype

Wela: Peronospora parasitica isolate compatible in the Weiningen ecotype of

Arabidopsis

Ws-O: Arabidopsis ecotype Issilewskija

WT: wild type YAC: Yeast Artificial Chromosome

EXAMPLES

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

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, etal., Molecular Cloning, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and by TJ. 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 π/t77 Mutants

Example 1: Plant Lines and Fungal Strains

Arabidopsis thaliana ecotype Isilewskija (Ws-O; stock number CS 2360) and fourth- generation (T₄) 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-0 plants were obtained from Lehle Seeds (Round Rock, TX).

Pseudomonas syringae pv. Tomato (Pst) strain DC3000 containing the cloned avrRpt2 gene [DC3000(awPpf2)] was obtained from B. Staskawicz, University of California, Berkeley. P. parasitica pathovars and their sources were as follows: Emwa from E. Holub and I.R. Crute, Horticultural Research Station, East Mailing, Kent; Wela from A. Slusarenko and B. Mauch-Mani, Institut fur Pflanzenbiologie, 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. Example 2: Mutant Screens

M₂ or T₄ 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°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, 75 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 (nim1-1) was isolated from the T-DNA population and five (nim1-2, nim1-3, nim1-4, nim1-5, and nim1-6) from the EMS population.

Example 3: Disease Resistance of nimi 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. Wild-type seeds and seeds for each of the nimi alleles (nim1-1, -2, -3, -4, -5, -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 2 weeks post-planting, germinated seedlings had produced 4 true leaves. Plants were then treated with H₂0, 5mM SA, 300 μM BTH ,or 300 μM 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 19°C day/17° C night temperatures and 8h light/16h dark cycles.

To determine the relative strength of the different t7/^' 7 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 nimi 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.

Table 1

I ³ercent Infection - 1 Emwa/Control mutant Dav O Dav 5 Dav 6 Dav 7 Dav 11 Ws WT 0 10 25 100 90 nim1-1 0 75 95 100 100 nim 1-2 0 30 85 100 100 nim 1-3 0 30 90 100 100 nim 1-4 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 Dav O Day 5 Dav 6 Dav 7 Dav 11 Ws WT 0 5 30 70 100 nim 1-1 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 nim 1-6 0 80 100 100 100

Percent Infection - Emwa/INA mutant Dav O Dav 5 Dav 6 Day 7 Dav 11 Ws WT 0 0 0 0 0 nim 1-1 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 5 nim 1-6 0 1 50 90 100 Percent Infection • • Emwa/BTH mutant Dav O Dav 5 Dav 6 Day 7 Dav 11 Ws WT 0 0 0 0 0 nim1-1 0 1 5 30 100 nim 1-2 0 0 25 90 100 nim 1-3 0 15 70 100 100 nim 1-4 0 80 100 100 100 nim 1-5 0 0 1 1 10 nim 1-6 0 1 90 100 100 As shown in Table 1 , during normal growth, nim1-1, nim1-2, nim1-3, 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 nim1-5 plants where fungal growth was delayed by several days relative to both the other nimi mutants and the Ws-0 control, but eventually all of the nim1-5 plants succumbed to the fungus.

Following SA treatment, the mutants could be grouped into three classes: nim1-4 and nim 1-6 showed a relatively rapid fungal growth; nim1-1, nim1-2, nim1-3 plants exhibited a somewhat slower rate of fungal growth; and fungal growth in t7/^'m7-5 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; nim1-1, nim1-2, nim1-3, 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 nim1-4 being the most severely compromised, nim1-1, nim1-2, nim1-3 and nim 1-6 showing an intermediate inhibition of fungus and t7//777-5with only slightly impaired fungal resistance.

Table 2 shows the disease resistance assessment via infection rating of the various nimi 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 nimi alleles were found. The various nimi 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. Table 2

Infection Severity - Emwa/Water mutant Dav 7 Dav 11

Ws WT 3 3 nim1-1 4 4.5 nim 1-2 3 4 nim 1-3 4 4 nim 1-4 5 5 nim 1-5 1 3.5 nim 1-6 3 4.5

NahG 4 5

Infection Severity - Emwa/SA mutant Dav 7 Dav 11

Ws WT 3 4 nim1-1 3 4.5 nim 1-2 3 4 nim 1-3 3 4 nim 1-4 4 5 nim 1-5 3 3 nim 1-6 4 4.5

NahG 4 5

Infection Severity ^■ ^• Emwa INA mutant Dav 7 Dav 11

Ws WT 0 0 nim 1-1 2.5 4 nim 1-2 4 4 nim 1-3 3 3.5 nim 1-4 4 5 nim 1-5 1 2 nim 1-6 3 4.5

NahG 3 3

Infection Severity - Emwa/BTH mutant Day 7 Dav 11

Ws WT 0 0 nim 1-1 2.5 4 nim 1-2 3.5 4 nim 1-3 3 3.5 nim 1-4 4 5 nim 1-5 1.5 2 nim 1-6 3 4

NahG 0 0 From Table 2 it can be seen that the nim1-4 and n/m 7 -6 alleles had the most severe Peronospora parasitica infections; this was most easily observable at the earlier time points. In addition, the n/tn -5 allele showed the greatest response to both INA and BTH and therefore was deemed the weakest t7/^'m 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 INA- induced 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 nim1-1, nim1-2 and other nimi 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 nimi 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 t7/^'m alleles. Wild-type seeds and seeds for each of the nimi alleles (nim 1-1, -2, -3, -4, -5, -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 H₂0, 5mM SA, 300 μ M BTH ,or 300 μM 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 19°C day/17°C 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 the compatible P. parasitica Emwa isolate. The RNA was size-fractionated by agarose gel electrophoresis and transferred to GeneScreenP/us 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 nimi 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 nim1-1 plants.

As shown in Figure 2, pathogen infection (Emwa) of wild type Ws-0 plants induced PR-1 gene expression within 4 days after infection. In nim1-1 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 nim1-1 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 nim1-1, 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 nim1-4 and nim 1 -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 nimi alleles. PP-7 mRNA accumulation was elevated in the n/t-7- mutant, but only mildly induced following chemical treatment or P. parasitica infection. Based on both Pfi-7 mRNA accumulation and fungal infection, the mutants have been determined to fall into three classes: severely compromised alleles (nim1-4 and niml- 6); moderately compromised alleles (nim1-1, nim1-2, and nim1-3); and a weakly compromised allele (nim1-5).

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 nimi plants indicates a disruption in a component of the SAR pathway downstream of SA and upstream of SAR gene induction. t7/^'tn 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 nimi plants. The leaves were harvested 2 days later for SA analysis as described by Delaney et al, 1995, PNAS 92, 6602-6606. This analysis showed that the nimi 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 t7/^'tτ77 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 t7/tn7 phenotype, pollen from wild type plants was transferred to the stigmata of nim1-1, -2, -3, -4, -5, -6. If the mutation is dominant, then the nimi 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 nim1-1, -2, -3, -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 nim 1 -5 X wild type F1 descendants all exhibited the nimi 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 nimi phenotype in these F1 plants was less severe than observed when nimi -5 was homozygous.

To determine allelism, pollen from the kanamycin-resistant nim1-1 mutant plants was transferred to the stigmata of nim1-2, -3, -4, -5, -6. Seeds resulting from the cross were plated onto Murashige-Skoog B5 plates containing kanamycin at 25 μg/ml to verify the hybrid origin of the seed. Kanamycin resistant (F1 ) plants were transferred to soil and assayed for the n/tτ?7 phenotype. Because the F1 descendants of the cross of the nim1-5 mutant with the Ws wild type display a nimi phenotype, analysis of nim1-5X nim1-1 F2 was also carried out. As shown in Table 3, all of the resulting F1 plants exhibited the nimi phenotype.

Thus, the mutation in the nim1-2, -3, -4, -5, -6 was not complemented by the nim1-1; these plants all fall within the same complementation group and are therefore allelic. F2 descendants from the nim1-5 Xnim1-1 cross also displayed the nimi phenotype, confirming that nim1-5 \s a nimi allele.

Table 3. Genetic Segregation of nim Mutants

Phenotype

Mutant Generatio Female Male Wild type ^a nim1 ^b n nim 1-1 FI wild type ^c t7 tn7-7 24 0

F2 98 32 nim1-2 F1 nim1-2 Wild type 3 0 nim1-3 F1 nim1-3 Wild type 3 0 nim1-4 F1 nim1-4 Wild type 3 0 nim 1-5 F1 nim 1-5 Wild type 0 35

F1 Wild type nim1-5 0 18 nim 1-6 F1 nim 1-6 Wild type 3 0 nim1-2 F1 nim1-2 nim1-1 0 15 nim1-3 F1 nim1-3 nim1-1 0 10 nim1-4 F1 nim1-4 nim1-1 0 15 nim1-5 F1 nim1-5 nim1-1 0 14

F2 9 85 nim1-6 F1 nim1-6 nim1-1 0 12

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 nimi 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 NIM1, 74 F₂ nim plants from a cross between π/m7-7 (Ws-0) and Landsberg erecta (Let) were identified for their susceptibility to P. parasitica and lack of accumulation of P -7 mRNA following INA treatment. Using simple sequence length polymorphism (SSLP) markers (Bell and Ecker 1994), nim1-1 was determined to lie about 8.2 centimorgans (cM) from nga128 and 8.2 cM from ngal 11 on the lower arm of chromosome 1. In addition, nim1-1 was determined to lie between ngal 11 and about 4 cM from the SSLP marker ATHGENEA. (Figure 5A)

For fine structure mapping, 1138 nim plants from an F₂ population derived from a cross between nim 1-1 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 nim1-1, giving a genetic distance of approximately 4.1 cM (93 of 2276), and 239 recombinant chromosomes were identified between nga111 and nim1-1, 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 nim1-1) and W85.1 (0.6 cM from nim1-1) 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 nimi, 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 t7/^' was located between L84.Y1 and L84.8, representing a gap of about 0.09 cM. C. Isolation of the NIM1 Gene

Example 8: Construction of a Cosmid Contig

A cosmid library of the NIM1 region was constructed in the Agrobacterium-compaWb\e

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. coli strain 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 >90 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 NIM 1 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 kanamycin- sensitive nim1-1 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 plating and tested for the nimi phenotype as described below. Transformed plants that no longer had the nimi phenotype identified cosmid(s) containing a functional NIM1 gene.

Example 10: Complementation of the nimi Phenotype

Plants transferred to soil were grown in a phytotron for approximately one week after transfer. 300μm 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 19°C day/17^CC 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 t7/^'m7 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 ³²P-labeled PR-1 cDNA probe. The resulting blots were exposed to film to determine which transformants were able to induce PR-1 expression after INA treatment. The results are summarized in Table 4, which shows complementation of the nimi phenotype by cosmid clones D5, E1 , and D7.

Table 4

NA - not applicable 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 t?/m7 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 EcoRV- digested pBRKanF4 (a derivative of pBRKan_Fι (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, Ml), 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 nim1-1 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 /7/^'tn7 alleles and cDNAs was performed essentially as described above.

A region of approximately 9.9 kb defined by the overlap of cosmids E1 and D7 was identified by complementation analysis to contain the n/t777 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 E1 using a modification of the ammonium acetate method (Traynor, P.L., 1990. BioTechniques 9(6): 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 E1 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- digested pSGCGOI . 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 nimi allelic variants nim1-1, -2, -3, -4, -5, and -6. 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 1.0% 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 3.5 reads/base.

Sequences were edited and were assembled using Sequencher 3.0. Base changes specific to various t?/m 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.

Table 5

Positions listed in the table relate to SEQ ID NO:1. All alleles nim1-1 to nim1-6are 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 nimi alleles. At the same time, at least one of the nimi 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. The cosmid region containing the nimi gene was delineated by a BamHI-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. coli strain 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 (pSGCGOI) 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 t7/tr?7 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 5).

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 pSGCGOI 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 nim1-1 plants were infiltrated with this bacteria as previously described. The resulting seed was harvested and planted on GM agar containing 50μg/ml kanamycin. Plants surviving selection were transferred to soil and tested for complementation. Transformed plants and control Ws and nimi plants were sprayed with 300μm 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 evaluated 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 15 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 nim1-1 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 nim1-3 allele and the nim1-6 allele. The nim1-2 allele showed approximately normal levels in the untreated sample and showed similar induction to that of the wild type sample, as did the nim1-4 allele. The /7/m7- 5 allele seemed to show higher basal level expression of the NIM1 gene and much stronger expression when induced by chemical inducers. 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 oiigonucleotide 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, 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 NaP0₄, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride) at 55°C for 18-24h] with ³²P-radiolabelled Arabidopsis thaliana NIM1 cDNA. Following hybridization the blots were washed under low stringency conditions [6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C; 1XSSC is 0.15M NaCI, 15mM Na-citrate (pH7.0)] and exposed to X-ray film to visualize bands that correspond to NIML

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 (e.g., 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 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.

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' U.S. Patent Application Serial No. 08/773,554, 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 E1 cosmids (containing the NIM1 gene) were selfed and the T2 seed collected. Seeds from one E1 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 analyzed for fungal growth at 3-14 days, preferably 7-12 days, following infection. Plants showing 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

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 pSGCGOI 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 μg/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 demonstrated constitutive resistance to Emwa. This shows that plants overexpressing the NIM1 gene under its native promoter have constitutive immunity against pathogens.

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

Example 20: 35S Driven Overxpression of NIM 1

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 Xbal fragment 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 μg/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 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 (i.e. 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 non- experimental 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. 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 nim1-1, -2, -3, -4, -5, 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 with different pathogens and pathotypes that may be differentially virulent on the different crop plant cultivars. Furthermore, the use of Arabidopsis has advantages because of its small size and the possibility of thereby undertaking more tests with limited resources of space.

Example 26: NIM1 Is A Homolog Of lκBα

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 α (Figure 9). 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, Wl). The sequences used in the alignment were NIM1 (SEQ ID NO:3), mouse lκBα (SEQ ID NO:18, GenBank Accession #: 1022734), rat IKBCC (SEQ ID NO:19, GenBank accession Nos. 57674 and X63594; Tewari et al.,

Nucleic Acids Res. 20, 607 (1992)), and pig lκBα (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, M.O., Dayhoff, ed (National Biomedical Research Foundation, Washington, D.C.), 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, D.C.) 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-κB and IKB. The best overall homology is to IKB and related molecules (Figure 9). 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-κB. 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-κB molecules. The C-termini of IKB'S can be dissimilar. NIM1 has some homology to a QL-rich region (amino acids 491-499) found in the C-termini of some lκ 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 lκBα is required for stimulus-activated degradation of lκBα thereby activating NF-κB. Mutagenesis of the serine residues (S32- S36) in human lκBα to alanine residues inhibits stimulus-induced phosphorylation thus blocking lκBα proteosome-mediated degradation (E. Britta-Mareen Traenckner et al., EMBO J. 14: 2876-2883 (1995); Brown et al., Science 267:1485-1488 (1996); Brockman et al., Molecular and Cellular Biology -\ 5: 2809-2818 (1995); Wang et al., Science 274:784-787 (1996)).

This altered form of IKBCC functions as a dominant negative form by retaining NF-κB 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 lκBα. 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 I92-226): 5'-CAA CAG CTT CGA AGC CGT CTT TGA CGC GCC GGA TG-3' (SEQ ID NO:32) and 5'- 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.coli strain XL1-Blue. Plasmids from resulting colonies are extracted and sequenced to verify the presence of the mutated bases and to confirm that no other mutations occurred. The mutagenized NIM1 cDNA is digested with the restriction endonuclease EcoRI and cloned into pCGN1761 under the transcriptional regulation of the double 35S promoter of the cauliflower mosaic virus. The transformation cassette including the 35S promoter, NIM1 cDNA and tml terminator is released from pCGN1761 by partial restriction digestion with Xbal and ligated into the Xbal and ligated into the Xbal site of dephosphorylated pCIB200. SEQ ID NO's: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 NaPθ4, 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: 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 lκBα, which includes K21 , K22, S32 and S36, results in a dominant-negative lκBα 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 2). 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 1612- bp PCR product (SEQ ID NO:21 : 418 to 2011): 5'-gg aat tca-ATG GAT TCG GTT GTG ACT GTT TTG-3' (SEQ ID NO:34) and 5'-gga att cTA CAA ATC TGT ATA CCA TTG G-3' (SEQ ID NO:35) in which the synthetic start codon is underlined (ATG) and 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 MgCI₂/0.001 % gelatin/0.25 mM each dNTP/0.2 mM of each primer and 1 unit rTth DNA polymerase in a final volume of 50 mL and a Perkin Elmer Cetus 9600 PCR machine. PCR conditions are as follows: 94°C 3min: 35x (94°C 30 sec: 52°C 1 min: 72°C 2 min): 72°C 10 min. The PCR product is cloned directly into the pCR2.1 vector (Invitrogen). The PCR-generated insert in the PCR vector is released by restriction endonuclease digestion using EcoRI and ligated 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 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 NaPθ4, 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 lκBα 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 NIML 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. (I989), 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'-cggaattcGATCTCTTTAATTTGTGAATTT C-3' (SEQ ID NO:36) and 5'-ggaattcICAACAGTT 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: 52°C 1 min: 72°C 2 min); 72°C 10 min]. The PCR product is cloned directly into the pCR2.1 vector (Invitrogen). The PCR-generated insert in the PCR vector is released by restriction endonuclease digestion using EcoRI and ligated into the EcoRI site of dephosphorylated pCGN1761 , which contains the double 35S promoter. The construct is sequenced to verify the presence of the synthetic in-frame stop codon and to confirm that no other mutations occurred during PCR. The transformation cassette including the promoter, modified NIM1 cDNA, and tml 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 NaPθ4, 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: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 EcoRI to eliminate EcoRI concatemers and cloned into the EcoRI site of dephosphorylated pCGN1761. The N/C- terminal deletion form of NIM1 is under the transcriptional regulation of the double 35S 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 35S promoter, NIM1 chimera and tml 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 C- terminal amino acid deletions. 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 NaPθ4, 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°C 3 min:35x (94°C 30 sec: 62°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' ggaattcICAACCTTCCAAAGTTGCTTCTGATG-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 tml terminator is released from pCGN1761 by partial restriction digestion with Xbal and ligated into the XbalsWe 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 NIML

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 NaP04, 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 wild- type gene product. 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 (U.S. Pat. No. 5,614,395). Such expression permits strong expression of the altered NIM1 forms only under pathogen attack or other SAR- activating 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 (i.e., 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., Cell 78, 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. ^• 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/ml from two compatible P. parasitica isolates, Emwa and Noco (i.e. these fungal strains cause disease on wildtype Ws-0 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 (U.S. Pat. No. 5,614,395) and πdr7 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 al., Molecular Cloning , eds., Cold Spring Harbor Laboratory Press. (1989)) and amplification by PCR using oiigonucleotide primers (see, e.g. Innis etal., 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, 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 NaP0₄, pH7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride) at 55°C for 18-24h] with ³²P-radiolabelled Arabidopsis thaliana NIM1 cDNA. Following hybridization the blots were washed under low stringency conditions [6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C; 1XSSC is 0.15M NaCI, 15mM Na-citrate (pH7.0)] and exposed to X-ray film to visualize bands that correspond to NIML 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 (e.g., 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^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 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-0 or Ws-0 are transformed into crop plants for evaluation. Alternatively, altered native NIMI genes isolated from crops in the preceding example are put back into the respective crops. Although the NIM1 gene can be inserted into any plant cell falling within these broad classes, it is particularly useful in crop plant cells, such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane. Transformants are evaluated for enhanced disease resistance. In a preferred embodiment of the invention, the expression of the 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.

REFERENCES

The disclosures of each of the following references are hereby expressly incorporated by reference into the instant disclosure:

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SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Novartis AG

(B) STREET: Schwarzwaldallee 215

(C) CITY: Basel

(E) COUNTRY: Switzerland

(F) POSTAL CODE (ZIP) : 4002

(G) TELEPHONE: +41 61 69 11 11 (H) TELEFAX: + 41 61 696 79 76 (I) TELEX: 962 991

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

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

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(ii) TITLE OF INVENTION: METHODS OF USING THE NIMl GENE TO CONFER DISEASE RESISTANCE IN PLANTS

(iii) NUMBER OF SEQUENCES: 39

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9919 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

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

TGATCATGAA TTGCGTGTAG GGTTGTGTTT TAAAGATAGG GATGAGCTGA AGAAGGCGGT 60

GGACTGGTGT TCCATTAGAG GGCAGCAAAA GTGTGTAGTA CAAGAGATTG AGAAGGACGA 120

GTATACGTTT AAATGCATCA GATGGAAATG CAATTGGTCG CGTCGGGCAG ATTGAATAGA 180

AGAACATGGA CTTGTTAAGA TAACTAAGTG TAGTTGGTCC ACATACTTGT TGTTCTATTA 240

AGCCGGAAAA CTTCAACTTG TAATTTGCAG CAGAAGAGAT TGAGTGTCTG ATCAGGGTAC 300

AACCCACTCT AACAGCAGAG TTGAAAAGTT TGGTGACATG CTTAAAACTT CAAAGCTGCG 360

GGCAGCAGAA CAGGAAGTAA TCAAAGATCA GAGTTTCAGA GTATTGCCTA AACTAATTGG 420

CTGCATTTCA CTCATCTAAT GGGCTACTTG TGGACTGCAA TATGAGCTTT TCCCTAATCC 480

TGAATTTGCA TCCTTCGGTG GCGCGTTTTG GGCGTTTCCA CAGTCCATTG AAGGGTTTCA 540

ACACTGTAGA CCTCTGATCA TAGTGGATTC AAAAGACTTG AACGGCAAGT ACCCTATGAA 600

ATTGATGATT TCCTCAGGAC TCGACGCTGA TGATTGCTTT TTCCCGCTTG CCTTTCCGCT 660

TACCAAAGAA GTGTCCACTG ATAGTTGGCG TTGGTTTCTC ACTAATATCA GAGAGAAGGT 720

AACACAAAGG AAAGACGTTT GCCTCGTCTC CAGTCCTCAC CCGGACATAG TTGCTGTTAT 780

TAACGAACCC GGATCACTGT GGCAAGAACC TTGGGTCTAT CACAGGTTCT GTCTGGATTG 840 TTTTTGCTTA CAATTCCATG ATATTTTTGG AGACTACAAC CTGGTGAGCC TTGTGAAGCA 900

GGCTGGATCC ACAAGTCAGA AGGAAGAATT TGATTCCTAC ATAAAGGACA TCAAAAAGAA 960

GGACTCAGAA GCTCGGAAAT GGTTAGCCCA ATTCCCTCAA AATCAGTGGG CTCTGGCTCA 1020

TGACCAGTGG TCGGAGATAT GGAGTCATGA CGATAGAAAC AGAAGATTTG AGGGCAATTT 1080

GTGAAAGCTT TCAGTCTCTT GGTCTATCAG TGACAGCGAA CGCACCTGCA CATGTGGGAA 1140

GTTTCAATCG AAGAAGTTTC CATGTATGCA CCCAGAAATG GTGCAAAGGA TTGTTAACTT 1200

GTGTCATTCA CAAATGTTGG ATGCAATGGA GCTGACTAGG AGAATGCACC TTACACGCCC 1260

ACTCAGTGTT CTCTTATCTC TAGACCTGAA ACTAACTTGC TGTGTAATTC GAGTTACAAA 1320

AGGTTAAAGG AAGAATTAGG AAGATACA A TAACATGAAT GTTGCCAGAA GTTCAGGGAA 1380

CTTGAATATT CTTTTGGTTC TTGGTGGAAA ATATCCAACA GATGAACAAT TTGACATTAT 1440

TTCACACTTT GATTCTAGCA ACTCTGTAAC ACCATCATGG GTTATTGTTG ATGTACATAA 1500

ATATATATTA CAAATCTGTA TACCATTGGT TCAAATTGTT ACAACATTTG TTTGAAGCAC 1560

ACCTGCAGCA ATAATACACA GGATGCAAAA CGAAGAGCGA AACTATATGA CGCCAACGAT 1620

AGACATAAAC AGTTACAGTC ATCATGAAAA CAGAATTATA TGGTACAGCA AAAATTACAC 1680

TAAGAGGCAA GAGTCTCACC GACGACGATG AGAGAGTTTA CGGTTAGACC TCTTTCCACC 1740

GGTTGATTTC GATGTGGAAG AAGTCGAATC TGTCAGGGAC GAATTTCCTA ATTCCAAATT 1800

GTCCTCACTA AAGGCCTTCT TTAGTGTCTC TTGTATTTCC ATGTACCTTT GCTTCTTTTG 1860

TAGTCGTTTC TCAGCAGTGT CGTCTTCTCC GCAAGCCAGT TGAGTCAAGT CCTCACAGTT 1920 CATAATCTGG TCGAGCACTG CCGAACAGCG CGGGAAGAAT CGTTTCCCGA GTTCCACTGA 1980

TGATAAAAAA AACAAGGTCA GACAGCAAGT AACAAAACCA TGTTTAAAGA TCATTTAGTT 2040

TTGTTTTTTG TGATAAGGAG TCCGATGAAG TGGGTGAGAA TCCATACCGG TTTTAGAAAG 2100

CGCTTTTAGT CTACTTTGAT GCTCTTCTAG GATTCTGAAA GGTGCTATCT TTACACCCGG 2160

TGATGTTCTC TTCGTACCAG TGAGACGGTC AGGCTCGAGG CTAGTCACTA TGAACTCACA 2220

TGTTCCCTTC ATTTCGGCGA TCTCCATTGC AGCTTGTGCT TCCGTTGGAA AAAGACGTTG 2280

AGCAAGTGCA ACTAAACAGT GGACGACACA AAGAATAGTT ATCATTAGTT CACTCAGTTT 2340

CCTAATAGAG AGGACATAAA TTTAATTCAA ACATATAAGA AATAAGACTT GATAGATACC 2400

TCTATTTTCA AGATCGAGCA GCGTCATCTT CAATTCATCG GCCGCCACTG CAAAAGAGGG 2460

AGGAACATCT CTAGGAATTT GTTCTCGTTT GTCTTCTTGC TCTAGTATTT CTACACATAG 2520

TCGGCCTTTG AGAGAATGCT TGCATTGCTC CGGGATATTA TTACATTCAA CCGCCATAGT 2580

GGCTTGTTTT GCGATCATGA GTGCGGTTCT ACCTTCCAAA GTTGCTTCTG ATGCACTTGC 2640

ACCTTTTTCC AATAGAGATA GTATCAATTG TGGCTCCTTC CGCATCGCAG CAACATGAAG 2700

CACCGTATAT CCCCTCGGAT TCCTATGGTT GACATCGGCA AGATCAAGTT TTAAAAGATC 2760

TGTTGCGGTC TTCACATTGC AATATGCAAC AGCGAAATGA AGAGCACACG CATCATCTAG 2820

ATTGGTGTGA TCCTCTTTCA AAAGCAACTT GACTAACTCA ATATCATCCG AGTCAAGTGC 2880

CTTATGTACA TTCGAGACAT GTTTCTTTAC TTTAGGTACC TCCAAACCAA GCTCTTTACG 2940

TCTATCAATT ATCTCTTTAA CAAGCTCTTC CGGCAATGAC TTTTCAAGAC TAACCATATC 3000 TACATTAGAC TTGACAATAA TCTCTTTACA TCTATCCAAT AGCTTCATAC AAGCTTTACC 3060

ACATATATTA GCAAGCTTGA GTATAACCAA TGTGTCCTCT ATAACAACTT TGTCTACAAC 3120

GTCCAATAAG TGCCTCTGAA ATACAAATAC AAGTACTCAA GTAAGAACAT ATTCATGAAT 3180

GTGTAACCAT AGCTTAATGC AGATGGTGTT TTACCTGATA GAGAGTAATT AATTCAGGGA 3240

TCTTGAAGAT GAAAGCCAAA TAGAGAACCT CCAACATGAA ATCCACCGCC GGCCGGCAAG 3300

CCACGTGGCA GCAATTCTCG TCTGCGCATT CAGAAACTCC TTTAGGCGGC GGTCTCACTC 3360

TGCTGCTGTA AACATAAGCC AAAACAGTCA CAACCGAATC GAAACCGACT TCGTAATCCT 3420

TGGCAATCTC CTTAAGCTCG AGCTTCACGG CGGCGGTGTT GTTGGAGTCT TTCTCCTTCT 3480

TAGCGGCGGC TAAAGCGCTC TTGAAGAAAG AGCTTCTCGC TGACAAAACG CACCGGTGGA 3540

AAGAAACTTC CCGGCCGTCG GAGAGAACAA GCTTAGCGTC GCTGTAGAAA TCATCCGGCG 3600

AGTCAAAGAC GGATTCGAAG CTGTTGGAGA GCAATTGCAG AGCAGATACA TCAGGTCCGG 3660

TGAGTACTTG TTCGGCGGCC AGATAAACAA TAGAGGAGTC GGTGTTATCG GTAGCGACGA 3720

AACTAGTGCT GCTGATTTCA TAAGAATCGG CGAATCCATC AATGGTGGTG TCCATCAACA 3780

GGTTCCGATG AATTGAAATT CACAAATTAA AGAGATCTCT GCTAATCAAC GAAGAGACCT 3840

TATCAACTGG ATTTGGTTAA AGATCGAAGA TAACCATTGA CGAGCAGAGC CAAGTCAAGT 3900

CAACGAGAGT GGTGGTGAGA TATGAAGAAG CATCCTCGTC CCACGGTTTA CATTTCACCA 3960

AAACCGGTAA ATTTCCAGGA AAGGAATCTT TGTCAGAGAT CTTTTTTAAA AAGATATAAC 4020

AGGAAGCTAA ACCGGTTCGG GTTATAAATG TTAGTATTTA TACCGGAGAC ATTTTGTGTT 4080 GCTAATTTTT GTATATGAGA AGTTCAATCC GGTTCGGTAA GCCCCTGAAC CAAACTAGAT 4140

TTGGAGATGA TATAAATATA TAAAATTTAT TTTTCATCCG GTTCGTTATT TTCATATAAA 4200

TATATAAATA TTATTTTTTA AATTTAAGAA TTAGATTTAC ATGTGAAAGT TACATTTCTG 4260

TTTATTTTCT TTGAAGTAAA ATGATAAAGG GAACGTATAT TAAGTTTCAT GCTTTATTCA 4320

CATAAGTTTT GTAATGTATA TTATATTTTT CGTTTATTGA AAAAGTAATT TTCAGTGTTC 4380

AGCATGTTTA CACTATAATT AAATCAAGTC GAATATTTCC TGGAACTATT CTCCTTGTTC 4440

TATAGCAAAT GAAAACGCTC TTCACAACAA AATCATTATA GATATAGGAA TAAATTACAT 4500

TAAAAACATG AAAGTCATAA TGAATATATT TTTTTAATTA GGATTTGATT TAAAAACAAT 4560

TATTGTATAC ATATAAAAGA CTTCTTTAGT TATTTGCCTT CAACTTCTCG TTCTGAATCA 4620

TGCGATAAAT CAGCTTTTTC AATAACTACG ACGTAAAAGC AAATTCATAA CACGTCTAAA 4680

CAAATTTGGC TCATCCTTCA CTTGATTGGT GTTTTCCGGA CTCGATGTTG CTGGAAACTG 4740

AGAAGAAGAA GGAATCTGCA TAATCACCTC TTGGTTCCTC ACCGGTAGAC TCATTTTGTT 4800

GGATCGAAAA CGATCGAGAT CAGAAAATGA AAAGATAGGT TAAAGATGCC TATGAATACA 4860

ACAACGTAAG ATTATGTTGA ATAAACAGAG TACTTTATAT AGGAGTTATA A AAGGTAAA 4920

TAAATTATTG CTTTCCGCGT TTTTTACTTT TGTATTTCTT AAATGATAAG TTAAATTAGG 4980

ATAAGATTTG TATGATTTTA AGTAAATTTA CAATAACTCT CTATAACTCA A AGCATCAC 5040

ATATTTAATT AATTTTACTA ATTATCTTTT GAACAATTTT ATGAAATAGT TTTCTTTTAA 5100

TTAATTTTTT AAAATGA AT ATTA AAAAT TTAATTGAAT CAATCTGATA TAATTTTTTT 5160 ATCTTCTACC ATCTATTATA GTTGATAAAT ATTGTGATAA ACTTTAGATA AACACCCAAT 5220

_TGCCAAATAT TTAATAAATT TTGTGTACCA TGCGTTTTTT TTGGAGAATA TATATACGTG 5280

GACAGCATAC CGTACATATA TTGTATAAAA GCTTATAAAA CATAGATACG GGTTATATTG 5340

G_TAAGCTATA AATATATGTA AACAATAGTA AGATATTACG TGTTGTGTCT AAATATGTGT 5400

TGCTTTAGAT ATTATGTATA TCTAATATAT TAAAATATCT TTTATTAACT AATATATTAT 5460

TTAAGAGAGA AAATTGGGAC ACTATTTTCT ATACAGTAAC TGTTTTCAAC TATAAACAGG 5520

AACCCTTGAT AT ATAAAAT AACTAGCCAA AAAATCAGAT TAAATATTCA TAAAACAATG 5580

TTTGGTATTA TTACATAAAC CTAAGAAACA AAATTCAATA TTCCTTTTTA CCTTATAAAA 5640

AACAATTAAA CATCACTAGA TATATTTATG CCCCACAATG AGCGAGCCAA TTGAGACTTG 5700

AGACTTGAGA TCCTTGTCAA CTACGTTTGC ATTTGTCGGC CCATTTTTTT TATTTTTTTT 5760

TTAAAGTGTC GGCCCGTTGC TTCTTCCGTT CAGATCAACC CTCTCGTAAT CAGAACAAAA 5820

CGGAAAACAA ACGAAAGAAC AATCAGATCC CTCTTTTTTT GCATAAACTA AATTCAACTT 5880

CTCTGCGTTT ATGTTGTAGA GGCAACCACG ATCACTACTA CGAAACAATA CAACGTCGTT 5940

GCTTGGAGTC CACGTAATCA AATCTACTCC AATGCTTTTA ATATCTTTCA CTTTAACCCA 6000

CGACTTTTCA AAACTGCTCT TTAAAACCCA TAACTCGTGA ACATCTTCTT GATCTTTGTT 6060

TGTCCACTGA CGAATAGCAC CTAGCTTCCC TTCGTATCTG ACTAATCCTG AGAAAACATC 6120

AGAGTTCGGA GTATGGAAGA AGGACCAAGT TTCGGTTTTG AGACAAAACC GGATCACATT 6180

GTTGTTCCGT GATATCCAAT GCAAGAACCC CGAAACTTGT ATCGGGTTGG AAAAAATTAA 6240 TCTGTCTGTT TTTGGTAGAC GCAAATTTTC TAATCTCTTC CAGGTAAACG AATCAGAATC 6300

GAAAACTTCG CACATAAAAG TTCTGTGATT CAAATGGTAG ATACCCCGAG ACATACACAT 6360

ACGCCGAGAC TGCGAAAGCC TTTGTATTTT ATACCGGAAA GGGTTCAATC CGATTACCGC 6420

TAAACCCAAT GACATATCCC AACCCTTCAC TTCTGGCTTT GGTATGACCT GATACTGTTT 6480

AGTGGTTGGT TTGAAGACTA TGTATCCACG TGATGGTTTT GTATACTTAA CACAAAGCAA 6540

TATCCCATGA CTTGCATCAC AAGCTTCGAT CTTTATCATT CCGGGTGGCA GAAAGTCGAT 6600

GGAGACTCCA TTGTTTTGTA AATCACTCCT CTCATGGACA AAACTGGTTC GAAGTTCGTG 6660

TCCTTTTACT ATGTAGTGTT GTATGAAGTA TCCCGAAATA CGATTGGTTC TAAGGAGATT 6720

AAGATTGACA AACCATGACT CGTAGCTTCT CTTGTTGCAC TCTTTATTCA GGAGCCTGAA 6780

TTTTCCGATT TTTGACGCCG GAAGATAAGA AAGAAATTCT TGGATCATGT CTTGATTTAT 6840

CACCGGAGAA CTCATGATCC TGTCGGGAAT AAAGAGATGA GCACGATCAC TGAATGAGAA 6900

ATGAAAAAAT CAGGATCGGT AGAGAACAAC TTATGATGAA TAAAGTGTTT ATATATCCTT 6960

TCTTTGTTTA AGGAAAGTAT CAAAATTTGC CTTTTTCTTC GCTAGTCCTA AAACAAACAA 7020

ATTAACCAAA AGATAAAATC TTTCATGATT AATGTTACTT GTGATACCTT AAGCCAAAAC 7080

TTTATCTTTA GACTTTTAAC CAAATCTACA GTAATTTAAT TGCTAGACTT AGGAAACAAC 7140

TTTTTTTTTT ACCCAACAAT CTTTGGATTT TAATTGTTTT TTTTTCTACT AATAGATTAA 7200

CAACTCATTA TATAATAATG TTTCTATCAT AATTGACAAT TCTTTCTTTT TAATAAACAT 7260

CCAGCTTGTA TAATAATCCA CAAGTCAATT TCACCATTTT GGCCAATTTA TTTTCTTATA 7320 AAAATTAGCA CAAAAAAGAT TATCATTGTT TAGCAGATTT AATTTCTAAT TAACTTACGT 7380

_AA_TTTCCATT TTCCATAGAT TTATCTTTCT TTTTATTTCC TTAGTTATCT TAGTACTTTC 7440

TTAGTTTCCT TAGTAATTTT AAATTTTAAG ATAATATATT GAAATTAAAA GAAGAAAAAA 7500

AACTCTAGTT ATACTTTTGT TAAATGTTTC ATCACACTAA CTAATAATTT TTTTTAGTTA 7560

AATTACAATA TATAAACACT GAAGAAAGTT TTTGGCCCAC ACTTTTTTGG GATCAATTAG 7620

TACTATAGTT AGGGGAAGAT TCTGATTTAA AGGATACCAA AAATGACTAG TTAGGACATG 7680

AATGAAAACT TATAATCTCA ATAACATACA TACGTGTTAC TGAACAATAG TAACATCTTA 7740

CGTGTTTTGT CCATATATTT GTTGCTTATA AATATATTCA TATAACAATG TTTGCATTAA 7800

GCTTTTAAGA AGCACAAAAC CATATAACAA AATTAAATAT TCCTATCCCT ACCAAAAAAA 7860

AAAATTAAAT ATTCCTACAG CCTTGTTGAT TATTTTATGC CCTACGTTGA GCCTTGTTGA 7920

CTAGTTTGCA TTTGTCGGTC CATTTCTTCT TCCGTCCAGA TCAACCCTCT CGTAATCAGA 7980

ACAAAAGGGG AAACAAACGT AAGAGGCAAA ATCCTTGTTT GTATGAACTA AGTTTAACTT 8040

CTCTGTGTTT AAGTTGTAGA GGCAAACATG ATCCCAACTA GAAAGCATTA CGACGTCGTT 8100

GCTTGGTATC CACGTAATAT GCTCTACTCC AATGCTTTCA ATATCTTTCA CTTTTTCCCA 8160

CGACTTTTCA AAACTGCTCT TTAAAACCCA TAATCTGTGA ACATCTTCTT GATTGTTGTT 8220

TATCCAGTGA CGAATAACAC CTAGCTTCCC TTCGTAGCTG ACTAACTCTG GGAATAAACC 8280

AACGTTTGGA GTATGTAAGA AAGACCAAGT TTCGGTTTTG GGACATAACC GGATCACATT 8340

GTGGTTCCAT GATCTCCAAT GCAAGAACCC TGAAGCTTGT ACCGGGTTTG AAAGAATTAG 8400 ACCGTCTGTT CTCGGTAGAC GCAAATTTTT TAATCTCTTC CACATAAACG AATCGGAATC 8460

_AAAAACTTCG CACGCAAAAG TTCTGAGATT CCGAGTCATA CCAGGCGATT TCGAAAGCCT 8520

_AA_ATATTTTA TACCGGAAAG GCTGCAATCC GGTTACCGTT AGACCTAATG ACTTATCACA 8580

ACTCCTCACT TTTGGGTTTG GTATGATCTG ATACTGTTTT GTTGTTGGTT TGCAGACTAT 8640

GTATTCCGGT ATTGGTCTTG TATCATTATA ACAAAGCAAT ATCCCATGAC GTGCATCACA 8700

AGCTTTGATC TTTACCTCTC CTTGTGGCAG AAAATCGATG GAGACTCCTT TGTTATCCAA 8760

ATCTCTCCTC TCATGGAAAA AACTGGTATC AAGTTTGTAT CCTCTTTCGT AGCGTTCTAG 8820

GAAGTATCCA GAGATATTGT TGGTTCGATG GAGATTTAGG TTGACAAACC AAGACTCGTA 8880

GCTTCTCTTG TTGCACTCTT TATTGATGAG CCTCAATTTT CCGATTTCGG ACCCCCGAAG 8940

AAAGAAAGA ACCTCTTGGA TCGTGTCCTG ATTTATCACC GGAGAACTCA TGATCTTATT 9000

GGAAAAAAGA AAGAAAGAGA TGAGCACGAT CAGTGAATGA GATATATAGA AATCAGGATT 9060

GGTAGAGAAC CGACGATGAT GAATATACAA GTGTTTATAA GTATCACAAA TTGCCTTTTT 9120

CTTCGCTAGT CCCAAAACAA GCAAATTAAC CAAAGATAAA ATCTTCATTA ATGTTTTCCT 9180

TTTTCTTCGC CAGTCCCAGA TAAAAATATA TATAAAATAT TTCATTAGGT TACTTGTAGT 9240

ACCTTGAGCC CAAAGTTTCT CTTTTGACTT TTAACCAAAT TAACAGTAAT TTAATAGCTA 9300

GACTTAGAAA ACAACATTTT GTATATATAT TCTTTGACAT CAAAATTCAA CAATCTTTGG 9360

GTTTCTATAG TGTTTTTTTT CTTATTCTAA TAGATTACCA CTCATTATAT CATATACAAA 9420

GTGTTTCCTT TTCAATCAAC ATCCATTTTC TTTAAAAATT AGCAAGTTTG TTCTTATATC 9480 ATCATTCAGC AGATTTCTTA ATTAAACTTA GTGATTTCCA TTTTGCACCT ATATGTTTCT 9540

CTTTCTTAGT TTAGTACTTT AAATTTTCAT ATATATAATT TATTAAAATT AAAAGTAAAA 9600

ACTCCAGTTT AACTTATGTT AAATGTTTCA TCACACTAAA AGAGCATTAA GTAATAAATA 9660

TTTTAGCTTT ATGAAAAAAA ATATCAAATC ACTGAAGACA TTTGTTGGCC TATACTCTAT 9720

TTTTTATTTG GCCAATTAGT AATAGACTAA TAGTAACTCA TATGATATCT CTCTAATTCT 9780

GGCGAAACGA ATATTCTGAT TCTAAAGATA GTAAAAATGA ATTTTGATGA AGGGAATACT 9840

ATTTCACACA CCTAGAAAGA GTAAGGTAGA AACCTTTTTT TTTTTGGTCA GATTCTTGTA 9900

TCAAGAAGTT CTCATCGAT 9919

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5655 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: exon

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

(A) NAME/KEY: exon

(B) LOCATION: 3427..4162

(D) OTHER INFORMATION: /product_^ "2nd exon of NIMl"

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 4271..4474

(D) OTHER INFORMATION: /product= "3rd exon of NIMl"

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 4586.-4866

(D) OTHER INFORMATION: /product= "4th exon of NIMl"

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: join (2787..3347 , 3427..4162, 4271..4474, 4586..4866)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

TGTGATGCAA GTCATGGGAT ATTGCTTTGT GTTAAGTATA CAAAACCATC ACGTGGATAC 60

ATAGTCTTCA AACCAACCAC TAAACAGTAT CAGGTCATAC CAAAGCCAGA AGTGAAGGGT 120

TGGGATATGT CATTGGGTTT AGCGGTAATC GGATTGAACC CTTTCCGGTA TAAAATACAA 180

AGGCTTTCGC AGTCTCGGCG TATGTGTATG TCTCGGGGTA TCTACCATTT GAATCACAGA 240

ACTTTTATGT GCGAAGTTTT CGATTCTGAT TCGTTTACCT GGAAGAGATT AGAAAATTTG 300

CGTCTACCAA AAACAGACAG ATTAATTTTT TCCAACCCGA TACAAGTTTC GGGGTTCTTG 360

CATTGGATAT CACGGAACAA CAATGTGATC CGGTTTTGTC TCAAAACCGA AACTTGGTCC 420 TTCTTCCATA CTCCGAACTC TGATGTTTTC TCAGGATTAG TCAGATACGA AGGGAAGCTA 480

_GGTGCTATTC GTCAGTGGAC AAACAAAGAT CAAGAAGATG TTCACGAGTT ATGGGTTTTA 540

_AAGAGCAGTT TTGAAAAGTC GTGGGTTAAA GTGAAAGATA TTAAAAGCAT TGGAGTAGAT 600

TTGATTACGT GGACTCCAAG CAACGACGTT GTATTGTTTC GTAGTAGTGA TCGTGGTTGC 660

CTCTACAACA TAAACGCAGA GAAGTTGAAT TTAGTTTATG CAAAAAAAGA GGGATCTGAT 720

TGTTCTTTCG TTTGTTTTCC GTTTTGTTCT GATTACGAGA GGGTTGATCT GAACGGAAGA 780

AGCAACGGGC CGACACTTTA AAAAAAAAAT AAAAAAAATG GGCCGACAAA TGCAAACGTA 840

GTTGACAAGG ATCTCAAGTC TCAAGTCTCA ATTGGCTCGC TCATTGTGGG GCATAAATAT 900

ATCTAGTGAT GTTTAATTGT TTTTTATAAG GTAAAAAGGA ATATTGAATT TTGTTTCTTA 960

GGTTTATGTA AAATACCAA ACATTGTTTT ATGAATATTT AATCTGATTT TTTGGCTAGT 1020

TATTTTATTA TATCAAGGGT TCCTGTTTAT AGTTGAAAAC AGTTACTGTA TAGAAAATAG 1080

TGTCCCAATT TTCTCTCTTA AATAATATAT TAGTTAATAA AAGATATTTT AATATATTAG 1140

ATATACATAA TATCTAAAGC AACACATATT TAGACACAAC ACGTAATATC TTACTATTGT 1200

TTACATATAT TTATAGCTTA CCAATATAAC CCGTATCTAT GTTTTATAAG CTTTTATACA 1260

ATATATGTAC GGTATGCTGT CCACGTATAT ATATTCTCCA AAAAAAACGC ATGGTACACA 1320

AAATTTATTA AATATTTGGC AATTGGGTGT TTATCTAAAG TTTATCACAA TATTTATCAA 1380

CTATAATAGA TGGTAGAAGA TAAAAAAATT ATATCAGATT GATTCAATTA AATTTTATAA 1440

TATATCATTT TAAAAAATTA ATTAAAAGAA AACTATTTCA TAAAATTGTT CAAAAGATAA 1500 TTAGTAAAAT TAATTAAATA TGTGATGCTA TTGAGTTATA GAGAGTTATT GTAAATTTAC 1560

TTAAAATCAT ACAAATCTTA TCCTAATTTA ACTTATCATT TAAGAAATAC AAAAGTAAAA 1620

AACGCGGAAA GCAATAATTT ATTTACCTTA TTATAACTCC TATATAAAGT ACTCTGTTTA 1680

TTCAACATAA TCTTACGTTG TTGTATTCAT AGGCATCTTT AACCTATCTT TTCATTTTCT 1740

GATCTCGATC GTTTTCGATC CAACAAAATG AGTCTACCGG TGAGGAACCA AGAGGTGATT 1800

ATGCAGATTC CTTCTTCTTC TCAGTTTCCA GCAACATCGA GTCCGGAAAA CACCAATCAA 1860

GTGAAGGATG AGCCAAATTT GTTTAGACGT GTTATGAATT TGCTTTTACG TCGTAGTTAT 1920

TGAAAAAGCT GATTTATCGC ATGATTCAGA ACGAGAAGTT GAAGGCAAAT AACTAAAGAA 1980

GTCTTTTATA TGTATACAAT AATTGTTTTT AAATCAAATC CTAATTAAAA AAATATATTC 2040

ATTATGACTT TCATGTTTTT AATGTAATTT ATTCCTATAT CTATAATGAT TTTGTTGTGA 2100

AGAGCGTTTT CATTTGCTAT AGAACAAGGA GAATAGTTCC AGGAAAATT CGACTTGATT 2160

TAATTATAGT GTAAACATGC TGAACACTGA AAATTACTTT TTCAATAAAC GAAAAATATA 2220

ATATACATTA CAAAACTTAT GTGAATAAAG CATGAAACTT AATATACGTT CCCTTTATCA 2280

TTTTACTTCA AAGAAAATAA ACAGAAATGT AACTTTCACA TGTAAATCTA ATTCTTAAAT 2340

TTAAAAAATA ATATTTATAT ATTTATATGA AAATAACGAA CCGGATGAAA AATAAATTTT 2400

ATATATTTAT ATCATCTCCA AATCTAGTTT GGTTCAGGGG CTTACCGAAC CGGATTGAAC 2460

TTCTCATATA CAAAAATTAG CAACACAAAA TGTCTCCGGT ATAAATACTA ACATTTATAA 2520

CCCGAACCGG TTTAGCTTCC TGTTATATCT TTTTAAAAAA GATCTCTGAC AAAGATTCCT 2580 TTCCTGGAAA TTTACCGGTT TTGGTGAAAT GTAAACCGTG GGACGAGGAT GCTTCTTCAT 2640

ATCTCACCAC CACTCTCGTT GACTTGACTT GGCTCTGCTC GTCAATGGTT ATCTTCGATC 2700

TTTAACCAAA TCCAGTTGAT AAGGTCTCTT CGTTGATTAG CAGAGATCTC TTTAATTTGT 2760

GAATTTCAAT TCATCGGAAC CTGTTG ATG GAC ACC ACC ATT GAT GGA TTC GCC 2813

Met Asp Thr Thr lie Asp Gly Phe Ala 1 5

GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC GTC GCT ACC GAT AAC ACC 2861 Asp Ser Tyr Glu lie Ser Ser Thr Ser Phe Val Ala Thr Asp Asn Thr 10 15 20 25

GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA GTA CTC ACC GGA CCT 2909 Asp Ser Ser lie Val Tyr Leu Ala Ala Glu Gin Val Leu Thr Gly Pro 30 35 40

GAT GTA TCT GCT CTG CAA TTG CTC TCC AAC AGC TTC GAA TCC GTC TTT 2957 Asp Val Ser Ala Leu Gin Leu Leu Ser Asn Ser Phe Glu Ser Val Phe 45 50 55

GAC TCG CCG GAT GAT TTC TAC AGC GAC GCT AAG CTT GTT CTC TCC GAC 3005 Asp Ser Pro Asp Asp Phe Tyr Ser Asp Ala Lys Leu Val Leu Ser Asp 60 65 70

GGC CGG GAA GTT TCT TTC CAC CGG TGC GTT TTG TCA GCG AGA AGC TCT 3053 Gly Arg Glu Val Ser Phe His Arg Cys Val Leu Ser Ala Arg Ser Ser 75 80 85

TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAG GAG AAA GAC TCC AAC 3101 Phe Phe Lys Ser Ala Leu Ala Ala Ala Lys Lys Glu Lys Asp Ser Asn 90 95 100 105

AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG ATT GCC AAG GAT TAC 3149 Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu lie Ala Lys Asp Tyr 110 115 120

GAA GTC GGT TTC GAT TCG GTT GTG ACT GTT TTG GCT TAT GTT TAC AGC 3197 Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser 125 130 135

AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT GAA TGC GCA GAC GAG 3245 Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu 140 145 150

AAT TGC TGC CAC GTG GCT TGC CGG CCG GCG GTG GAT TTC ATG TTG GAG 3293 Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu 155 160 165

GTT CTC TAT TTG GCT TTC ATC TTC AAG ATC CCT GAA TTA ATT ACT CTC 3341 Val Leu Tyr Leu Ala Phe lie Phe Lys lie Pro Glu Leu lie Thr Leu 170 175 180 185

TAT CAG GTAAAACACC ATCTGCATTA AGCTATGGTT ACACATTCAT GAATATGTTC 3397 Tyr Gin

TTACTTGAGT ACTTGTATTT GTATTTCAG AGG CAC TTA TTG GAC GTT GTA GAC 3450 Arg His Leu Leu Asp Val Val Asp

190 195

AAA GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA 3498 Lys Val Val lie Glu Asp Thr Leu Val lie Leu Lys Leu Ala Asn lie 200 205 210

TGT GGT AAA GCT TGT ATG AAG CTA TTG GAT AGA TGT AAA GAG ATT ATT 3546 Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu lie lie 215 220 225

GTC AAG TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA 3594 Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu 230 235 240

GAG CTT GTT AAA GAG ATA ATT GAT AGA CGT AAA GAG CTT GGT TTG GAG 3642 Glu Leu Val Lys Glu lie lie Asp Arg Arg Lys Glu Leu Gly Leu Glu 245 250 255

GTA CCT AAA GTA AAG AAA CAT GTC TCG AAT GTA CAT AAG GCA CTT GAC 3690 Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp 260 265 270 275

TCG GAT GAT ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG GAT CAC ACC 3738 Ser Asp Asp lie Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr 280 285 290

AAT CTA GAT GAT GCG TGT GCT CTT CAT TTC GCT GTT GCA TAT TGC AAT 3786 Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn 295 300 305

GTG AAG ACC GCA ACA GAT CTT TTA AAA CTT GAT CTT GCC GAT GTC AAC 3834 Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn 310 315 320

CAT AGG AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG 3882 His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg 325 330 335

AAG GAG CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA 3930 Lys Glu Pro Gin Leu lie Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala 340 345 350 355

TCA GAA GCA ACT TTG GAA GGT AGA ACC GCA CTC ATG ATC GCA AAA CAA 3978 Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met lie Ala Lys Gin 360 365 370

GCC ACT ATG GCG GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT 4026 Ala Thr Met Ala Val Glu Cys Asn Asn lie Pro Glu Gin Cys Lys His 375 380 385

TCT CTC AAA GGC CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA 4074 Ser Leu Lys Gly Arg Leu Cys Val Glu lie Leu Glu Gin Glu Asp Lys 390 395 400

CGA GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TTT GCA GTG GCG GCC 4122 Arg Glu Gin lie Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala 405 410 415

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

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

GTATCTATCA AGTCTTATTT CTTATATGTT TGAATTAAAT TTATGTCCTC TCTATTAGGA 4222

AACTGAGTGA ACTAATGATA ACTATTCTTT GTGTCGTCCA CTGTTTAG TT GCA CTT 4278

Val Ala Leu 435

GCT CAA CGT CTT TTT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC 4326 Ala Gin Arg Leu Phe Pro Thr Glu Ala Gin Ala Ala Met Glu lie Ala 440 445 450

GAA ATG AAG GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC 4374 Glu Met Lys Gly Thr Cys Glu Phe lie Val Thr Ser Leu Glu Pro Asp 455 460 465

CGT CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAG ATA GCA CCT 4422 Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys lie Ala Pro 470 475 480

TTC AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA 4470 Phe Arg lie Leu Glu Glu His Gin Ser Arg Leu Lys Ala Leu Ser Lys 485 490 495 ACC G GTATGGATTC TCACCCACTT CATCGGACTC CTTATCACAA AAAACAAAAC 4524

Thr

500

TAAATGATCT TTAAACATGG TTTTGTTACT TGCTGTCTGA CCTTGTTTTT TTTATCATCA 4584

G TG GAA CTC GGG AAA CGA TTC TTC CCG CGC TGT TCG GCA GTG CTC 4629 Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu 505 510 515

GAC CAG ATT ATG AAC TGT GAG GAC TTG ACT CAA CTG GCT TGC GGA GAA 4677 Asp Gin lie Met Asn Cys Glu Asp Leu Thr Gin Leu Ala Cys Gly Glu 520 525 530

GAC GAC ACT GCT GAG AAA CGA CTA CAA AAG AAG CAA AGG TAC ATG GAA 4725 Asp Asp Thr Ala Glu Lys Arg Leu Gin Lys Lys Gin Arg Tyr Met Glu 535 540 545

ATA CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GAA TTA 4773 lie Gin Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn Leu Glu Leu 550 555 560

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

GGT GGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT CGT CGG TGA 4866

Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg Arg * 580 585 590

GACTCTTGCC TCTTAGTGTA ATTTTTGCTG TACCATATAA TTCTGTTTTC ATGATGACTG 4926

TAACTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT 4986

ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG 5046 ATTTGTAATA TATATTTATG TACATCAACA ATAACCCATG ATGGTGTTAC AGAGTTGCTA 5106

GAATCAAAGT GTGAAATAAT GTCAAATTGT TCATCTGTTG GATATTTTCC ACCAAGAACC 5166

AAAAGAATAT TCAAGTTCCC TGAACTTCTG GCAACATTCA TGTTATATGT ATCTTCCTAA 5226

TTCTTCCTTT AACCTTTTGT AACTCGAATT ACACAGCAAG TTAGTTTCAG GTCTAGAGAT 5286

AAGAGAAC C TGAGTGGGCG TGTAAGGTGC ATTCTCCTAG TCAGCTCCAT TGCATCCAAC 5346

ATTTGTGAAT GACACAAGTT AACAATCCTT TGCACCATTT CTGGGTGCAT ACATGGAAAC 5406

TTCTTCGATT GAAACTTCCC ACATGTGCAG GTGCGTTCGC TGTCACTGAT AGACCAAGAG 5466

ACTGAAAGCT TTCACAAATT GCCCTCAAAT CTTCTGTTTC TATCGTCATG ACTCCATATC 5526

TCCGACCACT GGTCATGAGC CAGAGCCCAC TGATTTTGAG GGAATTGGGC TAACCATTTC 5586

CGAGCTTCTG AGTCCTTCTT TTTGATGTCC TTTATGTAGG AATCAAATTC TTCCTTCTGA 5646

CTTGTGGAT 5655

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 594 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Met Asp Thr Thr lie Asp Gly Phe Ala Asp Ser Tyr Glu lie Ser Ser 1 5 10 15

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

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

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

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

Arg Cys Val Leu Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala

85 90 95

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

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

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

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

Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe lie

165 170 175

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

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

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

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

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

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

Ala Leu Asp Ser Asp Asp lie Glu Leu Val Lys Leu Leu Leu Lys Glu 275 280 285

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

Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala 305 310 315 320

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

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

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

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

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

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

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

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

Glu lie Ala Glu Met Lys Gly Thr Cys Glu Phe lie Val Thr Ser Leu 450 455 460

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

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

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

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

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

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

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

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

Arg

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : not relevant

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

He Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp He Glu Leu Val 1 5 10 15

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

Val His Tyr Ala Val Gin His Cys Asn 35 40

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Pro Thr Gly Lys Thr Ala Leu His Leu Ala Ala Glu Met Val Ser Pro 1 5 10 15

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

Thr Xaa Asp Gly Val Thr 35

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

He Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp He Glu Leu Val 1 5 10 15

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

Val His Tyr Ala Val Gin His Cys Asn 35 40

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) 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 1 5 10 15

Ser Pro Asp Met Val Ser Val Leu Leu Asp Gin 20 25

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

He Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp He Glu Leu Val 1 5 10 15

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

Val His Tyr Ala Val Gin His Cys Asn 35 40

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: not relevant

(D) 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 15

Ser Pro Asp Met Val Ser Val Leu Leu Asp Gin 20 25

(2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

He Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp He Glu Leu Val

1 5 10 15

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

Val His Tyr Ala Val Gin His Cys Asn 35 40

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: peptide (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 15

Asp Met Val

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "oiigonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

AATTCTAAAG CATGCCGATC GG 22

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oiigonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

AATTCCGATC GGCATGCTTT A 21

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "oiigonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

AATTCTAAAC CATGGCGATC GG 22

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oiigonucleotide' (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

AATTCCGATC GCCATGGTTT A 21

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "oiigonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

CCAGCTGGAA TTCCG 15

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oiigonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

CGGAATTCCA GCTGGCATG 19

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 314 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

Met Phe Gin Pro Ala Gly His Gly Gin Asp Trp Ala Met Glu Gly Pro 1 5 10 15

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

Gly Leu Asp Ser Met Lys Asp Glu Glu Tyr Glu Gin Met Val Lys Glu

35 40 45

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

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

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

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

Thr Pro Leu His Leu Ala Val He Thr Asn Gin Pro Gly He Ala Glu 115 120 125

Ala Leu Leu Lys Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly 130 135 140

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

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

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

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

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

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

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

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

Thr Leu Glu Asn Leu Gin 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

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 314 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant (D) 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 15

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

Gly Leu Asp Ser Met Lys Asp Glu Asp Tyr Glu Gin Met Val Lys Glu 35 40 45 Leu Arg Glu He Arg Leu Gin Pro Gin Glu Ala Pro Leu Ala Ala Glu 50 55 60

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

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

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

Thr Pro Leu His Leu Ala Val He Thr Asn Gin Pro Gly He Ala Glu 115 120 125

Ala Leu Leu Lys Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly 130 135 140

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

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

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

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

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

Val Asp Leu Gin Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly 225 230 235 240 Ala Asp Val Asn Arg Val Thr Tyr Gin Gly Tyr Ser Pro Tyr Gin Leu 245 250 255

Thr Trp Gly Arg Pro Ser Thr Arg He Gin Gin Gin Leu Gly Gin Leu

260 265 270

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

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

Cys Val Phe Gly Gly Gin Arg Leu Thr Leu 305 310

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 314 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

Met Phe Gin Pro Ala Glu Pro Gly Gin Glu Trp Ala Met Glu Gly Pro 1 5 10 15

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

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

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

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

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

Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gin Asn Asn Leu Gin Gin

100 105 110

Thr Pro Leu His Leu Ala Val He Thr Asn Gin Pro Glu He Ala Glu 115 120 125

Ala Leu Leu Glu Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly 130 135 140

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

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

Gin Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser He

180 185 190

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

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

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

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

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

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

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

Cys Val Leu Gly Gly Gin Arg Leu Thr Leu 305 310

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2011 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Arabidopsis thaliana

(ix) FEATURE:

(A) NAME/KEY: misc_feature (B) LOCATION : 1 . .2011

(D) OTHER INFORMATION: /note= "NIMl cDNA sequence"

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 43..1824

(D) OTHER INFORMATION: /product= "NIMl protein"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC 54

Met Asp Thr Thr 1

ATT GAT GGA TTC GCC GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC GTC 102 He Asp Gly Phe Ala Asp Ser Tyr Glu He Ser Ser Thr Ser Phe Val 5 10 15 20

GCT ACC GAT AAC ACC GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA 150 Ala Thr Asp Asn Thr Asp Ser Ser He Val Tyr Leu Ala Ala Glu Gin 25 30 35

GTA CTC ACC GGA CCT GAT GTA TCT GCT CTG CAA TTG CTC TCC AAC AGC 198 Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gin Leu Leu Ser Asn Ser

40 45 50

TTC GAA TCC GTC TTT GAC TCG CCG GAT GAT TTC TAC AGC GAC GCT AAG 246 Phe Glu Ser Val Phe Asp Ser Pro Asp Asp Phe Tyr Ser Asp Ala Lys 55 60 65

CTT GTT CTC TCC GAC GGC CGG GAA GTT TCT TTC CAC CGG TGC GTT TTG 294 Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His Arg Cys Val Leu 70 75 80

TCA GCG AGA AGC TCT TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAG 342 Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala Ala Ala Lys Lys 85 90 95 100

GAG AAA GAC TCC AAC AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG 390 Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu

105 110 115

ATT GCC AAG GAT TAC GAA GTC GGT TTC GAT TCG GTT GTG ACT GTT TTG 438 He Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu 120 125 130

GCT TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT 486 Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser 135 140 145

GAA TGC GCA GAC GAG AAT TGC TGC CAC GTG GCT TGC CGG CCG GCG GTG 534 Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val 150 155 160

GAT TTC ATG TTG GAG GTT CTC TAT TTG GCT TTC ATC TTC AAG ATC CCT 582 Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe He Phe Lys He Pro 165 170 175 180

GAA TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA 630 Glu Leu He Thr Leu Tyr Gin Arg His Leu Leu Asp Val Val Asp Lys

185 190 195

GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT 678 Val Val He Glu Asp Thr Leu Val He Leu Lys Leu Ala Asn He Cys 200 205 210

GGT AAA GCT TGT ATG AAG CTA TTG GAT AGA TGT AAA GAG ATT ATT GTC 726 Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu He He Val 215 220 225

AAG TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA GAG 774 Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu 230 235 240

CTT GTT AAA GAG ATA ATT GAT AGA CGT AAA GAG CTT GGT TTG GAG GTA 822 Leu Val Lys Glu He He Asp Arg Arg Lys Glu Leu Gly Leu Glu Val 245 250 255 260

CCT AAA GTA AAG AAA CAT GTC TCG AAT GTA CAT AAG GCA CTT GAC TCG 870 Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser 265 270 275

GAT GAT ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG GAT CAC ACC AAT 918 Asp Asp He Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn 280 285 290

CTA GAT GAT GCG TGT GCT CTT CAT TTC GCT GTT GCA TAT TGC AAT GTG 966 Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val 295 300 305

AAG ACC GCA ACA GAT CTT TTA AAA CTT GAT CTT GCC GAT GTC AAC CAT 1014 Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His 310 315 320

AGG AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG AAG 1062 Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys 325 330 335 340

GAG CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA 1110 Glu Pro Gin Leu He Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser 345 350 355

GAA GCA ACT TTG GAA GGT AGA ACC GCA CTC ATG ATC GCA AAA CAA GCC 1158 Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met He Ala Lys Gin Ala 360 365 370

ACT ATG GCG GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT TCT 1206 Thr Met Ala Val Glu Cys Asn Asn He Pro Glu Gin Cys Lys His Ser 375 380 385

CTC AAA GGC CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA CGA 1254 Leu Lys Gly Arg Leu Cys Val Glu He Leu Glu Gin Glu Asp Lys Arg 390 395 400

GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TTT GCA GTG GCG GCC GAT 1302 Glu Gin He Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala Asp 405 410 415 420

GAA TTG AAG ATG ACG CTG CTC GAT CTT GAA AAT AGA GTT GCA CTT GCT 1350 Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala 425 430 435

CAA CGT CTT TTT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC GAA 1398 Gin Arg Leu Phe Pro Thr Glu Ala Gin Ala Ala Met Glu He Ala Glu 440 445 450

ATG AAG GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC CGT 1446 Met Lys Gly Thr Cys Glu Phe He Val Thr Ser Leu Glu Pro Asp Arg 455 460 465

CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAG ATA GCA CCT TTC 1494 Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys He Ala Pro Phe 470 475 480

AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA ACC 1542 Arg He Leu Glu Glu His Gin Ser Arg Leu Lys Ala Leu Ser Lys Thr 485 490 495 500

GTG GAA CTC GGG AAA CGA TTC TTC CCG CGC TGT TCG GCA GTG CTC GAC 1590 Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu Asp 505 510 515

CAG ATT ATG AAC TGT GAG GAC TTG ACT CAA CTG GCT TGC GGA GAA GAC 1638 Gln He Met Asn Cys Glu Asp Leu Thr Gin Leu Ala Cys Gly Glu Asp 520 525 530

GAC ACT GCT GAG AAA CGA CTA CAA AAG AAG CAA AGG TAC ATG GAA ATA 1686 Asp Thr Ala Glu Lys Arg Leu Gin Lys Lys Gin Arg Tyr Met Glu He 535 540 545

CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GAA TTA GGA 1734 Gin Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn Leu Glu Leu Gly 550 555 560

AAT TTG TCC CTG ACA GAT TCG ACT TCT TCC ACA TCG AAA TCA ACC GGT 1782

Asn Leu Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser Lys Ser Thr Gly

565 570 575 580

GGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT CGT CGG TGA 1824

Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg Arg * 585 590

GACTCTTGCC TCTTAGTGTA ATTTTTGCTG TACCATATAA TTCTGTTTTC ATGATGACTG 1884

TAACTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT 1944

ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG 2004

ATTTGTA 2011

(2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2011 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION: 43..1824

(D) OTHER INFORMATION: /product= "altered form of NIMl' /note= "Serine residues at amino acid positions 55 and 59 in wild-type NIMl gene product have been changed to Alanine residues . "

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 205..217 (D) 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 54

Met Asp Thr Thr

1

GCT ACC GAT AAC ACC GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA 150 Ala Thr Asp Asn Thr Asp Ser Ser He Val Tyr Leu Ala Ala Glu Gin

25 30 35

GTA CTC ACC GGA CCT GAT GTA TCT GCT CTG CAA TTG CTC TCC AAC AGC 198 Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gin Leu Leu Ser Asn Ser 40 45 50 TTC GAA GCC GTC TTT GAC GCG CCG GAT GAT TTC TAC AGC GAC GCT AAG 246 Phe Glu Ala Val Phe Asp Ala Pro Asp Asp Phe Tyr Ser Asp Ala Lys 55 60 65

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

ATT GCC AAG GAT TAC GAA GTC GGT TTC GAT TCG GTT GTG ACT GTT TTG 438

He Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu

120 125 130

GAA TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA 630 Glu Leu He Thr Leu Tyr Gin Arg His Leu Leu Asp Val Val Asp Lys 185 190 195 GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT 678 Val Val He Glu Asp Thr Leu Val He Leu Lys Leu Ala Asn He Cys 200 205 210

AGG AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG AAG 1062 Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys 325 330 335 340 GAG CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA 1110 Glu Pro Gin Leu He Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser 345 350 355

CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAG ATA GCA CCT TTC 1494 Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys He Ala Pro Phe 470 475 480 AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA ACC 1542 Arg He Leu Glu Glu His Gin Ser Arg Leu Lys Ala Leu Ser Lys Thr 485 490 495 500

CAG ATT ATG AAC TGT GAG GAC TTG ACT CAA CTG GCT TGC GGA GAA GAC 1638 Gin He Met Asn Cys Glu Asp Leu Thr Gin Leu Ala Cys Gly Glu Asp 520 525 530

AAT TTG TCC CTG ACA GAT TCG ACT TCT TCC ACA TCG AAA TCA ACC GGT 1782 Asn Leu Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser Lys Ser Thr Gly 565 570 575 580

GGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT CGT CGG TGA 1824

Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg Arg * 585 590

GACTCTTGCC TCTTAGTGTA ATTTTTGCTG TACCATATAA TTCTGTTTTC ATGATGACTG 1884

TAACTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT 1944

ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG 2004

ATTTGTA 2011 (2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 594 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

Met Asp Thr Thr He Asp Gly Phe Ala Asp Ser Tyr Glu He Ser Ser 1 5 10 15

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

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

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

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

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

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

Glu Leu Lys Glu He Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val 115 120 125 Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro 130 135 140

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

Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe He 165 170 175

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

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

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

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

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

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

Ala Leu Asp Ser Asp Asp He Glu Leu Val Lys Leu Leu Leu Lys Glu 275 280 285

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

Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala 305 310 315 320 Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala 325 330 335

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

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

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

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

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

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

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

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

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

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

Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser 500 505 510 Ala Val Leu Asp Gin He Met Asn Cys Glu Asp Leu Thr Gin Leu Ala 515 520 525

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

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

Leu Glu Leu Gly Asn Leu Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser

565 570 575

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

Arg

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1597 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1410

(D) OTHER INFORMATION: /product_^ "Altered form of NIMl' /note= "N-terminal deletion compared to wild-type NIMl sequence . " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

ATG GAT TCG GTT GTG ACT GTT TTG GCT TAT GTT TAC AGC AGC AGA GTG 48 Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val 1 5 10 15

AGA CCG CCG CCT AAA GGA GTT TCT GAA TGC GCA GAC GAG AAT TGC TGC 96 Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys 20 25 30

CAC GTG GCT TGC CGG CCG GCG GTG GAT TTC ATG TTG GAG GTT CTC TAT 144 His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr 35 40 45

TTG GCT TTC ATC TTC AAG ATC CCT GAA TTA ATT ACT CTC TAT CAG AGG 192 Leu Ala Phe He Phe Lys He Pro Glu Leu He Thr Leu Tyr Gin Arg 50 55 60

CAC TTA TTG GAC GTT GTA GAC AAA GTT GTT ATA GAG GAC ACA TTG GTT 240 His Leu Leu Asp Val Val Asp Lys Val Val He Glu Asp Thr Leu Val 65 70 75 80

ATA CTC AAG CTT GCT AAT ATA TGT GGT AAA GCT TGT ATG AAG CTA TTG 288 He Leu Lys Leu Ala Asn He Cys Gly Lys Ala Cys Met Lys Leu Leu

85 90 95

GAT AGA TGT AAA GAG ATT ATT GTC AAG TCT AAT GTA GAT ATG GTT AGT 336 Asp Arg Cys Lys Glu He He Val Lys Ser Asn Val Asp Met Val Ser 100 105 110

CTT GAA AAG TCA TTG CCG GAA GAG CTT GTT AAA GAG ATA ATT GAT AGA 384 Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu He He Asp Arg 115 120 125

CGT AAA GAG CTT GGT TTG GAG GTA CCT AAA GTA AAG AAA CAT GTC TCG 432 Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser 130 135 140

AAT GTA CAT AAG GCA CTT GAC TCG GAT GAT ATT GAG TTA GTC AAG TTG 480 Asn Val His Lys Ala Leu Asp Ser Asp Asp He Glu Leu Val Lys Leu 145 150 155 160

CTT TTG AAA GAG GAT CAC ACC AAT CTA GAT GAT GCG TGT GCT CTT CAT 528 Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His 165 170 175

TTC GCT GTT GCA TAT TGC AAT GTG AAG ACC GCA ACA GAT CTT TTA AAA 576 Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys 180 185 190

CTT GAT CTT GCC GAT GTC AAC CAT AGG AAT CCG AGG GGA TAT ACG GTG 624 Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val 195 200 205

CTT CAT GTT GCT GCG ATG CGG AAG GAG CCA CAA TTG ATA CTA TCT CTA 672 Leu His Val Ala Ala Met Arg Lys Glu Pro Gin Leu He Leu Ser Leu 210 215 220

TTG GAA AAA GGT GCA AGT GCA TCA GAA GCA ACT TTG GAA GGT AGA ACC 720 Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr 225 230 235 240

GCA CTC ATG ATC GCA AAA CAA GCC ACT ATG GCG GTT GAA TGT AAT AAT 768 Ala Leu Met He Ala Lys Gin Ala Thr Met Ala Val Glu Cys Asn Asn 245 250 255

ATC CCG GAG CAA TGC AAG CAT TCT CTC AAA GGC CGA CTA TGT GTA GAA 816 He Pro Glu Gin Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu 260 265 270

ATA CTA GAG CAA GAA GAC AAA CGA GAA CAA ATT CCT AGA GAT GTT CCT 864 He Leu Glu Gin Glu Asp Lys Arg Glu Gin He Pro Arg Asp Val Pro 275 280 285

CCC TCT TTT GCA GTG GCG GCC GAT GAA TTG AAG ATG ACG CTG CTC GAT 912 Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp 290 295 300

CTT GAA AAT AGA GTT GCA CTT GCT CAA CGT CTT TTT CCA ACG GAA GCA 960 Leu Glu Asn Arg Val Ala Leu Ala Gin Arg Leu Phe Pro Thr Glu Ala 305 310 315 320

CAA GCT GCA ATG GAG ATC GCC GAA ATG AAG GGA ACA TGT GAG TTC ATA 1008 Gin Ala Ala Met Glu He Ala Glu Met Lys Gly Thr Cys Glu Phe He 325 330 335

GTG ACT AGC CTC GAG CCT GAC CGT CTC ACT GGT ACG AAG AGA ACA TCA 1056 Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser 340 345 350

CCG GGT GTA AAG ATA GCA CCT TTC AGA ATC CTA GAA GAG CAT CAA AGT 1104 Pro Gly Val Lys He Ala Pro Phe Arg He Leu Glu Glu His Gin Ser 355 360 365

AGA CTA AAA GCG CTT TCT AAA ACC GTG GAA CTC GGG AAA CGA TTC TTC 1152 Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe 370 375 380

CCG CGC TGT TCG GCA GTG CTC GAC CAG ATT ATG AAC TGT GAG GAC TTG 1200 Pro Arg Cys Ser Ala Val Leu Asp Gin He Met Asn Cys Glu Asp Leu 385 390 395 400

ACT CAA CTG GCT TGC GGA GAA GAC GAC ACT GCT GAG AAA CGA CTA CAA 1248 Thr Gin Leu Ala Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gin 405 410 415

AAG AAG CAA AGG TAC ATG GAA ATA CAA GAG ACA CTA AAG AAG GCC TTT 1296 Lys Lys Gin Arg Tyr Met Glu He Gin Glu Thr Leu Lys Lys Ala Phe 420 425 430

AGT GAG GAC AAT TTG GAA TTA GGA AAT TTG TCC CTG ACA GAT TCG ACT 1344 Ser Glu Asp Asn Leu Glu Leu Gly Asn Leu Ser Leu Thr Asp Ser Thr 435 440 445

TCT TCC ACA TCG AAA TCA ACC GGT GGA AAG AGG TCT AAC CGT AAA CTC 1392 Ser Ser Thr Ser Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu 450 455 460

TCT CAT CGT CGT CGG TGA GACTCTTGCC TCTTAGTGTA ATTTTTGCTG 1440

Ser His Arg Arg Arg * 465 470

TACCATATAA TTCTGTTTTC ATGATGACTG TAACTGTTTA TGTCTATCGT TGGCGTCATA 1500

TAGTTTCGCT CTTCGTTTTG CATCCTGTGT ATTATTGCTG CAGGTGTGCT TCAAACAAAT 1560

GTTGTAACAA TTTGAACCAA TGGTATACAG ATTTGTA 1597

(2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 470 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val 1 5 10 15 Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys 20 25 30

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

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

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

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

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

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

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

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

Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His 165 170 175

Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys 180 185 190

Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val 195 200 205 Leu His Val Ala Ala Met Arg Lys Glu Pro Gin Leu He Leu Ser Leu 210 215 220

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

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

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

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

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

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

Gin Ala Ala Met Glu He Ala Glu Met Lys Gly Thr Cys Glu Phe He 325 330 335

Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser 340 345 350

Pro Gly Val Lys He Ala Pro Phe Arg He Leu Glu Glu His Gin Ser 355 360 365

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

Pro Arg Cys Ser Ala Val Leu Asp Gin He Met Asn Cys Glu Asp Leu 385 390 395 400 Thr Gin Leu Ala Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gin 405 410 415

Lys Lys Gin Arg Tyr Met Glu He Gin Glu Thr Leu Lys Lys Ala Phe 420 425 430

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

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

Ser His Arg Arg Arg * 465 470

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1608 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 43..1608 (D) OTHER INFORMATION: /product= "Altered form of NIMl"

/note= "C-terminal deletion compared to wild-type NIMl."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC 54 Met Asp Thr Thr

1

_ATT GAT GGA TTC GCC GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC GTC 102 He Asp Gly Phe Ala Asp Ser Tyr Glu He Ser Ser Thr Ser Phe Val 5 10 15 20

GTA CTC ACC GGA CCT GAT GTA TCT GCT CTG CAA TTG CTC TCC AAC AGC 198 Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gin Leu Leu Ser Asn Ser 40 45 50

GAA TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA 630 Glu Leu He Thr Leu Tyr Gin Arg His Leu Leu Asp Val Val Asp Lys 185 190 195

GTT GTT ATA GAC GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT 678 Val Val He Glu Asp Thr Leu Val He Leu Lys Leu Ala Asn He Cys 200 205 210

AGG AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG AAG 1062

Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys 325 330 335 340

GAG CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA 1110

Glu Pro Gin Leu He Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser 345 350 355

CAG ATT ATG AAC TGT TGA 1608 Gin He Met Asn Cys * 520

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 522 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

Met Asp Thr Thr He Asp Gly Phe Ala Asp Ser Tyr Glu He Ser Ser 1 5 10 15

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

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

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

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

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

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

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

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

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

Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe He 165 170 175 Phe Lys He Pro Glu Leu He Thr Leu Tyr Gin Arg His Leu Leu Asp 180 185 190

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

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

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

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

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

Ala Leu Asp Ser Asp Asp He Glu Leu Val Lys Leu Leu Leu Lys Glu 275 280 285

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

Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala 305 310 315 320

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

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

Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met He 355 360 365 Ala Lys Gin Ala Thr Met Ala Val Glu Cys Asn Asn He Pro Glu Gin 370 375 380

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

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

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

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

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

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

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

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

Ala Val Leu Asp Gin He Met Asn Cys * 515 520

(2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1194 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1194

(D) OTHER INFORMATION: /product= "Altered form of NIMl" /note= "N-terminal/C-terminal chimera."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

AGA CCG CCG CCT AAA GGA GTT TCT GAA TGC GCA GAC GAG AAT TGC TGC 96 Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys

20 25 30

ATA CTC AAG CTT GCT AAT ATA TGT GGT AAA GCT TGT ATG AAG CTA TTG 288 He Leu Lys Leu Ala Asn He Cys Gly Lys Ala Cys Met Lys Leu Leu 85 90 95

CTT TTG AAA GAG GAT CAC ACC AAT CTA GAT GAT GCG TGT GCT CTT CAT 528 Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His

165 170 175

CCG CGC TGT TCG GCA GTG CTC GAC CAG ATT ATG AAC TGT TGA 1194

Pro Arg Cys Ser Ala Val Leu Asp Gin He Met Asn Cys * 385 390 395

(2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 398 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val 1 5 10 15

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

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

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

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

He Leu Lys Leu Ala Asn He Cys Gly Lys Ala Cys Met Lys Leu Leu 85 90 95 Asp Arg Cys Lys Glu He He Val Lys Ser Asn Val Asp Met Val Ser 100 105 110

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

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

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

Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His 165 170 175

Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys 180 185 190

Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val 195 200 205

Leu His Val Ala Ala Met Arg Lys Glu Pro Gin Leu He Leu Ser Leu 210 215 220

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

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

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

He Leu Glu Gin Glu Asp Lys Arg Glu Gin He Pro Arg Asp Val Pro 275 280 285 Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp 290 295 300

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

Gin Ala Ala Met Glu He Ala Glu Met Lys Gly Thr Cys Glu Phe He 325 330 335

Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser 340 345 350

Pro Gly Val Lys He Ala Pro Phe Arg He Leu Glu Glu His Gin Ser 355 360 365

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

Pro Arg Cys Ser Ala Val Leu Asp Gin He Met Asn Cys * 385 390 395

(2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 786 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 1..786

(D) OTHER INFORMATION: /product= "Altered form of NIMl' /note= "Ankyrin domains of NIMl.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

ATG GAC TCC AAC AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG ATT 48 Met Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu He 1 5 10 15

GCC AAG GAT TAC GAA GTC GGT TTC GAT TCG GTT GTG ACT GTT TTG GCT 96 Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Ala 20 25 30

TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT GAA 144 Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu 35 40 45

TGC GCA GAC GAG AAT TGC TGC CAC GTG GCT TGC CGG CCG GCG GTG GAT 192 Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp 50 55 60

TTC ATG TTG GAG GTT CTC TAT TTG GCT TTC ATC TTC AAG ATC CCT GAA 240 Phe Met Leu Glu Val Leu Tyr Leu Ala Phe He Phe Lys He Pro Glu 65 70 75 80

TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA GTT 288 Leu He Thr Leu Tyr Gin Arg His Leu Leu Asp Val Val Asp Lys Val 85 90 95

GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT GGT 336 Val He Glu Asp Thr Leu Val He Leu Lys Leu Ala Asn He Cys Gly 100 105 110

AAA GCT TGT ATG AAG CTA TTG GAT AGA TGT AAA GAG ATT ATT GTC AAG 384 Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu He He Val Lys 115 120 125 TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA GAG CTT 432 Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu 130 135 140

GTT AAA GAG ATA ATT GAT AGA CGT AAA GAG CTT GGT TTG GAG GTA CCT 480 Val Lys Glu He He Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro 145 150 155 160

AAA GTA AAG AAA CAT GTC TCG AAT GTA CAT AAG GCA CTT GAC TCG GAT 528 Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser Asp 165 170 175

GAT ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG GAT CAC ACC AAT CTA 576 Asp He Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Leu 180 185 190

GAT GAT GCG TGT GCT CTT CAT TTC GCT GTT GCA TAT TGC AAT GTG AAG 624 Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val Lys 195 200 205

ACC GCA ACA GAT CTT TTA AAA CTT GAT CTT GCC GAT GTC AAC CAT AGG 672 Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His Arg 210 215 220

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

CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA GAA 768 Pro Gin Leu He Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu 245 250 255

GCA ACT TTG GAA GGT TGA 786 Ala Thr Leu Glu Gly * 260 (2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 262 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

Met Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu He 1 5 10 15

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

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

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

Phe Met Leu Glu Val Leu Tyr Leu Ala Phe He Phe Lys He Pro Glu 65 70 75 80

Leu He Thr Leu Tyr Gin Arg His Leu Leu Asp Val Val Asp Lys Val 85 90 95

Val He Glu Asp Thr Leu Val He Leu Lys Leu Ala Asn He Cys Gly 100 105 110

Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu He He Val Lys 115 120 125 Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu 130 135 140

Val Lys Glu He He Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro 145 150 155 160

Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser Asp 165 170 175

Asp He Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Leu 180 185 190

Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val Lys 195 200 205

Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His Arg 210 215 220

Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys Glu 225 230 235 240

Pro Gin Leu He Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu 245 250 255

Ala Thr Leu Glu Gly * 260

(2) INFORMATION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "oiigonucleotide'

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

CAACAGCTTC GAAGCCGTCT TTGACGCGCC GGATG 35

(2) INFORMATION FOR SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "oiigonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

CATCCGGCGC GTCAAAGACG GCTTCGAAGC TGTTG 35

(2) INFORMATION FOR SEQ ID NO: 34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oiigonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

GGAATTCAAT GGATTCGGTT GTGACTGTTT TG 32

(2) INFORMATION FOR SEQ ID NO: 35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "oiigonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

GGAATTCTAC AAATCTGTAT ACCATTGG 28

(2) INFORMATION FOR SEQ ID NO: 36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:

CGGAATTCGA TCTCTTTAAT TTGTGAATTT C 31

(2) INFORMATION FOR SEQ ID NO: 37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "oiigonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

GGAATTCTCA ACAGTTCATA ATCTGGTCG 29

(2) INFORMATION FOR SEQ ID NO: 38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

GGAATTCAAT GGACTCCAAC AACACCGCCG C 31

(2) INFORMATION FOR SEQ ID NO: 39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "oiigonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:

GGAATTCTCA ACCTTCCAAA GTTGCTTCTG ATG 33

American Type Culture Collection 12 JO I r.rkl.wp Drive • Roekvll , MD 2085- USA • Telephone: (301)2.1-5520 Telex: 198459 ATCCNORTH • FAX: 301-770-1517

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF c i & fλ - ' ^

THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERN A TIONA L FORM

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT ISSUED PU RSUANT TO RULE 7.3 AND VIABILITY STATEMENT ISSUED PURSUANT TO RULE 10.2

^"HEIVED

To: (Name and Address of Depositor or Attorney)

Ciba-Geigy Corporation MAY 2 8 1936

Attn: Leslie B. Friedrich CIBA-GEIGY

P.O. Box 1 2257 ABRU PATENT DEPT.

Research Triangle Park, NC 27709

Deposited on Behalf of: Ciba-Geigy Corporation

Identification Reference by Depositor: ATCC Designation

Plasmid, BAC4 971543

The deposit was accompanied by: a scientific description _ a proposnd taxonomic description Indicated above.

The deposit was received May 8. I 99fi by this International Depository Authority and has been accepted.

AT YOUR REQUEST: X. We will Inform you of requests for the strain for 30 years.

The strain will be made available if a patent office signatory to the Budapest Treaty certifies one's right to receive or if a U.S. Patent is issued citing the strain, and ATCC is instructed by the United States Patent & Trademark Office or the depositor to release said strain

If the culture should die or be destroyed during the effective term of the deposit, it shall be your responsibility to replace it with living culture of the same .

The strain will be maintained for a period of at least 30 years from date of deposit, or five years after the most recent request for a sample, whichever is longer. The United States and many other countries are signatory to the Budapest Treaty.

The viability of the culture cited above was tested Ma 1 7. T 996. On that date, the culture was viable.

International Depository Authority: American Type Culture Collection. Bockville. Md. 20852 USA

S Siiggnnaattuurree ooff ppeerrssoonn hhaavviing authority to represent ATCC: Date: Mπy 70. 1 996

Barbara . Hailβy, Administrator, Pa nt Depository cc: Andrea C. Walsh, Ph.D. American Type Culture Collection 12301 P.rkl.wn DrUe • RockviUe, MD 2MSJ USA • Telephone: (301)231-5520 Telex: 191-tSS ATCCNORTH • FAX: 34I-770-2587

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERN A TIONAL FORM

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT ISSUED PURSUANT TO RULE 7.3 AND VIABILITY STATEMENT ISSUED PURSUANT TO RULE 10.2

RECEIVED

To: (Name and Address of Depositor or Attorney)

Deposited on Behalf of: Ciba-Geigy Corporation

Identification Reference by Depositor: ATCC Designation

Plasmid P1 -1 Θ 97606

The deposit was accompanied by: a scientific description _ a proposed taxonomic description indicated above.

The deposit was received June 13. 1996 by this International Depository Authority and has been accepted.

AT YOUR REQUEST: X. We will inform you of requests for the sti ain for 30 years.

The strain will be made available if a patent office signatory to the Budapest Treaty certifies one's right to receive, or if β U.S. Patent is issued citing the strain, end ATCC is Instructed by the United States Patent & Trademark Office or the depositor to release said strain.

If the culture should die or be destroyed during the effective term of the deposit, it shall be your responsibility to replace it with living culture of the same.

The viability of the culture cited above was tested June ?0. 1996. On ihat date, the culture was viable.

International Depository Authority: American Type Culture Collection, Rockville, Md. 20852 USA

Signature of person having authority to represent ATCC:

Date: Joins. ,21 , 1996

Barbara M. Hailey, Administrator, Patent Depository cc: Andrea C. Walsh. Ph.D.

12301 p.r.l.wn Drive • Roek-II , MD 20(52 USA • Telephone: <J0l):3l-SS20 Telex: WW-76- ATCCROVE * FAX: 30I-SK-UΛ6

BU DAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERN A TIONΛL FORM

RECEIPT IN TH E CASE OF AN ORIGINAL DEPOSIT ISSUED PURSUANT TO RULE 7 3 AND VIABILITY STATEMENT ISSUED PURSUANT TO RULE 10.2

To: (Name and Address of Depositor or Attorney)

Ciba-Geigy Corporation Attention: Leslie B. Friedrich P.O. Box 12257 Research Triangle Park, NC 27709

Deposited on Bohalf of: Ciba-Geigy Corporation

Identification Reference by Depositor: ATCC Designation

Cosmid, D7 97736

The deposit was accompanied by: a scientific description a proposed taxonomic description indicated above.

The deposit was received September 2 S. I 99fi by this International Depository Authority and has been accepted.

AT YOUR REQUEST:

_X_ We will inform you of requests for the strain for 30 years.

The strain will be made available if a patent office signatory to the Budapest 1 reaty certifies one's right to receive, or if a U.S. Patent is issued cuing the strain, and ATCC is instructed by th« United States Patent & Trademark Office or the depositor to release said strain.

If the culture should die or bo destroyed during the effective term of the deposit, it shall be your responsibility to replace it with living culture of the same

The strain will be meintained for a period of at least 30 years after the date oi deposit, and for a period of at least five years after the most recent request for a sample. Tho United States and many other countries are signatory to the Budapost Troaty.

The viability of the culture cited above was tested October 3, 1396. On that date, the culture was viable.

International Depository Authority: American Type Culture Collection, Roc ville, Md. 20852 USA

^Igna urβ of person having authority to represent

Date: October 7 1 996 cc: Andrea C. Walsh, Ph.D.

Claims

What is claimed is:

1. A DNA molecule that encodes an altered form of a NIM1 protein.

2. The DNA molecule according to claim 1 , that acts as a dominant-negative regulator of the SAR signal transduction pathway.

3. 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.

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

5. The DNA molecule according to claim 4, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:22 and all DNA.

6. The DNA molecule according to claim 1 , wherein the altered form of the NIM 1 protein is a truncated version of the NIM1 gene product.

7. 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.

8. The DNA molecule according to claim 7, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO:25.

9. The DNA molecule according to claim 8, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:24.

10. The DNA molecule according to claim 1 , 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.

11. The DNA molecule according to claim 22, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO:27.

12. The DNA molecule according to claim 23, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:26.

13. 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 ID NO:3.

14. The DNA molecule according to claim 13, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO:29.

15. The DNA molecule according to claim 14, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:28.

16. 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.

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

18. The DNA molecule according to claim 17, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:30.

19. The DNA molecule according to claiml , 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 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.

20. A chimeric gene comprising a promoter active in plants operatively linked to the DNA molecule according to anyone of claims 1 to 19.

21. A recombinant vector comprising the chimeric gene of claim 20, wherein said vector is capable of being stably transformed into a host cell.

22. A method of activating SAR in a plant, comprising transforming the plant with the recombinant vector of claim 21 , wherein said altered form of the NIM1 protein is expressed in said transformed plant and activates SAR in said plant.

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

24. A method of conferring a CIM phenotype to a plant, comprising transforming the plant with the recombinant vector of claim 21 , wherein said altered form of the NIM1 protein is expressed in said transformed plant and confers a CIM phenotype to said plant.

25. A host cell stably transformed with the vector of claim 21.

26. The host cell of claim 25, which is a plant cell.

27. A plant, plant cells and the descendants thereof comprising the chimeric gene of claim 19 which have a broad spectrum of disease resistance.

28. A plant, plant cells and the descendants thereof, wherein a 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.

29. A plant, plant cells and the descendants thereof of claim 27 or 28, wherein said plant is selected form the group consisting of gymnosperms, monocots, and dicots.

30. A plant, plant cells and the descendants thereof of claim 27 or 28, wherein said plant is a crop plant.

31. A plant, plant cells and the descendants thereof of claim 27 or 28, wherein said plant is selected form 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, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.

32. 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.

33. 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 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.

34. 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 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.

35. Use of a transgenic plant or the descendants thereof comprising a chimeric gene according to claim 20 in an agricultural method. .

36. 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.