Plants are constantly challenge 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.. 113, 5-12 (1997), incorporated by reference herein in its entirety ; Bi et al., Plant J. 8, 235-245 (1995), incorporated by reference herein in its entirety ; Mauch-Mani and Slusarenko, Plant Cell 8, 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., 1995PIant Cell7, 1691-1701 (1995), incorporated by reference herein in its entirety ; Vernooij et al., Plant Tell6, 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'Institut 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 Cell3, 1085-1094 (1991), incorporated by reference herein in its entirety). Transgenic disease-resistant plants have been created by transforming plants with various SAR genes (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 Isd (lesions simulating disease) mutants and acd2 (accelerated cell death) (Dietrich et al., Cell77, 551- 563 (1994), incorporated by reference herein in its entirety ; Greenberg et al., Ce//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 Isd mutants have been isolated and characterized (Dietrich et al., 1994 ; Weymann et al., Plant Cell7, 2013-2022 (1995), incorporated by reference herein in its entirety). Another interesting class of mutants are cim (constitutive immunity) mutants (Lawton et al., 1993"The molecular biology of systemic aquired resistance"in Mechanisms of Defence Responses in Plants, B. Fritig and M. Legrand, eds (Dordrecht, The Netherlands : Kluwer Academic Publishers), pp. 422-432 (1993), incorporated by reference herein in its entirety). See also, International PCT Application WO 94/16077, both of which are incorporated by reference entirety herein in their entireties. Like Isd mutants and acd2, cim mutants have elevated SA and SAR gene expression and resistance, but in contrast to Isd or acd2, do not display detectable lesions on their leaves. cprl (constitutive expresser of PR genes) may be a type of cim mutant ; however, because the presence of microscopic lesions on the leaves of cprl has not been ruled out, cprl might be a type of Isd mutant (Bowling et al., Plant Cell 6, 1845-1857 (1994), incorporated by reference herein in its entirety).

Mutants have also been isolated that are blocked in SAR signaling. ndrl Lon-race- specific disease resistance) is a mutant that allows growth of both Pseudomonas syringe containing various virulence genes and also normally virulent 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 NA 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 Cell8, 2033- 2046 (1996), incorporated by reference herein in its entirety). Certain eds mutants are phenotypically very similar to nprl, and, recently, eds5 and eds53 have been shown to be allelic to nprl (Glazebrook et al., 1996). niml (noninducible immunity) is a mutant that supports P. parasitica (i. e., causal agent of downy mildew disease) growth following INA treatment (Delaney et al., 1995 ; International PCT Application WO 94/16077). Although niml can accumulate SA following pathogen infection, it cannot induce SAR gene expression or disease resistance, suggesting that the mutation blocks the pathway downstream of SA. niml is also impaired in its ability to respond to INA or BTH, suggesting that the block exists downstream of the action of these chemicals (Delaney et al., 1995 ; Lawton et al., 1996).

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

NF-KB/IKB Signal Transduction Pathways NF-KB/IKB signaling pathways have been implicated in disease resistance responses in a range of organisms from Drosophila to mammals. In mammals, NF-KB/IKB signal transduction can be induced by a number of different stimuli including exposure of cells to lipopolysaccharide, tumor necrosis factor, interleukin 1 (IL-1), or virus infection (Baeuerle and Baltimore, Cell87, 13-20 (1996) ; Baldwin, Annu. Rev. Immunol. 14, 649-681 (1996)). The activated pathway leads to the synthesis of a number of factors involved in inflammation and immune responses, such as IL-2, IL-6, IL-8 and granulocyte/macrophage- colony stimulating factor (deMartin et al., Gene 152, 253-255 (1995)). In transgenic mouse studies, the knock out of NF-xB/IKB signal transduction leads to a defective immune

response including enhanced susceptibility to bacterial and viral pathogens (Beg and Baltimore, Science 274, 782-784 (1996) ; Van Antwerp et al., Science 274, 787-789 (1996) ; Wang et al., Science 274, 784-787 (1996) ; Baeuerle and Baltimore (1996)). In Arabidopsis, SAR is functionally analogous to inflammation in that normal resistance processes are potentiated following SAR activation leading to enhanced disease resistance (Bi et al., 1995 ; Cao et al., 1994 ; Delaney et al., 1995 ; Delaney et al., 1994 ; Gaffney et al., 1993 ; Mauch-Mani and Slusarenko 1996 ; Delaney, 1997). Furthermore, inactivation of the pathway leads to enhanced susceptibility to bacterial, viral and fungal pathogens.

Interestingly, SA has been reported to block NF-KB activation in mammalian cells (Kopp and Ghosh, Science 265, 956-959 (1994)), while SA activates signal transduction in Arabidopsis. Bacterial infection of Drosophila activates a signal transduction cascade leading to the synthesis of a number of antifungal proteins such as cercropin B, defensin, diptericin and drosomycin (Ip et al., Cell75, 753-763 (1993) ; Lemaitre et al., Cell86, 973- 983 (1996)). This induction is dependent on the gene product of dorsal and dif, two NF-KB homologs, and is repressed by cactus, an IxB 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 complexe 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 sequence : a DNA molecule which, when transcribed and translated, results in the formation of a polypeptide or protein.

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

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

Hence, 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 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 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 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 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 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 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, brocoli, 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-lcB/IKB 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 IxB subclass a. Mutations of IKB have been described that act as super-repressors or dominant-negatives of the NF-xB/IKB regulation scheme. The present invention encompasses altered forms of wild-type NIM1 gene (SEQ NO : 2) that act as dominant-negative regulators of the SAR signal transduction

pathway. These altered forms of NIM1 confer the opposite phenotype in plants transformed therewith as the niml 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 metting temperature Tm which can be easily calculated according to the formula provided in DNA PROBES, George H. Keller and Mark M. Manak, Macmillan Publishers Ltd, 1993, Section one : Molecular Hybridization Technology ; page 8 ff.

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

In one embodiment of the present invention, the NIM1 gene is altered so that the encoded product has alanines instead of serines in the amino acid positions corresponding to positions 55 and 59 of the wild-type Arabidopsis NIM1 amino acid sequence (SEQ ID NO : 3). An example of a preferred embodiment of this altered form of the NIM1 gene, which results in changes of these serine residues to alanine residues, is presented in SEQ ID NO : 22. An exemplary dominant-negative form of the NIM1 protein with alanines instead of serines at amino acid positions 55 and 59 is shown in SEQ ID NO : 23. The present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under moderate stringent conditions to the coding sequence set forth in SEQ ID NO : 22, especially preferred are the following conditions : hybridization in 1 % BSA ; 520mM 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 : 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 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 : 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 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 : 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 NO : 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 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 : 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 agronomicaliy 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 NO : 2 : 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.

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 NO : 3, wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID NO : 22 : hybridization in 1 % BSA ; 520mM 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 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 NO : 3, wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID NO : 26 : hybridization in 1% BSA ; 520mM 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.

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 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.

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 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.

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 niml Mutant An Arabidopsis thaliana mutant called niml (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 niml can accumulate SA following pathogen infection, neither SAR gene expression nor disease resistance can be induced, suggesting that the mutation blocks the pathway downstream of SA. niml is also impaired in its ability to respond to INA or BTH, suggesting that the block exists downstream of the action of these chemicals (Delaney et al., 1995 ; Lawton et al., 1996). This first Arabidopsis niml mutant (herein designated niml-1) was isolated from 80, 000 plants of a T-DNA tagged Arabidopsis ecotype Issilewskija (Ws-0) population by spraying two week old plants with 0. 33 mM INA followed by inoculation with P. parasitica (Delaney et al., 1995). Plants that supported fungal growth after INA treatment were selected as putative mutants. Five additional mutants (herein designated niml-2, niml-3, niml-4, niml-5, and niml-6) were isolated from 280, 000 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 Fi plants were then scored for their ability to support fungal growth following INA treatment. As shown in Table 3 of the Examples, all niml-1, niml-2, niml-3, niml-4, and niml-6Fi plants were phenotypically wild type, indicating a recessive mutation in each line. nim1-5 showed the nim phenotype in all 35 F1 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 Fi plants were phenotypically nim, confirming the dominance of the nim1-5 mutation.

To determine whether the nim 1-2 through nim 1-6 mutations were allelic to the previously characterized niml-1 mutation, pollen from niml-1 was used to fertilize niml-2 through niml-6. Because niml-1 carried resistance to kanamycin, Fi descendants were identified by antibiotic resistance. In all cases, the kanamycin-resistant F1 plants were nim, indicating they were all allelic to the niml-l mutant. Because the niml-5 mutant is dominant and apparently homozygous for the mutation, it was necessary to analyze niml-1 complementation in the F2 generation. If niml-1 and niml-5were 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 niml-5carries a point mutation in the NIM1 gene (infra), it is considered to be a niml 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, nim7-1, niml-2, niml-3, niml-4, and niml-6all supported approximately the same rate of fungal growth, which was somewhat faster than the Ws-0 control. The exception was the nim1-5 plants, in which fungal growth was delayed by several days relative to both the other nim 1 mutants and the Ws-0 control, but eventually all of the nim 1-5 plants succumbed to the fungus. Following SA treatment, the mutants could be grouped into three classes : niml-4 and niml-6 showed a relative rapid fungal growth ; niml-1, niml-2, niml-3 plants exhibited a somewhat slower rate of fungal growth ; and fungal growth in nim 1-5 plants was even slower than in the untreated Ws-0 controls. Following either INA or BTH treatment, the mutants also seemed to fall into three classes where niml-4 was the most severely compromised in its ability to restrict fungal growth following chemical treatment ; niml-1, nim 1-2, nim 1-3, and nim 1-6were alt 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 fall into three classes with nim1-4 being the most severely compromised, niml-1, niml-2, niml-3 and nimi-6 showing an intermediate inhibition of fungus and nie 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 niml-1, niml-2, and niml-3 plants, PR-1 mRNA accumulation was dramatically reduced relative to the wild type following chemical treatment. PR-1 mRNA was also reduced following P. parasitica infection, but there was still some accumulation in these mutants. In the niml-4 and niml-6 plants, PR-1 mRNA accumulation was more dramatically reduced than in the other alleles following chemical treatment (evident in longer exposures) and significantly less PR-1 mRNA accumulated following P. parasitica infection, supporting the idea that these could be particularly strong niml alleles. Interestingly, PR-1 mRNA accumulation was elevated in the nim1-5 mutant, but only mildly induced following chemical treatment or P. parasitica infection. Based on both PR-1 mRNA accumulation and fungal infection, the mutants fall into three classes : severely compromised alleles (niml-4 and niml-6) ; moderately compromised alleles (niml-1, niml-2, and niml-3) ; and a weakly compromised allele (niml-5).

Fine Structure Mapping of the niml Mutation To determine a rough map position for NIM1, 74 F2 nim phenotype plants from a cross between niml-1 (Ws-0) and Landsberg erecta (Leu were identified for their susceptibility to P. parasitica and lack of accumulation of PR-1 mRNA following INA treatment. After testing a number of simple sequence length polymorphism (SSLP) markers (Bell and Ecker 1994), niml was found to lie about 8. 2 centimorgans (cM) from nga128 and 8. 2 cM from nga111 on the lower arm of chromosome 1. In subsequent analysis, niml-1 was found to lie between nga111 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 niml-1 and LerDP23 were identified based on both their inability to accumulate PR-1 mRNA and their ability to support fungal growth following INA treatment.

DNA was extracted from these plants and scored for zygosity at both ATHGENEA and nga111. As shown in Figures 5A-5D, 93 recombinant chromosomes were identified between ATHGENEA and niml, giving a genetic distance of approximately 4. 1 cM (93 of 2276), and 239 recombinant chromosomes were identified between nga111 and niml, indicating a genetic distance of about 10. 5 cM (239 of 2276). Informative recombinants in the ATHGENEA to nga111 interval were further analyzed using amplified fragment length polymorphism (AFLP) analysis (Vos et al., 1995).

Initially, 10 AFLP markers between ATHGENEA and nga111 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 niml) and W85. 1 (0. 6 cM from niml) were used to isolate yeast artificial chromosome (YAC) clones from the CIC (for Centre d'Etude du Polymorphisme Humain, INRA and CNRS) library (Creusot et al., 1995). Two YAC clones, CIC12H07 and CIC12F04, were identified with W84. 2 and two YAC clones CIC7E03 and CIC10G07 (data not shown) were identified with the W85. 1 marker. However, it was determined that there was a gap between the two sets of flanking YAC clones. From this point, bacterial artificial chromosome (BAC) and P1 clones that overlapped CIC12H07 and CIC12F04 were isolated and mapped, and three sequential walking steps were then carried out extending the BAC/P1 contig toward NIM1 (Liu et al., 1995 ; Chio et al., 1995). At various times during the walk, new AFLPs were developed that were specific for BAC or P1 clones, and these were used to determine whether the NIM1 gene had been crossed. It was determined that NIM1 had been crossed when BAC and P1 clones were isolated that gave rise to both AFLP markers L84. 6a and L84. 8. The AFLP marker L84. 6a found on P1 clones P1-18, P1-17, and P1-21 identified three recombinants and L84. 8 found on P1 clones P1-20, P1- 22, P1-23, and P1-24 and BAC clones, BAC-04, BAC-05, and BAC-06 identified one

recombinant. Because these clones overlap to form a large contig (>100 kb), and include AFLP markers that flank niml, the gene was located on the contig. The BAC and P1 clones that comprised the contig were used to generate eight additional AFLP markers, which showed that niml was located between L84. Y1 and L84. 8, representing a gap of about 0. 09 cM.

A cosmid library was constructed in the Agrobacterium-compatible T-DNA cosmid vector pCLD04541 using DNA from BAC-06, BAC-04, and P1-18. A cosmid contig was developed using AFLP markers derived from these clones. Physical mapping showed that the physical distance between L84. Y1 and L84. 8 was greater than 90 kb, giving a genetic to physical distance of roughly 1 megabase per cM. To facilitate the later identification of the NIM1 gene, the DNA sequence of BAC-04 was determined.

Isolation of the NIM1 Gene To identify which cosmids contained the NIM1 gene, the 12 cosmids listed in Table 4 of the Examples were transformed into nit1-1, and transformants were evaluated for their ability to complement the mutant phenotype. Cosmids D5, E1, and D7 were all found to complement niml-1, as determined by the ability of the transformants to accumulate PR-1 mRNA following INA treatment. The ends of these cosmids were sequenced and found to be located on the DNA sequence of BAC-04. There were 9, 918 base pairs in the DNA region shared by D7 and D5 that contained the NIM1 gene. As shown in Figure 5D, four putative gene regions were identified in this 10-kb sequence. Region 1 could potentially encode a protein of 19, 105 D, region 3 could encode a protein of 44, 554 D, and region 4 could encode a protein of 52, 797 D. Region 2 had four open reading frames of various sizes located close together, suggesting a gene with three introns. Analysis using the NetPlantGene program (Hebsgaard et al., 1996) indicated a high probability that the open reading frames could be spliced together to form a large open reading frame encoding a protein of 66, 039 D.

To ascertain which gene region contained the NIM1 gene, gel blots containing RNA isolated from leaf tissue of Ws-0 and the different nim 1 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 niml 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 niml-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 nie7-1, a single adenosine is inserted at position 3579 that causes a frameshift resulting in a change in seven amino acids and a deletion of 349 amino acids. In niml-2, there is a cytidine-to-thymidine transition at position 3763 that changes a histidine to a tyrosine. In niml-3, a single adenosine is deleted at position 3301 causing a frameshift that altered 10 amino acids and deleted 412 from the predicted protein. Interestingly, both nit1-4 and niml-5have a guanosine-to-adenosine transition at position 4160 changing an arginine to a lysine, and in niml-6, there is a cytosine-to-thymidine transition resulting in a stop codon causing the deletion of 255 amino acids from the predicted protein. Although the mutation in niml-4 and niml-5alters 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 a/., 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 BamHl, Hindlll, Xbal, or Sall, 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 NIM1.

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

Homologues may also be obtained by PCR. In this method, comparisons are made between known homologues (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 3rd 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 collecte 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 IxBa The NIM1 gene is a key component of the systemic acquired resistance (SAR) pathway in plants (Ryals et a/., 1996). The NIM1 gene is associated with the activation of SAR by chemical and biological inducers and, in conjunction with such inducers, is required for SAR and SAR gene expression. The location of the NIM1 gene was determined by molecular biological analysis of the genome of mutant plants known to carry the mutant niml gene, which gives the host plants extreme sensitivity to a wide variety of pathogens and renders them unable to respond to pathogens and chemical inducers of SAR. The

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-KB and IKB (Michaely and Bennett, Trends Cell Biol. 2, 127-129 (1992)).

Beyond the ankyrin motif, however, conventional computer analysis did not detect other strong homologies, including homology to licha. 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 licb class of transcription regulators (Baeuerle and Baltimore 1996 ; Baldwin 1996). As shown in Figure 9, the NIM1 protein (SEQ ID NO : 3) shares significant homology with IKBa proteins from mouse, rat, and pig (SEQ ID NOs : 18, 19, and 20, respectively).

NIM1 contains several important structural domains of licba 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 llcba share identity at 30% of the residues and conservative replacements at 50% of the residues.

Thus, there is homology between iBa and NIM1 throughout the proteins, with an overall similarity of 80%.

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

The phosphorylated serine residues important in IxBa function are conserved in NIM1 within a large contiguous block of conserved sequence from amino acids 35 to 84 (Figure 9). In contrast to IKBa, where the double lysine is located about 15 amino acids toward the N-terminus of the protein, in NIM1 a double lysine is located about 40 amino acids toward the C-terminal end. Furthermore, a high degree of homology exists between NIM1 and licbcc in the serine/threonine rich carboxy terminal region which has been shown to be important in basal turnover rate (Sun etal., 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 licba function, NIM1 is expected to function like the licBa, having analogous effects on plant gene regulation.

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

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

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

Phosphorylation of serine residues in human KBa is required for stimulus activated degradation of IKBa thereby activating NF-KB. Mutagenesis of the serine residues (S32 and S36) in human licb to alanine residues inhibits stimulus-induced phosphorylation, thus blocking IKB (X proteosome-mediated degradation (Traenckner et al., 1995 ; Brown et al., 1996 ; Brockman et al., 1995 ; Wang et al., 1996). This altered form of llcba can function as a dominant-negative form by retaining NF-KB in the cytoplasm thereby blocking downstream signaling events. Based on the amino acid sequence comparison between NIM1 and IKB shown in Figure 9, serines 55 (S55) and 59 (S59) in NIM1 (SEQ ID NO : 3) are homologous to S32 and S36 in human licBa. 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 iysine 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 IKBa is expected to function as a dominant-negative form. A region rich in serine and threonine is present at amino acids 522-593 in the C-terminus of NIM1. Thus, in a preferred embodiment of the present invention, the NIM1 gene is altered so that the encoded product is missing approximately its C-terminal portion, including amino acides 522-593, compared to the native Arabidopsis NIM1 amino acid sequence. The

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 chieras. 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 addition 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 syringe and Pseudomonas tabaci ; 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 kgtl1, kgtlO 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 ieast 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 pcIB200 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 Acc/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)). Xhollinkers are ligated to the EcoRVfragment of PCIB7 which contains the left and right T-DNA borders, a plant selectable noslnptll chimeric gene and the pUC polylinker (Rothstein et al., Gene 53 : 153-161 (1987)), and the XhoE digested fragment are cloned into Sall digested pTJS75kan to create pCIB200 (see also EP 0 332 104, example 19). pCIB200 contains the following unique polylinker restriction sites : EcoRl, Sstl, Kpnl, Bglll, Xbal, and Sall. 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 EcoRl, Sstl, Kpnl, Bglll, Xbal, Sall, Mlul, Bcll, Avrll, Apal, Hpal, and Stul. pCIB2001, in addition to containing these unique restriction sites also has plant and bacterial kanamycin selection, left and right T-DNA borders for Agrobacterium-mediated transformation, the RK2-derived trfA function for mobilization between E. coli and other hosts, and the OriTand OriVfunctions also from RK2. The pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals. b. pCIB10 and Hygromycin Selection Derivatives thereof : The binary vector pCIB10 contains a gene encoding kanamycin resistance for selection in plants and T-DNA right and left border sequences and incorporates sequences from the wide host-range plasmid pRK252 allowing it to replicate in both E. coliand Agrobacterium. Its construction is described by Rothstein etal. (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 Sall 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 Sall 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 Sma/fragment containing the bargene 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. coll) 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. coligene 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 pSOG10. A 250-bp fragment encoding the E. colidihydrofolate reductase type 11 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 opaline 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 opaline 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 EcoRl 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 tml transcriptional terminator with a unique EcoRl 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 EcoRl 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, Sall, and Xbal sites 5'to the promoter and Xbal, BamHland Bgll sites 3'to the terminator for transfer to transformation vectors such as those described above.

Furthermore, the double 35S promoter fragment can be removed by 5'excision with Hindlll, Sphl, Sall, Xbal, or Pstl, and 3'excision with any of the polylinker restriction sites (EcoRl, Notl or Xho 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 pCGN1761ENX/Sph-. pCGN1761ENX/Sph-is cleaved with EcoRl, and ligated to an annealed molecular adaptor of the sequence 5'-AATTCTAAAGCATGCCGATCGG-3'/5'- AATTCCGATCGGCATGCTTTA-3' (SEQ ID NO's : 12 and 13). This generates the vector pCGNSENX, which incorporates the quasFoptimized 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 EcoRl, Notl, and Xholsites are retained.

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

This vector, designated pCGN1761NENX is made by inserting an annealed molecular adaptor of the sequence 5'-AATTCTAAACCATGGCGATCGG-3'/5'- AATTCCGATCGCCATGGTTTA-3' (SEQ ID NO's : 14 and 15) at the pCGN1761 ENX EcoRl site. Thus the vector includes the quassoptimized sequence TAAACC adjacent to the initiating ATG which is within the Ncol site. Downstream sites are EcoRl, Notl, and Xhot 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 replace 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-1 a promoter is cleaved from plasmid pCIB1004 (see EP 0 332 104, example 21 for construction which is hereby incorporated by reference) and transferred to plasmid pCGN1761ENX (Uknes et al. 1992). pCIB1004 is cleaved with 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-1 a-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-1 a promoter and the tm/terminator and an intervening polylinker with unique EcoRl 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-carboxylate, benzyl benzo-1, 2, 3-thiadiazole- 7-carboxylate, benzo-1, 2, 3-thiadiazole-7-carboxylic acid sec-butylhydrazide, and suitable derivatives thereof. Other chemical inducers may include, for example, benzoic acid, salicylic acid (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 Actl gene has been cloned and characterized (McElroy et al.

Plant Cell 2 : 163-171 (1990)). A 1. 3kb fragment of the promoter was found to contain all the 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 etal. Mol. Gen. Genet. 231 : 150-160 (1991)). These incorporate the Act/-intron 1, Adhl5'flanking sequence and Adh/-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 a/. (Mol. Gen. Genet. 231 : 150-160 (1991)) can be easily modified for the expression of cellulase genes and are particularly suitable for use in monocotyledonous hosts. For example, promoter-containing fragments is removed from the McElroy constructions and used to replace the double 35S promoter in pCGN1761 ENX, which is then available for the insertion of specific gene sequences. The fusion genes thus constructed can then be transferred to appropriate transformation vectors. In a separate report the rice Actl promoter with its first intron has also been found to direct high expression in cultured barley cells (Chibbar et al. Plant Cell Rep. 12 : 506-509 (1993)). e. Constitutive Expression, the Ubiquitin Promoter : Ubiquitin is another gene product known to accumulate in many cell types and its promoter has been cloned from several species for use in transgenic plants (e. g. sunflower -Binet etal. Plant Science 79 : 87-94 (1991) and maize-Christensen etal. 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 a/. (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-Inducible 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 a/. 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 et al. have described a wound-induced gene from the monocotyledon Asparagus officinalis which is expressed at local wound and pathogen invasion sites. Using cloning techniques well known in the art, these promoters can be transferred to suitable vectors, fused to the NIM1 genes of this invention, and used to express these genes at the sites of plant wounding. h. Pith-Preferred Expression : Patent Application WO 93/07278 (to Ciba-Geigy) which is herein incorporated by reference describes the isolation of the maize trpA gene which is preferentially expressed in pith cells. The gene sequence and promoter extending up to-1726 bp from the start of transcription are presented. Using standard molecular biological techniques, this promoter, or parts thereof, can be transferred to a vector such as pCGN1761 where it can replace the 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 a/. Mol. Gen. Genet. 205 : 193-200 (1986)). Using the restriction enzymes Dral and Sphl. pr Tsp5091 and Sphl the DNA sequence encoding this transit peptide can be excised from the plasmid prbcS-8B and manipulated for use with any of the constructions described above. The Dral-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 Tsp5091-Sphl fragment extends from-8 relative to the initiating rbcS ATG to, and including, the first amino acid of the mature peptide.

Thus, these fragments can be appropriately inserted into the polylinker of any chosen expression cassette generating a transcriptional fusion to the untranslated leader of the chosen promoter (e. g. 35S, PR-1 a, actin, ubiquitin etc.), while enabling the insertion of a NIM1 gene in correct fusion downstream of the transit peptide. Constructions of this kind are routine in the art. For example, whereas the Dral end is already blunt, the 5'Tsp5091 site may be rendered blunt by T4 polymerase treatment, or may alternatively be ligated to a linker or adaptor sequence to facilitate its fusion to the chosen promoter. The 3'Sphl site may be maintained as such, or may alternatively be ligated to adaptor of linker sequences to facilitate its insertion into the chosen vector in such a way as to make available appropriate restriction sites for the subsequent insertion of a selected NIM1 gene. Ideally the ATG of the Sphl site is maintained and comprises the first ATG of the selected NIM1 gene. Chen & Jagendorf provide consensus sequences for ideal cleavage for chloroplast import, and in each case a methionine is preferred at the first position of the mature protein.

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

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

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

A preferred vector is constructed by transferring the Dra/-Sph/transit peptide encoding fragment from prbcS-8B to the cloning vector pCGN1761 ENX/Sph-. This plasmid is cleaved with EcoRl 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'-termin-ally phosphorylated by treatment with T4 kinase. Subsequent cleavage with Dral releases the transit peptide encoding fragment which is ligated into the blunt-end ex-EcoR/sites of the modified vector described above.

Clones oriented with the 5'end of the insert adjacent to the 3'end of the 35S promoter are identified by sequencing. These clones carry a DNA fusion of the 35S leader sequence to the rbcS-8A promoter-transit peptide sequence extending from-58 relative to the rbcS ATG to the ATG of the mature protein, and including in that region a unique Sphl site, and a newly created EcoRl 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 Nlalll site at the amplified ATG, which following restriction enzyme cleavage with the appropriate enzyme is ligated into Sphl-cleaved pCGN1761/CT. To facilitate construction, it may be required to change the second amino acid of the product of the cloned gene ; however, in almost all cases the use of PCR together with standard site directed mutagenesis will enable the construction of any desired sequence around the cleavage site and first methionine of the mature protein.

A further preferred vector is constructed by replacing the double 35S promoter of 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 EcoRl 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 BamHl releases the promoter-transit peptide containing fragment which is treated with T4 DNA polymerase to render the BamHl

terminus blunt. The promoter-transit peptide fragment thus generated is cloned into the prepared pCGN1761ENX vector, generating a construction comprising the rbcS-8A promoter and transit peptide with an Sphl site located at the cleavage site for insertion of heterologous genes. Further, downstream of the Sphl site there are EcoRl (re-created), Notl, and Xho/cloning sites. This construction is designated pCGN1761rbcS/CT.

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

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

These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Appropriate transcriptional terminators are those which are known to function in plants and include the CaMV 35S terminator, the tm/terminator, the opaline 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 bronze1 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 etal. 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 a/. (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 a/. 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 a/. 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). 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. Appl.

Genet. 75 : 30-36 ; Klein et al., (1987) Nature 327 : 70-73 ; Howell et al., (1980) Science 208 : 1265 ; Horsch et al., (1985) Science 227 : 1229-1231 ; DeBlock et al., (1989) Plant Physioloav 91 : 694-701 ; Methods for Plant Molecular Bioloav (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. PI. Ce/// Rep. 6 : 321-325, 1987 ; Block, M. d. Genotype independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens. Theor. appLgenet. 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. PI. 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. PI. 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. BiolTechnol. 0 : 309-314. 1992 ; Dhir, S. K., Dhir, S., Savka, M. A., Belanger, F., Kriz, A. L., Farrand, S. K., and i5 ! idholm, J. M.

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

Plant Physiol 99 : 81-88, 1992 ; Ha, S. B., Wu, F. S., and Thorne, T. K. Transgenic turf-type tall fescue (Festuca arundinacea Schreb.) plants regenerated from protoplasts. Fl,', Cefl 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 : 1121211216, 1993 ; Christou, P. Philosophy and Practice of Variety Independent Gene Transfer into Recalcitrant Crops. In Vitro Cell Dev Biol-Plant 29P : 119-124, 1993 ; Damiani, 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. PI. Cell Rep. 12 : 245- 249, 1993 ; Franklin, C. I. and Trieu, T. N. Transformation of the Forage Grass Caucasian Bluestem via Biolistic Bombardment Mediated DNA Transfer. Plant Physiol 102 : 167, 1993 ; Golovkin, 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. PI. Cell Rep.

13, 1994 ; Ayres, N. M. and Park, W. D. Genetic Transformation of Rice. Crit Rev Plant Sci 13 : 219-239, 1994 ; Barcelo, P., Hagel, C., Becker, D., Martin, A., and Lorz, H. Transgenic Cereal (Tritordeum) Plants Obtained at High Efficiency by Microprojectile Bombardment of Inflorescence Tissue. PLANT J 5 : 583-592, 1994 ; Becker, D., Brettschneider, R., and Lorz, H. Fertile Transgenic Wheat from Microprojectile Bombardment of Scutellar Tissue. Plant J 5 : 299-307, 1994 ; Biswas, G. C. G., Iglesias, V. A., Datta, S. K., and Potrykus, 1. Transgenic Indica Rice (Oryza Sativa L) Plants Obtained by Direct Gene Transfer to Protoplasts. J Biotechnol32 : 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. Acta 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 Avances. Agro Food Ind Hi Tech 5 : 17-27, 1994 ; Chupeau, M. C., Pautot, V., and Chupeau, Y. Recovery of Transgenic Trees After Electroporation of Poplar Protoplasts.

Transgenic Res 3 : 13-19, 1994 ; Eapen, S. and George, L. Agrobacterium Tumefaciens Mediated Gene Transfer in Peanut (Arachis Hypogaea L). PI. Cell Rep. 13 : 582-586, 1994 ; Hartman, C. L., Lee, L., Day, P. R., and Tumer, 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 Biol24 : 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, 1. 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, 1. 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 Blechl, A. E.

Stable Transformation of Wheat (Triticum Aestivum L) by Microprojectile Bombardment. J Cell Biochem : 104, 1994 ; Ye, X. J., Brown, S. K., Scorza, R., Cordts, J., and Sanford, J. C.

Genetic Transformation of Peach Tissues by Particle Bombardment. Jamer Sochortsci 119 : 367-373, 1994 ; Spangenberg, G., Wang, Z. Y., Nagel, J., and Potrykus, l. 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.

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

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

Agrobacterium-mediated transformation is a preferred technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species. The many crop species which are alfalfa and poplar (EP 0 317 511 (cotton), EP 0 249 432 (tomato, to Calgene), WO 87/07299 (Brassica, to Calgene),

US 4, 795, 855 (polar)). 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 virgenes 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. colicarrying 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 (Hofgen & 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 a/. (Plant Ceil 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 11 : 194-200 (1993)) describe techniques for the transformation of elite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos of 1. 5-2. 5 mm length excised from a maize ear 14-15 days after pollination and a PDS-1000He Biolistics device for bombardment.

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

Patent Application EP 0 332 581 (to Ciba-Geigy) describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Furthermore, wheat transformation has been described by Vasil et al. (Biotechnology 10 : 667-674 (1992)) using particle bombardment into cells of type C long-ter 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 Breedina, Second Edition, Clarendon Press, Oxford (1987) ; Singh, D. P., Breeding for Resistance to Diseases and Insect Pests, Springer-Verlag, NY (1986) ; and Wricke and Weber, Quantitative Genetics and Selection Plant Breedina, 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 yieid, 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 abte to tolerate comparable adverse developmental conditions.

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

Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD), methalaxyl (Apron), and pirimiphos-methyl (Actellic). If desired these compounds are formulated together with further carriers, surfactants or 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 mi 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 syringe Resistance Assay Pseudomonas syringe 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 mi 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 niml plants. Chemical induction of SAR genes is diminished in niml plants. Water, SA, INA, or BTH is applied to wild type (WT) and niml plants. After 3 days, RNA is prepared from these plants and examined for expression of PR-1, PR-2, and PR-5.

FIGURE 2 depicts PR-1 gene expression in pathogen-infected Ws-O and niml plants.

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

FIGURE 3 shows the accumulation of PR-1 mRNA in niml mutants and wild-type plants after pathogen infection or chemical treatment. Plants containing niml alleles niml- 1,-2,-3,-4,-5, and-6 and Ws-O (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 et al., 1992).

FIGURE 4 shows the levels of SA accumulation in Ws-O and niml plants infected with P. syringe. niml plants accumulate SNA following pathogen exposure. Leaves of wild type and nim 1 plantsare infiltrated with PstDC3000 (avrRpt2) or carrier medium (10 mM MgCI2) alone. After 2 days, samples were collecte from untreated, MgCl2-treated, and DC3000 (avrRpt2)-treated plants. Bacteria-treated samples were separated into primary (infiltrated) and secondary (noninfiltrated) leaves. Free SA and total SA following hydrolysis with ß-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 NIM1.

(C) Cosmid clones that cover the NIM7 locus. The three cosmids that complement niml-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 a, le, es. 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 D 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 IxBa from mouse, rat, and pig. Vertical bars (I) above the sequences indicate amino acid identity between NIM1 and the IKBa 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 KBa 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 licBa proteins. The five ankyrin repeats in IicBa 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 Figure 5D.

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

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

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

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

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

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

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

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

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

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

SEQ ID NO : 12-Oligonudeotde.

SEQ ID NO : 13-Oligonucleotide.

SEQ D NO : 14-Oligonudeotide.

SEQ ID NO : 15-Oligonudeotide.

SEQ ID NO : 16-Oligonudeotde.

SEQ ID NO : 17-Oligonudeotde.

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

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

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

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

SEQ D 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 NIM1.

SEQ ID NOs : 32 through 39 are oligonucleotide primers.

Definitions acct accelerated cell death mutant plant AFLP : Amplified Fragment Length Polymorphism avrRpt2 : virulence gene Rpt2, isolated from Pseudomonas syringe 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 cprl : constitutive expresser of PR genes mutant plant Col-O : Arabidopsis ecotype Columbia ECs : Enzyme combinations Emwa : Peronospora parasitica isolate compatible in the Ws-O 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 niml : 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-O 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 detalled procedures, preparations, and examples. The examples are for illustration only, and are not to be construed as limiting the scope of the present invention.

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

Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and by Ausubel, F. M. et a/., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-lnterscience (1 987).

A. Characterization of niml 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-O plants were obtained from Lehle Seeds (Round Rock, TX).

Pseudomonas syringe pv. Tomato (Psf) strain DC3000 containing the cloned avrRpt2 gene [DC3000 (avrRpt2)] was obtained from B. Staskawicz, University of California, Berkeley. P. parasitica pathovars and their sources were as follows : Emwa from E. Holub and I. R. Crute, Horticultural Research Station, East Malling, 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 (niml-1) was isolated from the T-DNA population and five (niml-2, niml-3, niml-4, niml-5, and niml-6) from the EMS population.

Example 3 : Disease Resistance of niml Plants Salicylic acid (SA) and benzo (1, 2, 3) thiadiazole-7-carbothioic acid S-methyl ester (BTH) are chemicals that, like INA, induce broad spectrum disease resistance (SAR) in wild type plants. Mutant plants were treated with SA, INA, and BTH and then assayed for resistance to Peronospora parasitica. P. parasitica isolate'Emwa'is a P. p. isolate that is compatible in the Ws ecotype. Compatible isolates are those that are capable of causing disease on a particular host. The P. parasitica isolate'Noco'is incompatible on Ws but compatible on the Columbia ecotype. Incompatible pathogens are recognized by the potential host, eliciting a host response that prevents disease development.

Wild-type seeds and seeds for each of the niml alleles (niml-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, uM BTH, or 300 RM 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/l 6h dark cycles.

To determine the relative strength of the different niml alleles, each mutant was microscopically analyzed at various timepoints after inoculation for the growth of P. parasitica under normal growth conditions and following pretreatment with either SA, INA, or BTH. Under magnification, sporulation of the fungus could be observed at very early stages of disease development. The percentage of plants/pot showing sporulation at 5d, 6d, 7d, 11 d and 14d after inoculation was determined and the density of sporulation was also recorded.

Table 1 shows, for each of the niml mutant plant lines, the percent of plants that showed some surface conidia on at least one leaf after infection with the Emwa race of P. parasitica. P. parasitica was inoculated onto the plants three days after water or chemical treatment. The table indicates the number of days after infection that the disease resistance was rated.

Table 1 Percent Infection-Emwa/Control mutant Day 0 Day 5 Day 6 Day 7 Day 11 Ws WT 0 10 25 100 90 nit 1-1 0 75 95 100 100 nim 1-2 0 30 85 100 100 nim1-3 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 Day 0 Day 5 Day 6 Day 7 Day 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 Day 0 Day 5 Day 6 Day 7 Day 11 Ws WT 0 0 0 0 0 nim1-1 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 Day 0 Day 5 Day 6 Day 7 Day 11 Ws WT 0 0 0 0 0 time-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, niml-1, niml-2, niml-3, niml-4, and niml-6 all supported approximately the same rate of fungal growth, which was somewhat faster than the Ws-0 control. The exception was the niml-5 plants where fungal growth was delayed by several days relative to both the other niml mutants and the Ws-0 control, but eventually all of the nim 1-5 plants succumbed to the fungus.

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

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

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

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

Table 2 Infection Severity-Emwa/Water mutant Day 7 Day 11 Ws WT 3 3 niml-1 4 4. 5 nim1-2 3 4 nim1-3 4 4 nit1-4 5 5 nim 1-5 1 3. 5 nim1-6 3 4.5 NahG 4 5 Infection Severity-Emwa/SA mutant Day 7 Day 11 Ws WT 3 4 niml-1 3 4. 5 nim1-2 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 Day 7 Day 11 Ws WT 0 0 niml-1 2. 5 4 nim 1-2 4 4 nim1-3 3 3.5 nim1-4 4 5 niml-5 1 2 nim 1-6 3 4. 5 NahG 3 3 Infection Severity-Emwa/BTH mutant Day 7 Day 11 Ws WT 0 0 niml-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 nim 1-4 and nim 1-6 alleles had the most severe Peronospora parasitica infections ; this was most easily observable at the earlier time points.

In addition, the nim1-5allele showed the greatest response to both INA and BTH and therefore was deemed the weakest niml allele. The NahG plants showed very good response to both INA and BTH and looked very similar to the niml-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, nim1-2and other nim1 alleles showed virtually no response to SA or INA with regard to disease resistance.

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

Example 4 : Northern Analysis of SAR Gene Expression Since SA, INA and BTH did not induce SAR, or SAR gene expression in any of the niml plants, it was of interest to investigate whether pathogen infection could induce SAR gene expression in these plants, as it does in wild type plants. Thus, the accumulation of SAR gene mRNA was also used as a criterion to characterize the different nim1 a, leles.

Wild-type seeds and seeds for each of the nim1 alleles (niml-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 H2O, 5mM SA, 300 » M BTH, or 300 RM INA. Chemica, s 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 GeneScreenPlus membranes (DuPont).

Figures 1-3 present various RNA gel blots that indicate that SA, INA and BTH induce neither SAR nor SAR gene expression in niml plants. In Figure 1, replicate blots were hybridized to Arabidopsis gene probes PR-1, PR-2 and PR-5 as described in Uknes et aL (1992). In contrast to the case in wild type plants, the chemicals did not induce RNA accumulation from any of these 3 SAR genes in niml-1 ptants.

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

The RNA gel blot in Figure 3 shows that PR-1 mRNA accumulates to high levels following treatment of wild-type plants with SA, INA, or BTH or infection by P. parasitica. In the niml-l, niml-2, and niml-3 plants, PR-1 mRNA accumulation was dramatically reduced relative to the wild type following chemical treatment. PR-1 mRNA was also reduced following P. parasitica infection, but there was still some accumulation in these mutants. In the niml-4 and niml-6 plants, PR-1 mRNA accumulation was more dramatically reduced than in the other alleles following chemical treatment (evident in longer exposures) and significantly less PR-1 mRNA accumulated following P. parasitica infection, supporting the idea that these are particularly strong niml alieles. PR-1 mRNA accumulation was elevated in the nim 1-5 mutant, but only mildly induced following chemical treatment or P. parasitica infection. Based on both PR-1 mRNA accumulation and fungal infection, the mutants have been determined to fall into three classes : severely compromised alleles (niml-4 and niml- 6) ; moderately compromised alleles (niml-1, niml-2, and niml-3) ; and a weakly compromised allele (niml-5).

Example 5 : Determination of SA Accumulation in niml 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 nim1 plants indicates a disruption in a component of the SAR pathway downstream of SA and upstream of SAR gene induction.

niml plants were tested for their ability to accumulate SA following pathogen infection.

Pseudomonas syringe tomato strain DC 3000, carrying the avrRpt2 gene, was injected into leaves of 4-week-old niml plants. The leaves were harvested 2 days later for SA analysis as described by Delaney et al, 1995, PNAS 92, 6602-6606. This analysis showed that the niml plants accumulated high levels of SA in infected leaves, as shown in Figure 4.

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

Furthermore, INA and BTH (inactive in niml 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 niml plants from Emwa infection.

Example 6 : Genetic Analysis To determine dominance of the various mutants that display the niml phenotype, pollen from wild type plants was transferred to the stigmata of niml-1,-2,-3,-4,-5,-6. If the mutation is dominant, then the niml phenotype will be observed in the resulting F1 plants. If the mutation is recessive, then the resulting F1 plants will exhibit a wild type phenotype.

The data presented in Table 3 show that when 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 Xwild type F1 descendants all exhibited the niml phenotype, indicating that this is a dominant mutation. Following INA treatment, no P. parasitica sporulation was observed on wild type plants, while the F1 plants supported growth and some sporulation of P. parasitica. However, the niml phenotype in these F1 plants was less severe than observed when nim1-5was homozygous.

To determine allelism, pollen from the kanamycin-resistant niml-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 llg/ml to verify the hybrid origin of the seed. Kanamycin resistant (F1) plants were transferred to soil and assayed for the niml phenotype. Because the F1 descendants of the cross of the niml-5 mutant with the Ws wild type display a niml 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 nim1 phenotype.

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

Table 3. Genetic Seqreaation of nim Mutants Phenotype Mutant Generatio Female Male Wild typea nim1D n niml-1 FI wild type c niml-1 24 0 F2 98 32 nim1-2 F1 nim1-2 Wild type 3 0 nim1-3 F1 nim1-3 Wiid type 3 0 nim1-4 F1 nim1-4 Wild type 3 0 nim1-5 F1 nim1-5 Wild type 0 35 F1 Wild type nim 1-5 0 18 nim1-6 F1 nim1-6 Wild type 3 0 nimi-2 F1 nimi-2 niml-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 niml Mutation Mapping of the nim1 mutation is described in exhaustive detail in Applicants'U. S.

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

Example 7 : Identification of Markers in and Genetic Mapping of the NIM1 Locus To determine a rough map position for NIM1, 74 F2 nim plants from a cross between niml-1 (Ws-0) and Landsberg erecta (Ler) were identified for their susceptibility to P. parasitica and lack of accumulation of PR-1 mRNA following INA treatment. Using simple sequence length polymorphism (SSLP) markers (Bell and Ecker 1994), niml-1 was determined to lie about 8. 2 centimorgans (cM) from nga128 and 8. 2 cM from nga111 on the

lower arm of chromosome 1. In addition, niml-1 was determined to lie between nga111 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 niml-1 and LerDP23 were identified based on both their inability to accumulate PR-1 mRNA and their ability to support fungal growth following INA treatment.

DNA was extracted from these plants and scored for zygosity at both ATHGENEA and nga111. As shown in Figure 5A, 93 recombinant chromosomes were identified between ATHGENEA and niml-7, giving a genetic distance of approximately 4. 1 cM (93 of 2276), and 239 recombinant chromosomes were identified between nga111 and niml-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 nga111 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 niml-1) and W85. 1 (0. 6 cM from niml-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 Pl-20, Pl- 22, P1-23, and P1-24 and BAC clones, BAC-04, BAC-05, and BAC-06 identified one recombinant. Because these clones overlapped to form a large contig (>100 kb), and included AFLP markers that flanked niml, the gene was determined to be located on the contig. The BAC and P1 clones that comprised the contig were used to generate additional AFLP markers, which showed that niml was located between L84. Y1 and L84. 8, representing a gap of about 0. 09 cM.

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-compatible T-DNA cosmid vector pCLD04541 using CsCI-purified DNA from BAC-06, BAC-04, and P1- 18. The DNAs of the three clones were mixed in equimolar quantities and were partially digested with the restriction enzyme Sau3A. The 20-25 kb fragments were isolated using a sucrose gradient, pooled and filled in with dATP and dGTP. Plasmid pCLD04541 was used as T-DNA cosmid vector. This plasmid contains a broad host range pRK290-based replicon, a tetracycline resistance gene for bacterial selection and the nptll gene for plant selection. The vector was cleaved with Xhol and filled in with dCTP and dTTP. The prepared fragments were then ligated into the vector. The ligation mix was packaged and transduced into E. colistrain XL1-blue MR (Stratagene). Resulting transformants were screened by hybridization with the BAC04, BAC06 and P1-18 clones and positive clones isolated. Cosmid DNA was isolated from these clones and template DNA was prepared using the ECs EcoRl/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, Pst"BssHII and SgrAl. 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 NIM1 Gene.

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

Seedlings that survived the selection were transferred to soil approximately two weeks after

plating and tested for the niml phenotype as described below. Transformed plants that no longer had the nim1 phenotype identified cosmid (s) containing a functional NIM1 gene.

Example 10 : Complementation of the nim1 Phenotype Plants transferred to soil were grown in a phytotron for approximately one week after transfer. 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°C night temperatures and 8h light/16h dark cycles. Eight to ten days following fungal infection, plants were evaluated and scored positive or negative for fungal growth. Ws and niml plants were treated in the same way to serve as controls for each experiment.

Total RNA was extracted from the collecte 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 niml phenotype by cosmid clones D5, E1, and D7.

Table 4 Clone Name # of transformants # of plants with INA induced PR-1/# of plants tested (%) A8 3 0/3 0% A11 8 4/18 (22%) C2 10 1/10 10% C7 33 1/32 (3% D2 81 4/49 8% D5 6 5/6 (83%) E1 10 10/10 (100% D7 129 36/36 (100%) T E8 9 0/9 (0%) F12 6 0/6 (0% E6 1 0/1 (0%) E7 34 0/4 (0%) WS-control (wild-type) NA 28/28 (100%) nim1-1 phenotype control NA 0/34 (0% 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 niml mutants. BAC04 DNA was randomly sheared in a nebulizer to generate fragments with an average length of about 2 kb. Ends of the sheared fragments were repaired, and the fragments were purified. Prepared DNA was ligated with EcoRV- digested pBRKanF4 (a derivative of pBRKanF1 (Bhat 1993)). Resulting kanamycin-resistant colonies were selected for plasmid isolation using the Wizard Plus 9600 Miniprep System (Promega). Plasmids were sequenced using dye terminator chemistry (Applied BioSystems, Foster City, CA) with primers designed to sequence both strands of the plasmids (M13-21 forward and T7 reverse, Applied BioSystems). Data was collected on AB1377 DNA sequencers. Sequences were edited and assembled into contigs using Sequencher 3. 0 (GeneCodes Corp., Ann Arbor, MI), the Staden genome assembly programs, phred, phrap and crossmatch (Phil Green, Washington University, St. Louis, MO ) and consed (David Gordon, Washington University, St. Louis, MO). DNA from the cosmids found to complement the niml-1 mutation was sequenced using primers designed by Oligo 5. 0 Primer Analysis Software (National Biosciences, Inc., Plymouth, MN).

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

A region of approximately 9. 9 kb defined by the overlap of cosmids E1 and D7 was identified by complementation analysis to contain the niml region. Primers that flanked the insertion site of the vector and that were specific to the cosmid backbone were designed using Oligo 5. 0 Primer Analysis Software (National Biosciences, Inc.). DNA was isolated from cosmids D7 and 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 BamHl-EcoRV construct was completely digested with Spel, then was split into two separate reactions for double digestion. One aliquot was digested with BamHl, the other Hindlil. A BamHl-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 BamHl-Hindlll-

digested pSGCG01. DH5a was transformed with the ligation mix. Resulting colonies were screened for the correct insert by digestion with Hindlil following preparation of DNA using Wizard Magic MiniPreps (Promega). A clone containing the correct construct was electroporated into Agrobacterium strain GV3101 for transformation of Arabidopsis plants.

Example 12 : Sequence Analysis and Subcloning of the NIM1 Region The 9. 9 kb region containing the NIM1 gene was analyzed for the presence of open reading frames in all six frames using Sequencher 3. 0 and the GCG package. Four regions containing large ORF's were identified as possible genes (Gene Regions 1-4 in Figure 5D). These four regions were PCR amplified from DNA of the wild-type parent and the six different niml allelic variants niml-1,-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 QlAquick 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 niml alleles were identified only in the region designated Gene Region 2, as shown below in Table 5, which shows sequence variations among all six of the nim1 alleles.

Table 5 Gene Region Alle, e/2 (NIM1) 4 ecotype (bases 590- (bases 1380-4100) (bases 5870 (bases 8140- 1090)-6840) 9210) niml-1 no changes t inserted at 2981 : change no changes no changes of 7AA and premature termination of protein. nim1-2 no changes g to a at 2799 : His to Tyr no changes no changes nim1-3 no changes deletion of t at 3261 : no changes no changes change of 10AA and premature termination of protein. nim1-4 no changes c to t at 2402 : Arg to Iys no changes no changes nim1-5 no changes c to t at 2402 : Arg to Iys no changes no changes nim1-6 g to a at 734 : g to a at 2670 : Gln to Stop no changes no changes asp to I s WS no changes a to g at 1607 : Ile to Leu t to a at 5746 t to g at 8705 (compared a to c at 2344 : intron a to t at 5751 g to t at 8729 to t to g at 2480 : Gln to Pro t to a at 5754 g to t at 8739 Columbia) g to c at 2894 : Ser to Trp c to t at 6728 g to t at 8784 ggc deleted at 3449 : lose a to t at 6815 c to a at 8789 Ala ttocat6816 ctotat8812 c to t at 3490 : Ala to Thr a to g at 8829 c to t at 3498 : Ser to Asn t to g at 8856 a to t at 3873 : non-coding a to c at 9004 g to a at 3992 : non-coding a to t at 9011 g to a at 4026 : non-coding a to g at 8461 g to a at 4061: non-coding RNA No Yes No No detected I

Positions listed in the table relate to SEQ ID NO : 1. All al, eles nim1-1 to niml-6are WS strain. Columbia-0 represents the wild type It is apparent that the NIM1 gene lies within Gene Region 2, because there are amino acid changes or alterations of sequence within the open reading frame of Gene Region 2 in all six niml alieles. At the same time, at least one of the niml alleles shows no changes in the open reading frames within Gene Regions 1, 3 and 4. Therefore, the only gene region within the 9. 9 kb region that could contain the NIM1 gene is Gene Region 2.

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

The cosmid region containing the niml 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 BamHl and with EcoRV (NEB). The 5. 3 kb fragment from D7 was isolated from agarose gels and was purified using the Q, Aquick gel extraction kit (# 28796, Qiagen). The fragment was ligated overnight to the Bam-EcoRV-digested pBSll and the ligation mixture was transformed into E. colistrain DH5a. Colonies containing the insert were selected, DNA was isolated, and confirmation was made by digestion with Hindi". The Bam-EcoRV fragment was then engineered into a binary vector (pSGCG01) for transformation into Arabidopsis.

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

These blots were hybridized with PCR products generated from the four gene regions identified in the 9. 9 kb NIM1 gene region (SEQ ID NO : 1). Only the gene region containing the NIM1 gene (Gene Region 2) had detectable hybridization with the RNA samples, indicating that only the NIM1 region contains a detectable transcribed gene (Figure 5D and Table 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 pSGCG01 containing the gene for kanamycin resistance. The resulting plasmid was transformed into the Agrobacterium strain GV3101 and positive colonies were selected on kanamycin. PCR was used to verify that the selected colony contains the plasmid. Kanamycin-sensitive niml-1 plants were infiltrated with this bacteria as previously described. The resulting seed was harvested and planted on GM agar containing 501lg/m, kanamycin. Plants surviving selection were transferred to soil and tested for complementation. Transformed plants and control Ws and niml plants were sprayed with 300go NA. 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 niml 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 niml 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 EcoRl, 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 niml-3 allele and the time-6 allele. The nim 1-2 allele showed approximately normal levels in the untreated sample and showed similar induction to that of the wild type sample, as did the nim1-4 allele. The niml- 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, CAB ! OS 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 a/., Molecular Cloning, eds., Cold Spring Harbor Laboratory Press. (1989)) and amplification by PCR using oligonucleotide primers (see, e. g. Innis et a/., 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 BamHl, Hindlll, Xbal, or Sall, 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 ; 1 mM 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 ow 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 NIM1.

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

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

Homologues may be obtained by PCR. In this method, comparisons are made between known homologues (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 3rd 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 3rd 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 collecte.

Seeds from one E1 line, four D5 lines and 95 D7 fines 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 ess than normal fungal growth and several showed no visible fungal growth at all. RNA was prepared from collected samples and analyzed as previously described (Delaney et al, 1995). Blots were hybridized to the Arabidopsis gene probe PR-1 (Uknes et al, 1992).

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

Table 6 Line P. parasitica Line P. parasitica Line P. parasitica growth growth rowth D7-2 negative52+90+ 3 + 53 + 91 + 9 + 54 +/-92 + 11 + 56 + 93 + 12 + 57 + 94 + 13 + 58 + 95 + 14 + 59 + 96 + 17 + 60 + 97 + 18 + 61 + 98 +/- 19 + 62 + 100 +/- 20 + 63 + 101 +/- 21 + 64 + 102 +/- 22 + 66 + 103 + 23 + 67 + 104 + 24 + 68 + 106 + 25 + 69 + 107 + 28 + 70 + 108 + 29 + 71 + 114 + 31 + 72 + 115 + 32 + 73 + 118 + 33 +74negative119+ 34 + 75 + 122 + 35 + 77 + 123 + 36 + 78 + 124 + 38 + 79 + 125 + 39 + 80 +/-126 + 42 + 81 + 128 + 43 + 82 + 129 + 46 + 83 + __ 130 + 47 + 84 + D5-1 + 48 + 85 + 2 + 4g + 86 + 4 + 50 +-87 +/-6 +/- 51 + 89 negative E1-1 negative

Plants were treated with P. parasitica isolate Emwa and scored 10 days ater.

+, normal fungal growth +/-, less than normal fungal growth negative, no visible fungal growth Example 19 : NIM1 Overexpression Under Its Native Promoter Plants constitutively expressing the NIM1 gene were generated from transformation of Ws wild type plants with the BamHI-Hindlll NIM1 genomic fragment (SEQ ID NO : 2-bases 1249-5655) containing 1. 4 kb of promoter sequence. This fragment was cloned into pSGCG01 and transformed into the Agrobacterium strain GV3101 (pMP90, Koncz and Schell (1986) Mol. Gen. Genet. 204 : 383-396). Ws plants were infiltrated as previously described. The resulting seed was harvested and plated on GM agar containing 50 ug/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 syringe 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 NIM1 The full-length NIM1 cDNA (SEQ ID NO : 21) was cloned into the EcoR/site of pCGN1761 ENX (Comai et al. (1990) Plant Mol. BioL 15, 373-381). From the resulting plasmid, an Xba/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 ug/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, brocoli, 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 challenge 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 niml-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 licba A multiple sequence alignment between the protein gene products of NIM1 and IkB was performed by which it was determined that the NIM1 gene product is a homolog of IKB a (Figure 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, WI). The sequences used in the alignment were NIM1 (SEQ ID NO : 3), mouse IxBa (SEQ ID NO : 18, GenBank Accession # : 1022734), rat licba (SEQ ID NO : 19, GenBank accession Nos. 57674 and X63594 ; Tewari et al., NucleicAcids Res. 20, 607 (1992)), and pig licBa (SEQ ID NO : 20, GenBank accession No.

Z21968 ; de Martin et al., EMBO J. 12, 2773-2779 (1993) ; GenBank accession No. 517193, de Martin et al., Gene 152, 253-255 (1995)). Parameters used in the Clustal analysis were gap penalty of 10 and gap length penalty of 10. Evolutionary divergence distances were calculated using the PAM250 weight table (Dayhoff et al.,"A model of evolutionary change in proteins. Matrices for detecting distant relationships."In Atlas of Protein Sequence and Structure, Vol. 5, Suppl. 3, 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-lcB 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 IxBs have these N-terminal serines and they are required for inactivation of licb and subsequent release of NF-KB. NIM1 has ankyrin domains (amino acids 262-290 and 323-371). Ankyrin domains are believed to be involved in protein-protein interactions and are a ubiquitous feature for IKB and NF-KB molecules. The C-termini of IKB's can be dissimilar. NIM1 has

some homology to a QL-rich region (amino acids 491-499) found in the C-termini of some IK Bs.

Example 27 : Generation Of Altered Forms Of NIM1- Changes Of Serine Residues 55 and 59 To Alanine Residues Phosphorylation of serine residues in human licba is required for stimulus-activated degradation of licba thereby activating NF-KB. Mutagenesis of the serine residues (S32- S36) in human lcBa to alanine residues inhibits stimulus-induced phosphorylation thus blocking licb proteosome-mediated degradation (E. Britta-Mareen Traenckner et al., EM80 J. 14 : 2876-2883 (1995) ; Brown et al., Science 267 : 1485-1488 (1996) ; Brockman et al., Molecular and Cellular Biology 15 : 2809-2818 (1995) ; Wang et al., Science 274 : 784-787 (1996)).

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

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

Using a full length NIM1 cDNA (SEQ ID NO : 21) including 42 bp of 5'untranslated sequence (UTR) and 187 bp of 3'UTR, the mutagenized construct can be made per the manufacturer's instructions using the following primers (SEQ ID NO : 21, positions 192-226) : 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 EcoRl 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 tm/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 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 : 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 licha, which includes K21, K22, S32 and S36, results in a dominant-negative llcba 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 EcoRl 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 MgC, 2/0. 001% gelatin/0. 25 mM each dNTP/0. 2 mM of each primer and 1 unit rTth DNA polymerase in a final volume of 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 EcoRl and ligated into the EcoRl 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 tm/terminator is released from pCGN1761 ENX 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 D 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. 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 licba is believed to result in enhanced intrinsic stability by blocking the constitutive phosphorylation of serine and threonine residues in the C-terminus. A region rich in serine and threonine is present at amino acids 522-593 in the C-terminus of NIM1. The C-terminal coding region of the NIM1 gene may be modified by deleting the nucleotide sequences which encode amino acids 522-593. Using the method of Ho et al. (1989), the C-terminal coding region and 3'UTR of the NIM1 cDNA (SEQ ID N0 : 21 : 1606-2011) is deleted by PCR, generating a 1623 bp fragment using the following primers : 5'-cggaattcGATCTCTTTAATTTGTGAATTT C-3' (SEQ ID N0 : 36) and 5'-ggaattcTCAACAGTT CATAATCTGGTCG-3' (SEQ ID NO : 37) in which a synthetic stop codon is underlined (TGA on complementary strand) and EcoRl 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 EcoRl and ligated into the EcoRl 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 tm/ terminator is released from pCGN1761 by partial restriction digestion with Xbaland ligated into the Xbal site of dephosphorylated pCIB200. SEQ ID NO's : 26 and 27 show the DNA coding sequence and encoded amino acid sequence, respectively, of an altered form of the NIM1 gene having a C-terminal amino acid deletion.

The present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an a, lele hybridizes under the following conditions to the coding sequence set forth in SEQ ID NO : 26 : 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 : 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 EcoRllKpnl 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 EcoRllKpnl 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 EcoRl to eliminate EcoRl concatemers and cloned into the EcoRl 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 tm/terminator are released from pCGN1761 by partial restriction digestion with Xbal and ligated into the Xbal site of dephosphorylated pCIB200.

SEQ ID NO's : 28 and 29 show the DNA coding sequence and encoded amino acid sequence, respectively, of an altered form of the NIM1 gene having both N-terminal and 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 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 : 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'ggaattcTCAACCTTCCAAAGTTGCTTCTGATG-3' (SEQ ID NO : 39). The resulting product is restriction endonuclease digested with EcoRl and then spliced into the EcoRl 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 tm/terminator is released from pCGN1761 by partial restriction digestion with Xba/and ligated into the Xba/site of dephosphorylated pCIB200. SEQ ID NO's : 30 and 31 show the DNA coding sequence and encoded amino acid sequence, respectively, of the ankyrin domain of NIM1.

The present invention also encompasses altered forms of alleles of NIM1, wherein the coding sequence of such an allele hybridizes under the following conditions to the coding sequence set forth in SEQ ID NO : 30 : hybridization in 1 % BSA ; 520mM 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/BamHl fragment (SEQ D NO : 2 : bases 1249-5655) and/or a 5655 bp EcoRV/BamHl 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., Cell78, 1089- 1099 (1994)) or by standard root transformation. Seed from these plants is harvested and allowed to germinate on agar plates with kanamycin (or another appropriate antibiotic) as selection agent. Only plantlets that are transformed with cosmid DNA can detoxify the selection agent and survive. Seedlings that survive the selection are transferred to soil and tested for a CIM (constitutive immunity) phenotype. Plants are evaluated for observable phenotypic differences compared to wild type plants.

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-O and Col-0 plants, respectively), are prepared, and transformants are sprayed with the appropriate isolate depending on the ecotype of the transformant. Inoculated plants are incubated under high humidity for 7 days. Plants are disease rated at day 7 and a single leaf is harvested for RNA blot analysis utilizing a probe which provides a means to measure fungal infection.

Transformants that exhibit a CIM phenotype are taken to the T1 generation and homozygous plants are identified. Transformants are subjected to a battery of disease resistance tests as described beiow. 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 syringe 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 ndrl to establish the epistatic relationship of the resistance phenotype to other mutants and evaluate how these dominant-negative mutants of NIM1 may influence the SA-dependent feedback loop.

Example 35 : Isolation Of NIM1 Homologs Using the NIM1 cDNA (SEQ ID N0 : 21) as a probe, homologs of Arabidopsis NIMI 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 a/., Molecular Cloning, eds., Cold Spring Harbor Laboratory Press. (1989)) and amplification by PCR using oligonucleotide primers (see, e. g. Innis et a/., 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 BamHl, Hindlil, Xbal, or Sall, 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 ; 1 mM 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 NIM1.

In addition, expressed sequence tags (EST) identified with similarity to the NIM1 gene can be used to isolate homologues. For example, several rice expressed sequence tags (ESTs) have been identified with similarity to the NIM1 gene. A multiple sequence alignment was constructed using Clustal V (Higgins, Desmond G. and Paul M. Sharp (1989), Fast and sensitive multiple sequence alignments on a microcomputer, CABIOS 5 : 151-153) as part of the DNA* (1228 South Park Street, Madison Wisconsin, 53715) Lasergene Biocomputing Software package for the Macintosh (1994). Certain regions of the NIM1 protein are homologous in amino acid sequence to 4 different rice cDNA protein products. The homologies were identified using the NIM1 sequences in a GenBank BLAST search. Comparisons of the regions of homology in NIM1 and the rice cDNA products are shown in Figure 8 (See also, SEQ ID NO : 3 and SEQ D 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 MM ? homoiogues 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 3rd 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 3rd 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, brocoli, 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.

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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 NIM1 GENE TO CONFER DISEASE RESISTANCE IN PLANTS (iii) NUMBER OF SEQUENCES : 39 (2) INFORMATION FOR SEQ ID NO : 1 : (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 : 1 : TGATCATGAA TTGCGTGTAG GGTTGTGTTT TAAAGATAGG GATGAGCTGA AGAAGGCGGT 60 GGACTGGTGT TCCATTAGAG GGCAGCAAAA GTGTGTAGTA CAAGAGATTG AGAAGGACGA 120 GTATACGTTT AAATGCATCA GATGGAAATG CAATTGGTCG CGTCGGGCAG ATTGAATAGA 180 AGAACATGGA CTTGTTAAGA TAACTAAGTG TAGTTGGTCC ACATACTTGT TGTTCTATTA 240 AGCCGGAAAA CTTCAACTTG TAATTTGCAG CAGAAGAGAT TGAGTGTCTG ATCAGGGTAC 300 AACCCACTCT AACAGCAGAG TTGAAAAGTT TGGTGACATG CTTAAAACTT CAAAGCTGCG 360 GGCAGCAGAA CAGGAAGTAA TCAAAGATCA GAGTTTCAGA GTATTGCCTA AACTAATTGG 420 CTGCATTTCA 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 AAGATACATA TAACATGAAT GTTGCCAGAA GTTCAGGGAA 1380 CTTGAATATT CTTTTGGTTC TTGGTGGAAA ATATCCAACA GATGAACAAT TTGACATTAT 1440 TTCACACTTT GATTCTAGCA ACTCTGTAAC ACCATCATGG GTTATTGTTG ATGTACATAA 1500 ATATATATTA CAAATCTGTA TACCATTGGT TCAAATTGTT ACAACATTTG TTTGAAGCAC 1560 ACCTGCAGCA ATAATACACA GGATGCAAAA CGAAGAGCGA AACTATATGA CGCCAACGAT 1620 AGACATAAAC AGTTACAGTC ATCATGAAAA CAGAATTATA TGGTACAGCA AAAATTACAC 1680 TAAGAGGCAA GAGTCTCACC GACGACGATG AGAGAGTTTA CGGTTAGACC TCTTTCCACC 1740 GGTTGATTTC GATGTGGAAG AAGTCGAATC TGTCAGGGAC GAATTTCCTA ATTCCAAATT 1800 GTCCTCACTA AAGGCCTTCT TTAGTGTCTC TTGTATTTCC ATGTACCTTT GCTTCTTTTG 1860 TAGTCGTTTC TCAGCAGTGT CGTCTTCTCC GCAAGCCAGT TGAGTCAAGT CCTCACAGTT 1920

CATAATCTGG TCGAGCACTG CCGAACAGCG CGGGAAGAAT CGTTTCCCGA GTTCCACTGA 1980 TGATAAAAAA AACAAGGTCA GACAGCAAGT AACAAAACCA TGTTTAAAGA TCATTTAGTT 2040 TTGTTTTTTG TGATAAGGAG TCCGATGAAG TGGGTGAGAA TCCATACCGG TTTTAGAAAG 2100 CGCTTTTAGT CTACTTTGAT GCTCTTCTAG GATTCTGAAA GGTGCTATCT TTACACCCGG 2160 TGATGTTCTC TTCGTACCAG TGAGACGGTC 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 ATAAGGTAAA 4920 TAAATTATTG CTTTCCGCGT TTTTTACTTT TGTATTTCTT AAATGATAAG TTAAATTAGG 4980 ATAAGATTTG TATGATTTTA AGTAAATTTA CAATAACTCT CTATAACTCA ATAGCATCAC 5040 ATATTTAATT AATTTTACTA ATTATCTTTT GAACAATTTT ATGAAATAGT TTTCTTTTAA 5100 TTAATTTTTT AAAATGATAT ATTATAAAAT TTAATTGAAT CAATCTGATA TAATTTTTTT 5160

ATCTTCTACC ATCTATTATA GTTGATAAAT ATTGTGATAA ACTTTAGATA AACACCCAAT 5220 TGCCAAATAT TTAATAAATT TTGTGTACCA TGCGTTTTTT TTGGAGAATA TATATACGTG 5280 GACAGCATAC CGTACATATA TTGTATAAAA GCTTATAAAA CATAGATACG GGTTATATTG 5340 GTAAGCTATA AATATATGTA AACAATAGTA AGATATTACG TGTTGTGTCT AAATATGTGT 5400 TGCTTTAGAT ATTATGTATA TCTAATATAT TAAAATATCT TTTATTAACT AATATATTAT 5460 TTAAGAGAGA AAATTGGGAC ACTATTTTCT ATACAGTAAC TGTTTTCAAC TATAAACAGG 5520 AACCCTTGAT ATAATAAAAT AACTAGCCAA AAAATCAGAT TAAATATTCA TAAAACAATG 5580 TTTGGTATTA TTACATAAAC CTAAGAAACA AAATTCAATA TTCCTTTTTA CCTTATAAAA 5640 AACAATTAAA CATCACTAGA TATATTTATG CCCCACAATG AGCGAGCCAA TTGAGACTTG 5700 AGACTTGAGA TCCTTGTCAA CTACGTTTGC ATTTGTCGGC CCATTTTTTT TATTTTTTTT 5760 TTAAAGTGTC 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 AATTTCCATT TTCCATAGAT TTATCTTTCT TTTTATTTCC TTAGTTATCT TAGTACTTTC 7440 TTAGTTTCCT TAGTAATTTT AAATTTTAAG ATAATATATT GAAATTAAAA GAAGAAAAAA 7500 AACTCTAGTT ATACTTTTGT TAAATGTTTC ATCACACTAA CTAATAATTT TTTTTAGTTA 7560 AATTACAATA TATAAACACT GAAGAAAGTT TTTGGCCCAC 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 AAATATTTTA TACCGGAAAG GCTGCAATCC GGTTACCGTT AGACCTAATG ACTTATCACA 8580 ACTCCTCACT TTTGGGTTTG GTATGATCTG ATACTGTTTT GTTGTTGGTT TGCAGACTAT 8640 GTATTCCGGT ATTGGTCTTG TATCATTATA ACAAAGCAAT ATCCCATGAC GTGCATCACA 8700 AGCTTTGATC TTTACCTCTC CTTGTGGCAG AAAATCGATG GAGACTCCTT TGTTATCCAA 8760 ATCTCTCCTC TCATGGAAAA AACTGGTATC AAGTTTGTAT CCTCTTTCGT AGCGTTCTAG 8820 GAAGTATCCA GAGATATTGT TGGTTCGATG GAGATTTAGG TTGACAAACC AAGACTCGTA 8880 GCTTCTCTTG TTGCACTCTT TATTGATGAG CCTCAATTTT CCGATTTCGG ACCCCCGAAG 8940 ATAAGAAAGA ACCTCTTGGA TCGTGTCCTG ATTTATCACC GGAGAACTCA TGATCTTATT 9000 GGAAAAAAGA AAGAAAGAGA TGAGCACGAT CAGTGAATGA GATATATAGA AATCAGGATT 9060 GGTAGAGAAC CGACGATGAT GAATATACAA GTGTTTATAA GTATCACAAA TTGCCTTTTT 9120 CTTCGCTAGT CCCAAAACAA GCAAATTAAC CAAAGATAAA ATCTTCATTA ATGTTTTCCT 9180 TTTTCTTCGC CAGTCCCAGA TAAAAATATA TATAAAATAT TTCATTAGGT TACTTGTAGT 9240 ACCTTGAGCC CAAAGTTTCT CTTTTGACTT TTAACCAAAT TAACAGTAAT TTAATAGCTA 9300 GACTTAGAAA ACAACATTTT GTATATATAT TCTTTGACAT CAAAATTCAA CAATCTTTGG 9360 GTTTCTATAG TGTTTTTTTT CTTATTCTAA TAGATTACCA CTCATTATAT CATATACAAA 9420 GTGTTTCCTT TTCAATCAAC ATCCATTTTC TTTAAAAATT AGCAAGTTTG TTCTTATATC 9480

ATCATTCAGC AGATTTCTTA ATTAAACTTA GTGATTTCCA TTTTGCACCT ATATGTTTCT 9540 CTTTCTTAGT TTAGTACTTT AAATTTTCAT ATATATAATT TATTAAAATT AAAAGTAAAA 9600 ACTCCAGTTT AACTTATGTT AAATGTTTCA TCACACTAAA AGAGCATTAA GTAATAAATA 9660 TTTTAGCTTT ATGAAAAAAA ATATCAAATC ACTGAAGACA TTTGTTGGCC TATACTCTAT 9720 TTTTTATTTG GCCAATTAGT AATAGACTAA TAGTAACTCA TATGATATCT CTCTAATTCT 9780 GGCGAAACGA 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 NIM1"

(ix) FEATURE : (A) NAME/KEY : exon (B) LOCATION : 3427.. 4162 (D) OTHER INFORMATION :/product="2nd exon of NIM1" (ix) FEATURE : (A) NAME/KEY : exon (B) LOCATION : 4271.. 4474 (D) OTHER INFORMATION :/product="3rd exon of NIM1" (ix) FEATURE : (A) NAME/KEY : exon (B) LOCATION : 4586.. 4866 (D) OTHER INFORMATION :/product="4th exon of NIM1" (ix) FEATURE : (A) NAME/KEY : CDS (B) LOCATION : 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 ATAATACCAA 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 TATATAA. AGT 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 AGGAAATATT CGACTTGATT 2160 TAATTATAGT GTAAACATGC TGAACACTGA AAATTACTTT TTCAATAAAC GAAAAATATA 2220 ATATACATTA CAAAACTTAT GTGAATAAAG CATGAAACTT AATATACGTT CCCTTTATCA 2280 TTTTACTTCA AAGAAAATAA ACAGAAATGT AACTTTCACA TGTAAATCTA ATTCTTAAAT 2340 TTAAAAAATA ATATTTATAT ATTTATATGA AAATAACGAA CCGGATGAAA AATAAATTTT 2400 ATATATTTAT ATCATCTCCA AATCTAGTTT GGTTCAGGGG CTTACCGAAC CGGATTGAAC 2460 TTCTCATATA CAAAAATTAG CAACACAAAA TGTCTCCGGT ATAAATACTA ACATTTATAA 2520 CCCGAACCGG TTTAGCTTCC TGTTATATCT TTTTAAAAAA GATCTCTGAC AAAGATTCCT 2580

TTCCTGGAAA TTTACCGGTT TTGGTGAAAT GTAAACCGTG GGACGAGGAT GCTTCTTCAT 2640 ATCTCACCAC CACTCTCGTT GACTTGACTT GGCTCTGCTC GTCAATGGTT ATCTTCGATC 2700 TTTAACCAAA TCCAGTTGAT AAGGTCTCTT CGTTGATTAG CAGAGATCTC TTTAATTTGT 2760 GAATTTCAAT TCATCGGAAC CTGTTG ATG GAC ACC ACC ATT GAT GGA TTC GCC 2813 Met Asp Thr Thr Ile 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 Ile 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 Ile Val Tyr Leu Ala Ala Glu Gln 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 Gln 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 Ile 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 Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu 170 175 180 185 TAT CAG GTAAAACACC ATCTGCATTA AGCTATGGTT ACACATTCAT GAATATGTTC 3397 Tyr Gln TTACTTGAGT ACTTGTATTT GTATTTCAG AGG CAC TTA 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 Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile 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 Ile Ile 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 Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu 245 250 255 GTA CCT AAA GTA AAG AAA CAT GTC TCG AAT GTA CAT AAG GCA CTT GAC 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 Ile 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 Gln Leu Ile 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 Ile Ala Lys Gln 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 Ile Pro Glu Gln Cys Lys His 375 380 385 TCT CTC AAA GGC CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA 4074 Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln 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 Gln Ile 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 Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile 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 Ile Val Thr Ser Leu Glu Pro Asp 455 460 465 CGT CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAG ATA GCA CCT 4422 Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys Ile Ala Pro 470 475 480 TTC AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA 4470 Phe Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys 485 490 495

ACC G GTATGGATTC TCACCCACTT CATCGGACTC CTTATCACAA AAAACAAAAC 4524 Thr 500 TAAATGATCT TTAAACATGG TTTTGTTACT TGCTGTCTGA CCTTGTTTTT TTTATCATCA 4584 G 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 Gln Ile Met Asn Cys Glu Asp Leu Thr Gln Leu Ala Cys Gly Glu 520 525 530 GAC GAC ACT GCT GAG AAA CGA CTA CAA AAG AAG CAA AGG TAC ATG GAA 4725 Asp Asp Thr Ala Glu Lys Arg Leu Gln Lys Lys Gln Arg Tyr Met Glu 535 540 545 ATA CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GAA TTA 4773 Ile Gln 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 AAGAGAACAC TGAGTGGGCG TGTAAGGTGC ATTCTCCTAG TCAGCTCCAT TGCATCCAAC 5346 ATTTGTGAAT GACACAAGTT AACAATCCTT TGCACCATTT CTGGGTGCAT ACATGGAAAC 5406 TTCTTCGATT GAAACTTCCC ACATGTGCAG GTGCGTTCGC TGTCACTGAT AGACCAAGAG 5466 ACTGAAAGCT TTCACAAATT GCCCTCAAAT CTTCTGTTTC TATCGTCATG ACTCCATATC 5526 TCCGACCACT GGTCATGAGC CAGAGCCCAC TGATTTTGAG GGAATTGGGC TAACCATTTC 5586 CGAGCTTCTG AGTCCTTCTT TTTGATGTCC TTTATGTAGG AATCAAATTC TTCCTTCTGA 5646 CTTGTGGAT 5655 (2) INFORMATION 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 Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser

1 5 10 15 Thr Ser Phe Val Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu 20 25 30 Ala Ala Glu Gln Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gln Leu 35 40 45 Leu Ser Asn Ser Phe Glu Ser Val Phe Asp Ser Pro Asp Asp Phe Tyr 50 55 60 Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His 65 70 75 80 Arg Cys Val Leu Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala 85 90 95 Ala Ala Lys Lys Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu 100 105 110 Glu Leu Lys Glu Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val 115 120 125 Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro 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 Ile 165 170 175 Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp 180 185 190 Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu

195 200 205 Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys 210 215 220 Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser 225 230 235 240 Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu 245 250 255 Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys 260 265 270 Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu 275 280 285 Asp His Thr Asn Leu Asp Asp 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 Gln Leu Ile 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 Ile 355 360 365 Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln 370 375 380 Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln

385 390 395 400 Glu Asp Lys Arg Glu Gln Ile 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 Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met 435 440 445 Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu 450 455 460 Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys 465 470 475 480 Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln 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 Gln Ile Met Asn Cys Glu Asp Leu Thr Gln Leu Ala 515 520 525 Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gln Lys Lys Gln Arg 530 535 540 Tyr Met Glu Ile Gln 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 : Ile Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile 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 Gln 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 N0 : 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 : Ile Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile 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 Gln 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 Gln 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 : Ile Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile 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 Gln 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 Gln 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 : Ile Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile 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 Gln 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 ="oligonucleotide" (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 ="oligonucleotide"

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 13 : AATTCCGATC GGCATGCTTT A 21 (2) INFORMATION FOR SEQ ID NO : 14 : (i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 22 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear (ii) MOLECULE TYPE : other nucleic acid (A) DESCRIPTION :/desc ="oligonucleotide" (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 ="oligonucleotide"

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 15 : AATTCCGATC GCCATGGTTT A 21 (2) INFORMATION FOR SEQ ID NO : 16 : (i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 15 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear (ii) MOLECULE TYPE : other nucleic acid (A) DESCRIPTION :/desc ="oligonucleotide" (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 ="oligonucleotide"

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 17 : CGGAATTCCA GCTGGCATG 19 (2) INFORMATION FOR SEQ ID NO : 18 : (i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 314 amino acids (B) TYPE : amino acid (C) STRANDEDNESS : not relevant (D) TOPOLOGY : not relevant (ii) MOLECULE TYPE : protein (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 18 : Met Phe Gln Pro Ala Gly His Gly Gln 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 Gln Met Val Lys Glu 35 40 45 Leu Arg Glu Ile Arg Leu Gln Pro Gln Glu Ala Pro Leu Ala Ala Glu 50 55 60 Pro Trp Lys Gln Gln Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu

65 70 75 80 Ala Ile Ile His Glu Glu Lys Pro Leu Thr Met Glu Val Ile Gly Gln 85 90 95 Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln 100 105 110 Thr Pro Leu His Leu Ala Val Ile Thr Asn Gln Pro Gly Ile Ala Glu 115 120 125 Ala Leu Leu Lys Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly 130 135 140 Asn Thr Pro Leu His Leu Ala Cys Glu Gln Gly Cys Leu Ala Ser Val 145 150 155 160 Ala Val Leu Thr Gln Thr Cys Thr Pro Gln His Leu His Ser Val Leu 165 170 175 Gln Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser Thr 180 185 190 His Gly Tyr Leu Ala Ile Val Glu His Leu Val Thr Leu Gly Ala Asp 195 200 205 Val Asn Ala Gln Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala 210 215 220 Val Asp Leu Gln Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly 225 230 235 240 Ala Asp Val Asn Arg Val Thr Tyr Gln Gly Tyr Ser Pro Tyr Gln Leu 245 250 255 Thr Trp Gly Arg Pro Ser Thr Arg Ile Gln Gln Gln Leu Gly Gln Leu

260 265 270 Thr Leu Glu Asn Leu Gln 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 Gln 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 Gln Pro Ala Gly His Gly Gln 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 Gln Met Val Lys Glu 35 40 45

Leu Arg Glu Ile Arg Leu Gln Pro Gln Glu Ala Pro Leu Ala Ala Glu 50 55 60 Pro Trp Lys Gln Gln Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu 65 70 75 80 Ala Ile Ile His Glu Glu Lys Thr Leu Thr Met Glu Val Ile Gly Gln 85 90 95 Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln 100 105 110 Thr Pro Leu His Leu Ala Val Ile Thr Asn Gln Pro Gly Ile Ala Glu 115 120 125 Ala Leu Leu Lys Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly 130 135 140 Asn Thr Pro Leu His Leu Ala Cys Glu Gln Gly Cys Leu Ala Ser Val 145 150 155 160 Ala Val Leu Thr Gln Thr Cys Thr Pro Gln His Leu His Ser Val Leu 165 170 175 Gln Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser Ile 180 185 190 His Gly Tyr Leu Gly Ile Val Glu His Leu Val Thr Leu Gly Ala Asp 195 200 205 Val Asn Ala Gln Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala 210 215 220 Val Asp Leu Gln Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly 225 230 235 240

Ala Asp Val Asn Arg Val Thr Tyr Gln Gly Tyr Ser Pro Tyr Gln Leu 245 250 255 Thr Trp Gly Arg Pro Ser Thr Arg Ile Gln Gln Gln Leu Gly Gln Leu 260 265 270 Thr Leu Glu Asn Leu Gln 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 Gln 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 Gln Pro Ala Glu Pro Gly Gln 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 Gln Met Val Lys Glu 35 40 45 Leu Arg Glu Ile Arg Leu Glu Pro Gln Glu Ala Pro Arg Gly Ala Glu 50 55 60 Pro Trp Lys Gln Gln Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu 65 70 75 80 Ala Ile Ile His Glu Glu Lys Ala Leu Thr Met Glu Val Val Arg Gln 85 90 95 Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln 100 105 110 Thr Pro Leu His Leu Ala Val Ile Thr Asn Gln Pro Glu Ile 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 Gln Gly Cys Leu Ala Ser Val 145 150 155 160 Gly Val Leu Thr Gln Pro Arg Gly Thr Gln His Leu His Ser Ile Leu 165 170 175 Gln Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser Ile 180 185 190 His Gly Tyr Leu Gly Ile Val Glu Leu Leu Val Ser Leu Gly Ala Asp 195 200 205 Val Asn Ala Gln Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala

210 215 220 Val Asp Leu Gln Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly 225 230 235 240 Ala Asp Val Asn Arg Val Thr Tyr Gln Gly Tyr Ser Pro Tyr Gln Leu 245 250 255 Thr Trp Gly Arg Pro Ser Thr Arg Ile Gln Gln Gln Leu Gly Gln Leu 260 265 270 Thr Leu Glu Asn Leu Gln 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 Gln 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="NIM1 cDNA sequence" (ix) FEATURE : (A) NAME/KEY : CDS (B) LOCATION : 43.. 1824 (D) OTHER INFORMATION :/product="NIM1 protein" (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 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 Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val 5 10 15 20 GCT ACC GAT AAC ACC GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA 150 Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu Ala Ala Glu Gln 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 Gln 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 Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu 120 125 130 GCT TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA 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 Ile Phe Lys Ile Pro 165 170 175 180 GAA TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA 630 Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys 185 190 195 GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT 678 Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile 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 Ile Ile 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 Ile Ile 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 Ile 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 Gln Leu Ile 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 Ile Ala Lys Gln 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 Ile Pro Glu Gln Cys Lys His Ser 375 380 385 CTC AAA GGC CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA CGA 1254 Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys Arg 390 395 400 GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TTT GCA GTG GCG GCC GAT 1302 Glu Gln Ile 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 Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile 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 Ile 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 Ile Ala Pro Phe 470 475 480 AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA ACC 1542 Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys Thr 485 490 495 500 GTG GAA CTC GGG AAA CGA TTC TTC CCG 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 Ile Met Asn Cys Glu Asp Leu Thr Gln Leu Ala Cys Gly Glu Asp 520 525 530 GAC ACT GCT GAG AAA CGA CTA CAA AAG AAG CAA AGG TAC ATG GAA ATA 1686 Asp Thr Ala Glu Lys Arg Leu Gln Lys Lys Gln Arg Tyr Met Glu Ile 535 540 545 CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GAA TTA GGA 1734 Gln Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn Leu Glu Leu Gly 550 555 560 AAT TTG TCC CTG ACA GAT 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 NIM1" /note="Serine residues at amino acid positions 55 and 59 in wild-type NIM1 gene product have been changed to Alanine residues." (ix) FEATURE : (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 ATT GAT GGA TTC GCC GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC GTC 102 Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val 5 10 15 20 GCT ACC GAT AAC ACC GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA 150 Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu Ala Ala Glu Gln 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 Gln 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 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 8Q 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 Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu 120 125 130 GCT TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA 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 Ile Phe Lys Ile Pro 165 170 175 180 GAA TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA 630 Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys 185 190 195

GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT 678 Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile 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 Ile Ile 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 Ile Ile 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 Ile 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 Gln Leu Ile 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 Ile Ala Lys Gln 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 Ile Pro Glu Gln Cys Lys His Ser 375 380 385 CTC AAA GGC CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA CGA 1254 Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys Arg 390 395 400 GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TTT GCA GTG GCG GCC GAT 1302 Glu Gln Ile 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 Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile 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 Ile 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 Ile Ala Pro Phe 470 475 480

AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA ACC 1542 Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys Thr 485 490 495 500 GTG GAA CTC GGG AAA CGA TTC TTC CCG 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 Ile Met Asn Cys Glu Asp Leu Thr Gln Leu Ala Cys Gly Glu Asp 520 525 530 GAC ACT GCT GAG AAA CGA CTA CAA AAG AAG CAA AGG TAC ATG GAA ATA 1686 Asp Thr Ala Glu Lys Arg Leu Gln Lys Lys Gln Arg Tyr Met Glu Ile 535 540 545 CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GAA TTA GGA 1734 Gln Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn Leu Glu Leu Gly 550 555 560 AAT TTG TCC CTG ACA GAT 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 Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser 1 5 10 15 Thr Ser Phe Val Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu 20 25 30 Ala Ala Glu Gln Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gln 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 Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val 115 120 125

Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro 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 Ile 165 170 175 Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp 180 185 190 Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu 195 200 205 Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys 210 215 220 Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser 225 230 235 240 Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu 245 250 255 Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys 260 265 270 Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu 275 280 285 Asp His Thr Asn Leu Asp Asp 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 Gln Leu Ile 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 Ile 355 360 365 Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln 370 375 380 Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln 385 390 395 400 Glu Asp Lys Arg Glu Gln Ile 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 Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met 435 440 445 Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu 450 455 460 Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys 465 470 475 480 Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln 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 Gln Ile Met Asn Cys Glu Asp Leu Thr Gln Leu Ala 515 520 525 Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gln Lys Lys Gln Arg 530 535 540 Tyr Met Glu Ile Gln 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 NIM1" /note="N-terminal deletion compared to wild-type NIM1 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 Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg 50 55 60 CAC TTA TTG GAC GTT GTA GAC AAA GTT GTT ATA GAG GAC ACA TTG GTT 240 His Leu Leu Asp Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val 65 70 75 80 ATA CTC AAG CTT GCT AAT ATA TGT GGT AAA GCT TGT ATG AAG CTA TTG 288 Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu 85 90 95 GAT AGA TGT AAA GAG ATT ATT GTC AAG TCT AAT GTA GAT ATG GTT AGT 336 Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser 100 105 110 CTT GAA AAG TCA TTG CCG GAA GAG CTT GTT AAA GAG ATA ATT GAT AGA 384 Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg 115 120 125 CGT AAA GAG CTT GGT TTG GAG GTA CCT AAA GTA AAG AAA CAT GTC TCG 432

Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser 130 135 140 AAT GTA CAT AAG GCA CTT GAC TCG GAT GAT ATT GAG TTA GTC AAG TTG 480 Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu 145 150 155 160 CTT TTG AAA GAG GAT CAC ACC AAT CTA GAT GAT GCG TGT GCT CTT CAT 528 Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His 165 170 175 TTC GCT GTT GCA TAT TGC AAT GTG AAG ACC GCA ACA GAT CTT TTA AAA 576 Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys 180 185 190 CTT GAT CTT GCC GAT GTC AAC CAT AGG AAT CCG AGG GGA TAT ACG GTG 624 Leu Asp Leu 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 Gln Leu Ile Leu Ser Leu 210 215 220 TTG GAA AAA GGT GCA AGT GCA TCA GAA GCA ACT TTG GAA GGT AGA ACC 720 Leu Glu Lys Gly 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 Ile Ala Lys Gln 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 Ile Pro Glu Gln 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

Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile 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 Gln 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 Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile 325 330 335 GTG ACT AGC CTC GAG CCT GAC CGT CTC ACT GGT ACG 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 Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser 355 360 365 AGA CTA AAA GCG CTT TCT AAA ACC GTG GAA CTC GGG AAA CGA TTC TTC 1152 Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe 370 375 380 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 Gln Ile Met Asn Cys Glu Asp Leu 385 390 395 400 ACT CAA CTG GCT TGC GGA GAA GAC GAC ACT GCT GAG AAA CGA CTA CAA 1248 Thr Gln Leu Ala Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gln 405 410 415 AAG AAG CAA AGG TAC ATG GAA ATA CAA GAG ACA CTA AAG AAG GCC TTT 1296

Lys Lys Gln Arg Tyr Met Glu Ile Gln 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 Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg 50 55 60 His Leu Leu Asp Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val 65 70 75 80 Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu 85 90 95 Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser 100 105 110 Leu Glu Lys Ser Leu Pro Glu Glu Leu Va-1 Lys Glu Ile Ile 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 Ile 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 Gln Leu Ile 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 Ile Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn 245 250 255 Ile Pro Glu Gln Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu 260 265 270 Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile 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 Gln Arg Leu Phe Pro Thr Glu Ala 305 310 315 320 Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile 325 330 335 Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser 340 345 350 Pro Gly Val Lys Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser 355 360 365 Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe 370 375 380 Pro Arg Cys Ser Ala Val Leu Asp Gln Ile Met Asn Cys Glu Asp Leu 385 390 395 400

Thr Gln Leu Ala Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gln 405 410 415 Lys Lys Gln Arg Tyr Met Glu Ile Gln 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 NIM1" /note="C-terminal deletion compared to wild-type NIM1." (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 26 : GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC 54

Met Asp Thr Thr 1 ATT GAT GGA TTC GCC GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC GTC 102 Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val 5 10 15 20 GCT ACC GAT AAC ACC GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA 150 Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu Ala Ala Glu Gln 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 Gln 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 Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu 120 125 130 GCT TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA 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 Ile Phe Lys Ile Pro 165 170 175 180 GAA TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA 630 Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys 185 190 195 GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT 678 Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile 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 Ile Ile 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 Ile Ile 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 Ile 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 Gln Leu Ile 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 Ile Ala Lys Gln 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 Ile Pro Glu Gln Cys Lys His Ser 375 380 385 CTC AAA GGC CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA CGA 1254 Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys Arg 390 395 400 GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TTT GCA GTG GCG GCC GAT 1302 Glu Gln Ile 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 Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile 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 Ile 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 Ile Ala Pro Phe 470 475 480 AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA ACC 1542 Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys Thr 485 490 495 500 GTG GAA CTC GGG AAA CGA TTC TTC CCG CGC TGT TCG GCA GTG CTC GAC 1590 Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu Asp 505 510 515 CAG ATT ATG AAC TGT TGA 1608 Gln Ile 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 Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser 1 5 10 15 Thr Ser Phe Val Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu 20 25 30 Ala Ala Glu Gln Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gln Leu 35 40 45 Leu Ser Asn Ser Phe Glu Ser Val Phe Asp Ser Pro Asp Asp Phe Tyr 50 55 60 Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His 65 70 75 80 Arg Cys Val Leu Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala 85 90 95 Ala Ala Lys Lys Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu 100 105 110 Glu Leu Lys Glu Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val 115 120 125 Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro 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 Ile 165 170 175

Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp 180 185 190 Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu 195 200 205 Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys 210 215 220 Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser 225 230 235 240 Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu 245 250 255 Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys 260 265 270 Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu 275 280 285 Asp His Thr Asn Leu Asp Asp 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 Gln Leu Ile 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 Ile 355 360 365

Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln 370 375 380 Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln 385 390 395 400 Glu Asp Lys Arg Glu Gln Ile 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 Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met 435 440 445 Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu 450 455 460 Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys 465 470 475 480 Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln 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 Gln Ile 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 NIM1" /note="N-terminal/C-terminal chimera." (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 28 : ATG GAT TCG GTT GTG ACT GTT TTG GCT TAT GTT TAC 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 Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg 50 55 60 CAC TTA TTG GAC GTT GTA GAC AAA GTT GTT ATA GAG GAC ACA TTG GTT 240 His Leu Leu Asp Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val 65 70 75 80 ATA CTC AAG CTT GCT AAT ATA TGT GGT AAA GCT TGT ATG AAG CTA TTG 288 Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu

85 90 95 GAT AGA TGT AAA GAG ATT ATT GTC AAG TCT AAT GTA GAT ATG GTT AGT 336 Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser 100 105 110 CTT GAA AAG TCA TTG CCG GAA GAG CTT GTT AAA GAG ATA ATT GAT AGA 384 Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg 115 120 125 CGT AAA GAG CTT GGT TTG GAG GTA CCT AAA GTA AAG AAA CAT GTC TCG 432 Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser 130 135 140 AAT GTA CAT AAG GCA CTT GAC TCG GAT GAT ATT GAG TTA GTC AAG TTG 480 Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu 145 150 155 160 CTT TTG AAA GAG GAT CAC ACC AAT CTA GAT GAT GCG TGT GCT CTT CAT 528 Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His 165 170 175 TTC GCT GTT GCA TAT TGC AAT GTG AAG ACC GCA ACA GAT CTT TTA AAA 576 Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys 180 185 190 CTT GAT CTT GCC GAT GTC AAC CAT AGG AAT CCG AGG GGA TAT ACG GTG 624 Leu Asp Leu 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 Gln Leu Ile Leu Ser Leu 210 215 220 TTG GAA AAA GGT GCA AGT GCA TCA GAA GCA ACT TTG GAA GGT AGA ACC 720 Leu Glu Lys Gly 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 Ile Ala Lys Gln 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 Ile Pro Glu Gln 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 Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile 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 Gln 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 Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile 325 330 335 GTG ACT AGC CTC GAG CCT GAC CGT CTC ACT GGT ACG 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 Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser 355 360 365 AGA CTA AAA GCG CTT TCT AAA ACC GTG GAA CTC GGG AAA CGA TTC TTC 1152 Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe

370 375 380 CCG CGC TGT TCG GCA GTG CTC GAC CAG ATT ATG AAC TGT TGA 1194 Pro Arg Cys Ser Ala Val Leu Asp Gln Ile 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 Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg 50 55 60 His Leu Leu Asp Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val 65 70 75 80 Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu 85 90 95

Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser 100 105 110 Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile 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 Ile 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 Gln Leu Ile 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 Ile Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn 245 250 255 Ile Pro Glu Gln Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu 260 265 270 Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile 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 Gln Arg Leu Phe Pro Thr Glu Ala 305 310 315 320 Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile 325 330 335 Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser 340 345 350 Pro Gly Val Lys Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser 355 360 365 Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe 370 375 380 Pro Arg Cys Ser Ala Val Leu Asp Gln Ile 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 NIM1"

/note="Ankyrin domains of NIM1." (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 Ile 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 Ile Phe Lys Ile Pro Glu 65 70 75 80 TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA GTT 288 Leu Ile Thr Leu Tyr Gln 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 Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile 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 Ile Ile 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 Ile Ile 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 Ile 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 Gln Leu Ile 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 Ile 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 Ile Phe Lys Ile Pro Glu 65 70 75 80 Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys Val 85 90 95 Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Gly 100 105 110 Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val Lys 115 120 125

Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu 130 135 140 Val Lys Glu Ile Ile 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 Ile 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 Gln Leu Ile 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 ="oligonucleotide" (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 ="oligonucleotide" (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 ="oligonucleotide" (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 ="oligonucleotide" (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 (ii) MOLECULE TYPE : other nucleic acid

(A) DESCRIPTION :/desc ="oligonucleotide" (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 36 : CGGAATTCGA TCTCTTTAAT TTGTGAATTT C 31 (2) INFORMATION FOR SEQ ID NO : 37 : (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 ="oligonucleotide" (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 37 : GGAATTCTCA ACAGTTCATA ATCTGGTCG 29 (2) INFORMATION FOR SEQ ID NO : 38 : (i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 31 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear (ii) MOLECULE TYPE : other nucleic acid

(A) DESCRIPTION :/desc ="oligonucleotide" (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 ="oligonucleotide" (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 39 : GGAATTCTCA ACCTTCCAAA GTTGCTTCTG ATG 33

American jfpcCwjrCoHection 12301 l'arki@wn Drive @ Rockvlite. MD 20852 USA @ Telephonc: (301)231-5520 Telex; @98-055 ATCCNORTH # FAX: 301-770-2587 BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF CIBA@@@@ THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL FORM RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT ISSUED PURSUANT TO RULE 7. 3 AND VIABILITY STATEMENT ISSUED PURSUANT TO RULE 10. 2 -CEtVED To : (Name and Address of Depositor or Attorney) Cibe-Gelgy Corporation <BR> <BR> Attn: Leslie B. Friedrich MAY 2 8 1996<BR> <BR> P.O. Box 12257 CIBA-GEIGY<BR> <BR> <BR> <BR> Research Triangle Park, NC 27709 ABRU PATENT DEPT, Deposited on Behalf of : Ciba-Geigy Corporation Identification Reference by Depositor : ATCC Designation Plasmid, BAC4 97 : i43 The deposit was accompanied by : _ a scientific description a proposed taxonomic description Indicated above.

The deposit was received May 8. 1996 by this Intemetional Depository Authorlty 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 receivo, 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 responsibiiity 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 May 17. 1996. On that date, the culture was viable.

International Depository Authority : American Type Culture Collection, Rockville, Md. 20852 USA American2jC/7rCoHection<BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> 12301 Parkawn Drive @ Rockville, MD 20852 USA @ Telephone: (301)231-5520 Telex: 898-855 ATCCNORTH # FAX; 301-770-2587 BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNA 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) Ciba-Geigy Corporation JUN 2 7 1996 <BR> <BR> Attn: Leslie B. Friedrich<BR> <BR> <BR> P.O. Box 12257 CIBA-GEIGY<BR> <BR> <BR> Research Triangle Park, NC 27709 ABRU PATENT DEPT Oaposttod on Behalf of : Cjba-Geigy Corporation Identification Reference by Depositor: ATCC Designation Plasmid P1-18 97606 The deposit was accompanied by a scientific description-a proposed taxonomic description indicated above. <BR> <BR> <BR> <BR> <BR> <P>The deposit was received June 13. 1996 by this International Depository Authority and has been accepte.

AT YOUR REQUEST : X We will inform you of requests for the suain 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 Instruc : ted 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 June 20. 1996. On that date, the culture was viable.

International Depository Authority : American Type Culture Collection, Rockville, Md. 20852 USA american Type Culture Collection 12301 Parkiawn Drive # Rochwille, MD 10852 USA # Telephone: (301)231-5520 Teiex; 908-768 ATCCROVE # FAX: 301-816-4166 BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PIIRPOSES OF PATENT PROCéDURE INTERNATIONAL FORM RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT ISSUED PURSUANT TO RULE 7. 3 AND VIABILITY S' (ATEMENT ISSUED PURSUANT TO RULE 10. 2 To : (Name and Address of Daposltor or Attorney) Ciba-Geigy Corporation Attention : Leslie B. Friedrich P. O. Box 12257 Research Triangle Park, NC 27709 Deposited on Behalf of: Ciba-Gelgy 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 25, 1996 by this International Depository Authority and has been accepte.

AT YOUR REQUEST : W 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 & Trademerk Office or the depositor to release said strain.

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

The strain will be maintained for e period of at least 30 years after the date or doposit, and for a period of at least five years after the most recent request for a sampte. Tho United States and many other countries are signatory to the Budapost Treaty.

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

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