PL191355B1 - Method of protecting plants - Google Patents

Abstract

FIELD: agriculture. SUBSTANCE: invention relates to method for protection of plants against pathogens. After treatment of immunomodulated plants with usual bactericidal agent the resistance of plants to diseases enhances synergetically, i. e. these plants show activated (systemic acquired resistance; ASR). Immunomodulation, in turn, can be achieved by treatment of plants with chemical inducing agent of ASR, using selective selection program based on selection of constitutive expression of ASR genes and/or phenotype resistant to diseases, or by transformation of plants with one or some ASR genes. EFFECT: enhanced effectiveness for protection. 30 cl, 3 dwg, 40 tbl, 35 ex

Description

Description of the invention

The present invention relates to a method of protecting a plant against attack by a pathogen. This protection is achieved by applying a microbicide to a previously immunomodulated plant.

Plants are constantly exposed to various pathogenic organisms, including viruses, bacteria, fungi and nematodes. Crops are particularly susceptible to disease, as the farming is generally carried out as genetically homogeneous monocultures and when disease attacks this type of crop, losses are usually very severe. However, most plants have innate defense mechanisms against pathogens. This natural resistance to plant pathogens, genetically encoded, is known to plant breeders and pathologists and is used to protect many crops. Natural disease resistance genes often result in high levels of resistance or resistance to pathogens.

Systemic acquired resistance (SAR) is one component of a complex system that plants use to defend themselves against pathogens (Hunt and Ryals, Crit. Rev. in Plant Sci. 15, 583-606 (1996); Ryals et al., Plant Cell 8, 1809-1819 (1996); U.S. Patent No. 5,614,395).

SAR is a particularly important aspect of the plant-pathogen response because it is pathogen-inducible systemic resistance that encompasses a wide range of infectious agents, including viruses, bacteria, and fungi. When the SAR signal transduction pathway is blocked, plants become more sensitive to disease-causing pathogens, and they also become sensitive to certain infectious agents that do not normally cause disease (Gaffney et al., Science 261, 754-756 (1993); Delaney et al. et al., Science 266, 1247-1250 (1994); Delaney et al., Proc. Natl. Acad. Sci. USA 92, 6602-6606 (1995); Delaney, Plant Phys. 113, 5-12 (1997); Bi et al., Plant J. 8, 235-245 (1995); Mauch-Mani and Slusarenko, Plant Cell 8, 203-212 (1996)). These observations indicate that the SAR signal transduction pathway is extremely important in maintaining plant health.

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

Accumulation of salicylic acid (SA) 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 that specifically degrades SA, are also unable to induce SAR gene expression or disease resistance (Gaffney et al., 1993; Delaney et al., 1994; Mauch-Mani and Slusarenko 1996; Mahrer et al., Proc. Natl. Acad. Sci. USA 91, 7802-7806 (1994)). While it has been suggested that SA may act as a systemic signal, this is currently controversial, and so far it is only known for certain that if SA cannot accumulate then SAR signal transduction is blocked (Pallas et al., 1996; Shulayev et al., Plant Cell 7, 1961-1701 (1995); Vernooij et al., Plant Cell 6, 959-965 (1994)).

More recently, Arabidopsis has proven to be a model system for the SAR study (Uknes et al., Plant Cell 4, 645-656 (1992); Uknes et al., Mol. Plant-Microbe Interact. 6, 692-698 (1993); Cameron et al., et al., Plant J. 5 715-725 (1994); Mauch-Mani and Slusarenko, Mol. Plant-Microbe Interact. 7, 376-383 (1994),

Dempsey and Klessig, Bulletin de Hnstitut Pasteur, 167-186 (1995)). It has been shown that SAR can be activated in Arabidopsis by both pathogens and chemicals such as SA, 2,6-dichloro-isonicotinic acid (INA) and benzo [1,2,3] thiadiozole-7-carbothioic acid S-methyl ester (BTH) (Uknes et al., 1992; Vernooij et al., Mol. Plant-Microbe Interact. 8, 228-234 (1995); Lawdon et al., Plant J. 10, 71-82 (1996)). Upon treatment with either INA or BTH, or by infection with a pathogen, at least three pathogenesis-related (PR) protein genes, namely PR-1, PR-2 and PR-5, undergo co-ordinated induction with the onset of resistance (Uknes et al., 1992, 1993). In tobacco, the species best characterized, treatment with a pathogen or immunizing compound induces at least nine sets of genes (Ward et al., Plant Cell3, 1085-1094 (1991)). Transgenic disease resistant plants have been obtained by transforming plants with various SAR genes (US Patent 5,614,395).

A number of Arabidopsis mutants have been obtained 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) mutants (Dietrich et al., Cell 77, 565-577 (1994); Greenberg et al. , Cell 77, 551-563 (1994)). All these mutants have some degree of spontaneous production of necrotic lesions on their own

In leaves, elevated SA levels, mRNA accumulation for SAR genes, and significantly increased disease resistance. At least seven different mutants have been isolated and characterized (Dietrich et al., 1994; Weymann et al., Plant Cell 7, 2013-2022 (1995)). Another interesting class of mutants are the cim (constitutive immunity) mutants (Lawton et al., "The molecular biology of systemic acquired resistance" in The Mechanisms of Defense Responses in Plants, B. Fritig and M. Legrand, eds. (Dordrecht , The Netherlands, Kluwer Academic Publishers, pp. 422-432 (1993) and WO 94/16077 Similar to the Isd and acd2 mutants, the cim mutants have elevated SA and SAR gene expression and resistance but unlike Isd or acd2, they show no detectable damage on its leaves, cpr1 (constitutive expresser of PR genes - constitutively expressing PR genes) may be a type of cim mutant, but due to the presence of microscopic lesions on leaves, cpr1 has not been ruled out. cpr1 may be a type of Isd mutant (Bowling et al., Plant Cell 6, 1845-1857 (1994)).

Mutants that are blocked in SAR signaling have also been isolated. ndr1 (non-race specific disease resistance) is a mutant that allows the growth of both Pseudomonas syringae containing different avirulence genes and also normal avirulent isolates of Peronospora parasitica (Century et al., Proc. Natl. Acad. Sci. USA 92, 6597-6601 (1995)). This mutant appears to be blocked early in SAR signaling. nprl (nonexpresser of PR genes) is a mutant that fails to induce the expression of the SAR signaling pathway after INA treatment (Cao et al., Plant Cell 6, 1583-1592 (1994)). Eds (enhanced disease susceptibility) mutants were isolated on the basis of their ability to sustain infection by bacteria after inoculation with low bacterial concentrations (Glazebrook et al., Genetics 143, 973-982 (1996); Parker et al., Plant Cell 8, 2033-2046 (1996)). Certain eds mutants are phenotypically very similar to npr1, and recently it has been shown that eds5 and eds53 are allelic with nprl (Glazebrook et al., 1996). nim1 (noninducible immunity) is a mutant that maintains the growth of P. parasitica (the causative agent of downy mildew disease) after INA treatment (Delaney et al., 1995; WO 94/16077). While nim1 may accumulate SA following pathogen infection, it cannot induce SAR gene expression or disease resistance, suggesting that the mutation blocks the pathway downstream of SA. nim1 is also defective in its ability to respond to INA or BTH, suggesting that a block exists downstream of these chemicals (Delaney et al., 1995; Lawton et al., 1996).

Recently, two allelic Arabidopsis genes have been isolated and characterized in which mutants are responsible for the nim1 and npr1 phenotypes, respectively (Ryals et al., Plant Cell 9, 425-439 (1997); Cao et al., Cell 88, 57-63 (1997) ). The wild-type NIM1 gene product is involved in the signal transduction cascade leading to both SAR and gene-to-gene disease resistance in Arabidopsis (Ryals et al., 1997). Ryals et al., 1997 also report the isolation of five additional nim1 alleles, which display a phenotype range from poorly damaged in chemically induced PR-1 gene expression and fungal resistance to very strongly blocked. Transformation of the wild-type NPR1 gene into npr1 mutants not only complemented the mutants, restoring the SAR induction response for PR gene expression and disease resistance, but also made the transgenic plants more resistant to P. syringae infection in the absence of SAR induction (Cao et al., 1997).

NF-κΒ / ΙκΒ signal transduction pathways have been implicated in disease resistance responses ranging from Drosophila to mammals. In mammals, NF-kB / IkB signal transduction can be induced by a variety of stimuli, including cell contact with lipopolysaccharide, tumor necrosis factor, interleukin 1 (IL-1), or viral infection (Bauerle 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 related to inflammatory and immune responses, such as IL-2, IL-6, IL-8 and granulocyte / macrophage colony stimulating factor (deMartin et al., Gene 152, 253-255 (1995) ). In studies of transgenic mice, the knockout of NF-kB / IkB signal transduction leads to a defective immune response, including increased sensitivity 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, 764-787 (1996); Bauerle and Baltimore (1996)). In Arabidopsis, SAR is functionally analogous to inflammation except that the normal processes of resistance are enabled following SAR activation leading to increased disease resistance (Bi et al., 1995; Cao et al., 1994; Cao et al., 1995; Delaney et al. et al., 1995; Delaney et al., 1994; Gaffney et al., 1993; Mauch-Mani and Slusarenko 1996; Delaney, 1997). Moreover, inactivation of the pathway leads to increased sensitivity 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. Drosophila bacterial infection activates the signal transduction cascade leading to the synthesis of some antifungal proteins such as cercropin B, defensin, dipterisine and drosomycin (Ip et al., Cell 75, 753-763 (1993); Lemaitre et al., Cell 86, 973- 983 (1996)). This induction depends on the product of the dorsal and dif gene, two NF-κΒ homologs, and is repressed by the cactus, the IkB homolog in the fly. Mutants that have decreased synthesis of antifungal and antibacterial proteins have dramatically decreased resistance to infection.

Despite extensive research and the use of advanced and intensive plant protection products, including genetic transformation of plants, disease-related losses are still billions of dollars. Thus, there is a continuing need to develop new plant protection products based on the ever-growing understanding of the genetic basis of disease resistance in plants.

In view of the above, an advantageous aspect of the present invention relates to a new method of protecting plants against pathogen attack by conferring on the plant synergistic disease resistance, obtained by applying a microbicide to immunomodulated plants. Immunomodulated plants are those in which SAR is activated, typically showing greater SAR gene expression than that of the wild-type. These plants are referred to as SAR-enabled plants (SAR-on). Immunomodulated plants for use in the method of the invention can be obtained: by applying to the plants a chemical SAR inducer such as BTH, INA, or SA; through a selective breeding program in which plants are selected based on the selective expression of SAR genes and / or disease resistant phenotype; or by genetically engineering plants by transforming them with one or more SAR genes such as a functional form of the NIM1 gene. The microbicide applied to immunomodulated plants may either be a conventional microbicide such as a metalaxyl fungicide, or if it is applied to immunomodulated plants obtained by selective breeding or genetic engineering, the microbicide may be a chemical SAR inducer such as BTH, INA or SA.

Immunomodulation provides a certain level of disease resistance in the plant. Likewise, a certain level of disease resistance is provided by the application of a microbicide to the plant. The expected result of combining immunomodulation with a microbicide would be the sum of these resistances. However, in fact, application of a microbicide to an immunomodulated plant surprisingly increases disease resistance synergistically, i.e. the level of disease resistance is much greater than the expected additive increase in the level of resistance.

According to the invention, a method of protecting a plant from pathogen attack by imparting synergistic disease resistance to the plant consists in providing an immunomodulated plant having a first level of disease resistance, and applying to said immunomodulated plant at least one microbicide which imparts a second level of disease resistance to it, wherein application of a microbicidal agent to an already immunomodulated plant confers a synergistically enhanced third disease resistance level which is greater than the sum of the first and second disease resistance levels and is characterized in that the immunomodulated plant is:

(i) a mutant (cim) plant with constitutive resistance, (ii) a mutant mutant mutant plant, or (iii) a plant obtained by recombinant expression of the SAR gene.

Preferably, a cim mutant of a plant is selected from the plant population, whereby: (a) assessing the expression of SAR genes in uninfected plants that are phenotypically normal in that said uninfected plants do not have a lesion mimicking phenotype, and then (b) selecting uninfected plants plants that constitutively express SAR genes in the absence of viral, bacterial or fungal infection.

Preferably, a mutant mutant plant is selected from the plant population in the following sequence: (a) assessing the expression of SAR genes in uninfected plants that have a damage mimicking phenotype, and then (b) selecting uninfected plants that constitutively express SAR genes under absence of viral, bacterial or fungal infection.

According to the invention, a SAR gene is preferably used, which is a functional form of the NIM1 gene encoding the NIM1 protein, associated with the signal transduction cascade leading to acquired systemic resistance in plants, and in particular a NIM1 protein is produced having the amino acid sequence shown as SEQ ID NO: 2 or produced a NIM1 protein, which is encoded by a DNA molecule containing the coding sequence shown as SEQ ID NO: 1.

According to the invention, it is also preferred to use a SAR gene which codes for an altered form of the NIM1 protein which expresses the SAR gene constitutively in a transgenic plant, in particular:

An altered form of the NIM1 protein is produced that has alanines instead of serines at the amino acid positions 55 and 59 in SEQ ID NO: 2, and in particular an altered form of the NIM1 protein which includes the amino acid sequence shown in SEQ ID NO: 8. or which is encoded by a DNA molecule which comprises the nucleotide sequence shown as SEQ ID NO: 7; or an altered form of the NIM1 protein is produced which has cleaved amino acids at the N-terminus corresponding approximately to amino acid positions 1-125 in SEQ ID NO: 2, in particular an altered form of the NIM1 protein which comprises the amino acid sequence shown in SEQ ID NO: 10 or producing an altered form of the NIM1 protein that is encoded by a DNA molecule that comprises the nucleotide sequence shown as SEQ ID NO: 9; or an altered form of the NIM1 protein is produced which has cleaved amino acids at the C-terminus corresponding approximately to amino acid positions 522-593 in SEQ ID NO: 2, in particular an altered form of the NIM1 protein which comprises the amino acid sequence shown in SEQ ID NO: 12; or an altered form of the NIM1 protein which is encoded by the DNA molecule which comprises the nucleotide sequence shown as SEQ ID NO: 11; or an altered form of the NIM1 protein is produced which has a cleaved amino acid at the N-terminus corresponding to approximately amino acid positions 1-125 in SEQ ID NO: 2 and a cleaved amino acid at the C-terminus corresponding to approximately amino acid positions 522-593 in SEQ ID NO: 2 NO: 2, in particular, an altered form of the NIM1 protein is produced which comprises the amino acid sequence shown as SEQ ID NO: 14, or which is encoded by a DNA molecule which comprises the nucleotide sequence shown as SEQ ID NO: 13; or an altered form of the NIM1 protein is produced which consists essentially of ankyrin motifs corresponding to approximately amino acid positions 103-362 in SEQ ID NO: 2, and in particular, an altered form of the NIM1 protein is produced which comprises the amino acid sequence shown in SEQ ID NO: 16 or an altered form of the NIM1 protein which is encoded by the DNA molecule comprising the nucleotide sequence shown as SEQ ID NO: 15.

In all the above-mentioned embodiments of the process according to the invention, it is preferred that the microbicide is a fungicide selected from the group consisting of:

4- [3- (4-chlorophenyl) -3- (3,4-dimethoxyphenyl) acryloyl] morpholine ("dimethomorph"); 5-methyl-1,2,4-triazolo [3,4-b] [1,3] benzothiazole ("tricyclazole"); 3-allyloxy-1,2-benzothiazole-1,1-dioxide ("probonazole"); α- [2- (4-chloro-phenyl) ethyl] -a (1,1-dimethylethyl) -1H-1,2,4-triazole-1-ethanol ("tebuconazole"); 1 - {[3- (2-chlorophenyl) -2- (4-fluoro-phenyl) oxiran-2-yl] methyl} 1H-1,2,4-triazole ("epoxiconazole"); α- (4-chlorophenyl) -α- (1-cyclopropylethyl-1H-1,2,4-triazole-1-ethanol ("cyproconazole"); 5- (4-chlorobenzyl) -2-2-dimethyl-1- (1H-1,2,4-triazole-1-1-methyl) -cyclopentanol ("metconazole"); 2- (2,4-dichlorophenyl) -3- (1H-1,2,4-triazol-1-yl ) propyl-1,1,2,2-tetrafluoroethyl-ether ("tetraconazole"); methyl- (E) -2- {2- [6- (2-cyanophenoxy) pyrimidin-4-yl-oxy] phenyl} - 3-methoxyacrylate ("ICI A 5504", "azoxystrobin"); methyl (E) -2-methoxyimino-2- [α- (o-tolyloxy) -o-tolyl] acetate ("BAS 490 F", "Methylcresoxime "); 2- (2-phenoxyphenyl) - (E) -2-methoxyimino-N-methylacetamide; [2- (2,5-dimethylphenoxymethyl) -phenyl] - (E) -2-methoxyimino-N-methylacetamide; ( 1R, 3S / 1S, 3R) -2,2-dichloro-N - [(R) -1- (4-chlorophenyl) ethyl] -1-ethyl-3-methylcyclopropanecarboxamide ("KTU 3616"); polymeric ethylene bis ( manganese dithiocarbamate) with zinc salts ("mancozeb"); 1- [2- (2,4-dichlorophenyl) -4-propyl-1,3-dioxo-2-ylmethyl] -1H-1,2, 4-triazole ("propiconazole"); 1- [2- [2-chloro-4- (4-chlorophenoxy) phenyl] -4-methyl-1,3-di- xolan-2-yl-methyl] -1H-1,2,4-triazole ("diphenoconazole"); 1- [2- (2,4-dichlorophenyl] pentyl-1H-1,2,4-triazole ("penconazole"); cis-4- [3- (4-tert-butylphenyl) -2-methylpropyl] -2 , 6-dimethylmorpholine ("fenpropimorph"); 1- [3- (4-tert-butylphenyl) -2-methylpropyl] -piperidine ("fenpropidine"); 4-cyclopropyl-6-methyl-N-phenyl-2-pyrimidine -amine ("cyprodinyl"); (RS) -N- (2,6-dimethylphenyl-N- (methoxyacetyl) -alanine ("metalaxyl") methyl ester; (R) -N- (2,6-dimethylphenyl) methyl ester -N- (methoxyacetyl) -alanine ("R-metalaxyl"); 1,2,5,6-tetrahydro-4H-pyrrolo [3,2,1, ij] quinoline-4-one ("pyroquilone") and a phosphonate ethyl hydride ("fosetyl"), with metalaxyl being used particularly preferably as the fungicide.

It is also advantageous if either a benzothiadiazole compound or an isonicotinic acid compound or a salicylic acid compound is used as the microbicide in all of the above-mentioned embodiments of the process according to the invention, but it is also equally preferred that two microbicides are simultaneously applied to an immunomodulated plant, one of which is a fungicide. selected from the group consisting of 4- [3- (4-chlorophenyl) -3- (3,4-dimethoxyphenyl) acryloylmorpholine ("dimethomorph"); 5-methyl-1,2,4-triazolo [3,4-b] [1,3] benzothiazole ("tricyclazole"); 3-allyloxy-1,2-benzothiazole-1,1-dioxide ("probonazole");

Α- [2- (4-chlorophenyl) ethyl] - α (1,1-dimethylethyl) -1H-1,2,4-triazole-1-ethanol ("tebucanozole"); 1 - {[3- (2-chloro-phenyl) -2- (4-fluorophenyl) oxiran-2-yl] methyl} -1H-1,2,4-triazole ("epoxiconazole"); a- (4-chlorophenyl) -a- (1-cyclopropylethyl) -1H-1,2,4-triazole-1-ethanol ("cyproconazole"); 5- (4-chlorobenzyl) -2-2-dimethyl-1- (1H-1,2,4-triazol-1-ylmethyl) cyclopentanol, ("metconazole"); 2- (2,4-dichlorophenyl) -3- (1H-1,2,4-triazol-1-yl) -propyl-1,1,2,2-tetrafluoroethylether ("tetraconazole"); methyl- (E) -2- {2- [6- (2-cyanophenoxy) pyrimidin-4-yloxy] phenyl} -3-methoxyacrylate, ("ICI A 5504", "azoxystrobin"); methyl (E) -2-methoxyimino-2- [α- (o-tolyloxy) -o-tolyl] acetate ("BAS 490 F", "methyl cresoxime"); 2- (2-phenoxyphenyl) - (E) -2-methoxyimino N-methylacetamide; [2- (2,5-dimethylphenoxymethyl) phenyl] - (E) -2-methoxymino-N-methylacetamide; (1R, 3S / 1S, 3R) 2,2-dichloro-N - [(R) -1- (4-chlorophenyl) ethyl] -1-ethyl-3-methylcyclopropanecarboxamide ("KTU 3616"); polymeric manganese ethylene bis (dithiocarbamate) complex with zinc salts ("mancozeb"); 1- [2- (2,4-dichlorophenyl) -4-propyl-1,3-dioxolan-2-ylmethyl] -1H-1,2,4-triazole ("propiconazole"); 1- {2- [2-chloro-4- (4-chlorophenoxy) phenyl] -4-methyl-1,3-dioxolan-2-yl-methyl} -1H-1,2,4-triazole ("diphenoconazole" ); 1- [2- (2,4-dichlorophenyl} pentyl-1H-1,2,4-triazole ("penconazole"); cis-4- [3- (4-tert-butylphenyl) -2-methyl-propyl] -2,6-dimethylmorpholine ("fenpropimorph"); 1- [3- (4-tert-butylphenyl) -2-methylpropylpiperidine ("fenpropidine"); 4-cyclopropyl-6-methyl-N-phenyl-2- pyrimidinamine ("cyprodinyl"); (RS) -N- (2,6-dimethylphenyl-N- (methoxyacetyl) -alanine ("metalaxyl") methyl ester; (R) -N- (2,6-dimethyl) methyl ester lphenyl-N- (methoxyacetyl) -alanine ("R-metalaxyl"); 1,2,5,6-tetrahydro-4H-pyrrolo [3,2,1, ij] quinoline-4-one ("pyroquilone") and ethyl hydride phosphonate ("fosetyl").

It is especially preferred that said fungicide is metalaxyl and the other microbicide is a benzothiadiazole compound.

The method of the invention preferably produces the NIM1 protein or an altered form thereof using a DNA molecule which hybridizes to the nucleotide sequence shown as SEQ ID NO: 1, 7, 9, 11, 13 or 15 under the following conditions: 1% BSA; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM NaCl at 55 ° C for 18-24 hours. and washing in 6xSSC for 15 min (X3) 3xSSC for 15 min (X1) at 55 ° C. In this case, especially a fungicide selected from the group consisting of: 4- [3- (4-chlorophenyl) -3- (3,4-dimethoxyphenyl) acryloylmorpholine ("dimethomorph"); 5-methyl-1,2,4-triazolo [3,4-b] [1,3] benzothiazole ("tricyclazole"); 3-allyloxy-1,2-benzothiazole-1,1-dioxide ("probonazole"); α- [2- (4-chlorophenyl) ethyl] -α (1,1-dimethylethyl) -1H-1,2,4-triazole-1-ethanol ("tebucanozole"); 1 - {[3- (2-chlorophenyl) -2- (4-fluorophenyl) oxiran-2-yl] methyl} -1H-1,2,4-triazole ("epoxiconazole"); α- (4-chlorophenyl) -α- (1-cyclopropylethyl) -1H-1,2,4-triazole-1-ethanol ("cyproconazole"); 5- (4-chloro-benzyl) -2-2-dimethyl-1- (1H-1,2,4-triazol-1-ylmethyl) cyclopentanol, ("metconazole"); 2- (2,4-dichlorophenyl) -3- (1H-1,2,4-triazol-1-yl) -propyl-1,1,2,2-tetrafluoroethyl ether, ("tetraconazole"); methyl- (E) -2- {2- [6- (2-cyano-phenoxy) pyrimidin-4-yloxy] phenyl} -3-methoxyacrylate, ("ICI A 5504", "azoxystrobin"); methyl (E) -2-methoximino-α-α-o-tolyloxy-o-tolylphacetane ("BAS 490 F", "methyl cresoxime"); 2- (2-phenoxyphenyl) - (E) -2-methoxyimino-N-methylacetamide; [2- (2,5-dimethylphenoxymethyl) phenyl] - (E) -2-methoxymino-N-methyl acetamide; (1R, 3S / 1S, 3R) 2,2-dichloro-N - [(R) -1- (4-chlorophenyl) ethyl] -1-ethyl-3-methylcyclopropane carboxamide ("KTU 3616"); polymeric manganese ethylene bis (dithiocarbamate) complex with zinc salts ("mancozeb"); 1- [2- (2,4-dichlorophenyl) -4-propyl-1,3-dioxolan-2-ylmethyl] -1H-1,2,4-triazole ("propiconazole"); 1- {2- [2-chloro-4- (4-chlorophenoxy) phenyl] -4-methyl-1,3-dioxolan-2-yl-methyl} -1H-1,2,4-triazole ("diphenoconazole "); 1- [2- (2,4-dichlorophenyl} pentyl-1H-1,2,4-triazole ("penconazole"); cis-4- [3- (4-tert-butylphenyl) -2-methylpropyl] -2,6-dimethylmorpholine ("fenpropimorph"); 1- [3- (4-tert-butylphenyl) -2-methylpropylpiperidine ("fenpropidine"); 4-cyclopropyl-6-methyl-N-phenyl-2- pyrimidinamine ("cyprodinyl"); (RS) -N- (2,6-dimethylphenyl-N- (methoxyacetyl) -alanine ("metalaxyl") methyl ester; (R) -N- (2,6-dimethylphenyl) methyl ester N- (methoxyacetyl) -alanine ("R-metalaxyl"); 1,2,5,6-tetrahydro-4H-pyrrolo [3,2,1, ij] -quinoline-4-one ("pyroquilone") and ethyl hydride phosphonate ("fosetyl").

A particularly preferred fungicide among those mentioned above is metalaxyl, and particularly preferred microbicides are either a benzothiadiazole compound or an isonicotinic acid compound or a salicylic acid compound. It is also preferred that two microcybides are simultaneously applied to the immodulated plant, one of which is a fungicide selected from the group consisting of: 4- [3- (4-chlorophenyl) -3- (3,4-dimethoxyphenyl) acryloylmorpholine ("dimethomorph"); 5-methyl-1,2,4-triazolo [3,4-b] [1,3] benzothiazole ("tricyclazole"); 3-allyloxy-1,2-benzothiazole-1,1-dioxide ("probonazole"); α- [2- (4-chlorophenyl) ethyl] -α (1,1-dimethylethyl) -1H-1,2,4-triazole-1-ethanol ("tebucanozole"); 1 - {[3- (2-chloro-phenyl) -2- (4-fluorophenyl) oxiran-2-yl] methyl} -1H-1,2,4-triazole ("epoxiconazole"); α- (4-chlorophenyl) -α- (1-cyclopropylethyl) -1H-1,2,4-triazole-1-ethanol ("cyproconazole"); 5- (4-chlorobenzyl) -2-2-dimethyl-1- (1H-1,2,4-triazol-1-ylmethyl) cyclopentanol, ("metconazole"); 2- (2,4-dichlorophenyl) -3- (1H-1,2,4-triazol-1-yl) -propyl-1,1,2,2-tetrafluoroethyl ether, ("tetraconazole"); methyl- (E) -2- {2- [6- (2-cyanophenoxy) pyrimidin-4PL 191 355 B1

-yloxy] phenyl} -3-methoxyacrylate, ("ICI A 5504", "azoxystrobin"); methyl (E) -2-methoxyimino-2- [α- (o-tolyloxy) -o-tolyl] acetate ("BAS 490 F", "methyl cresoxime"); 2- (2-phenoxyphenyl) - (E) -2-methoxyimino-N-methylacetamide; [2- (2,5-dimethylphenoxymethyl) phenyl] - (E) -2-methoxymino-N-methylacetamide; (1R, 3S / 1S, 3R) 2,2-dichloro-N - [(R) -1- (4-chlorophenyl) ethyl] -1-ethyl-3-methylcyclopropanecarboxamide ("KTU 3616"); polymeric manganese ethylene bis (dithiocarbamate) complex with zinc salts ("mancozeb"); 1- [2- (2,4-dichlorophenyl) -4-propyl-1,3-dioxolan-2-ylmethyl] -1H-1,2,4-triazole ("propiconazole"); 1- {2- [2-chloro-4- (4-chlorophenoxy) phenyl] -4-methyl-1,3-dioxolan-2-yl-methyl} -1H-1,2,4-triazole ("diphenoconazole" ); 1- [2- (2,4-dichlorophenyl} pentyl-1H-1,2,4-triazole ("penconazole"); cis-4- [3- (4-tert-butylphenyl) -2-methylpropyl] -2 , 6-dimethylmorpholine ("fenpropimorph"); 1- [3- (4-tert-butyl-phenyl) -2-methylpropylpiperidine ("fenpropidine"); 4-cyclopropyl-6-methyl-N-phenyl-2-pyrimidinamine ( "Cyprodinyl"); (RS) -N- (2,6-dimethylphenyl-N- (methoxyacetyl) -alanine ("metalaxyl") methyl ester; (R) -N- (2,6-dimethyl-phenyl) methyl ester N- (methoxyacetyl) -alanine ("R-metalaxyl"); 1,2,5,6-tetrahydro-4H-pyrrolo [3,2,1, ij] quinoline-4-one ("pyroquilone") and hydride phosphonate ethyl ("fosetyl") and the second microbicide is either a benzothiadiazole compound or an isonicotinic acid compound or a salicylic acid compound Particularly preferably said fungicide is metalaxyl and the second microbicide is a benzothiadiazole compound.

Thus, the invention relates to the protection of plants against pathogen attack, the cultivation of immunomodulated plants and the application of an appropriate amount of a conventional microbicide to them. Particularly preferred embodiments of the invention relate to plants genetically manipulated to contain and express a functional form of the NIM1 gene or a homologue or variant thereof.

Examples of target plants for the indications disclosed herein include, but are not limited to, the following plant species: cereals (corn, wheat, barley, rye, oats, rice, sorghum, and related crops); beetroot (sugar beet and fodder beet); hard fruit, stone fruit and soft fruit (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and blackberries); legume plants (beans, lentils, peas, soybeans); oil plants (rapeseed, mustard, poppy seeds, olives, sunflowers, coconut, ricin plants, cocoa beans, peanuts); cucumber plants (squashes, cucumbers, melons); fiber plants (cotton, linen, hemp, jute); citrus fruits (oranges, lemons, grapefruits, mandarins); vegetables (spinach, lettuce, asparagus, cabbage, carrots, onions, tomatoes, potatoes, peppers); laureaceae (avocado, cinnamon, camphor); or plants such as tobacco, nuts, coffee, sugarcane, tea, vines, hops, bananas, natural gum plants, as well as ornamental plants (flowers, shrubs, broad-leaved trees and evergreens such as cones). This list is not intended to be limiting.

The method of the present invention can be used to confer resistance to a wide variety of plant pathogens, which include, but are not limited to: viruses or viroids such as tobacco or cucumber mosaic viruses, ringspot or necrosis virus, pelargonium leaf roll virus, corn red spot virus. , tomato bush virus and similar viruses; fungi from the group of asocysts such as the genera Venturia, Podosphaera, Erysiphe, Monolinia, Mycosphaerella, and Uncinula; fungi from Basidiomycetes such as from the genera Hemileia, Rhizoctonia, and Puccinia; Fungi imperfecti such as the genera Botyris, Herlminthosporium, Rhynchosporium, Fusarium (including F. monoforme), Septoria, Cercospora, Alternaria, Pyricularia and Pseudocercosporella (including P. herpotrichoides); Oomycetest fungi such as from the genera Phytophthora (including P. parasitica), Peronospora (including P. tabacina), Bremia, Pythium, and Plasmopara; as well as other fungi such as Sclerophthora macrospora, Sclerophthora rayissiae,

Sclerospora graminicola, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora sacchari and Peronosclerospora maydis, Physopella zeae, Cercospora zeae-maydis, Colletotrichum graminicola, Gibberella zeae, Exserohilum turipluicaris, and Kabberella zeae, Exserohilum turipluicaris, and Kabberella zeae; bacteria such as Pseudomonas syringae, Pseudomonas tabaci, and Erwinia stewartii; insects such as aphids such as Myzus persicae and lepidoptera such as Heliothus spp., and nematodes such as Meloidogyne incognita.

Obtaining immunomodulated plants

All three of the following three general methods for obtaining immunomodulated plants are related because they all fit the SAR transduction model given in Ryals et al. (1996). Once the SAR signal transduction pathway is activated to develop disease resistance, the same set of SAR genes are turned on and disease resistance arises, regardless of which of the three following routes is chosen. The differences between these three routes are only up to which point the SAR is enabled in the route, the final result is the same for these three routes. Thus, the analyzes and results observed with immunomodulated plants obtained in one way can be extrapolated and applied to immunomodulated plants obtained in another way.

PL 191 355 B1

A. Use of a chemical inducer of acquired systemic resistance

The first way to obtain immunomodulated plants involves administering to the plant a chemical compound capable of inducing SAR. Particularly strong chemical inducers of SAR are benzothiodizaols such as benzo [1,2,3] thiadiazole-7-carbothioic acid S-methyl ester (BTH). Benzothiadizaole derivatives that may further be used as regulators are described in US Patent Nos. 5,523,311 and 5,614,395, both of which are incorporated herein by reference. The BTH-induced SAR which confers protection in the field against a wide range of diseases in a variety of plants is described in detail in Freidrich et al., Plant Journal 10 (1), 61-70 (1996); Lawton et al. Plant Journal 10 (1), 71-82 (1996); and Gorlach et al., Plant Cell8, 629-643 (1996), each of which is incorporated herein by reference. Other chemical SAR inducers that can be used to obtain an immunomodulated plant for use in the method of the invention include isonicotinic acid compounds such as 2,6-dichloroisonicotinic acid (INA) and its lower alkyl esters, as well as salicylic acid (SA) compounds. Examples of suitable INA and SA compounds are described in US Patent 5,614,395.

B. Breeding of constitutive resistance (ClM) mutant plants

A second way of obtaining immunomodulated plants is through selective breeding programs described on the constitutive expression of the SAR gene and / or the disease resistance phenotype. Numerous data show a close correlation between SAR gene expression and systemic acquired resistance itself (Ward et al. (1991); Uknes et al. (1992); Ukres et al. (1993); Lawton et al. (1993); and Alexander et al. (1993) PNAS USA 90, 7327-7331, incorporated herein by reference In Arabidopsis, examples of well-characterized SAR genes are PR-1, PR-2, PR-5, with PR-1 being expressed at the highest level with the lowest level. background.

To identify and select plants that constitutively express SAR genes, Northern blot analysis is performed to detect detection of the SAR genes. Known SAR DNA sequences can be used by cross-hybridization as described in Uknes et al. (1992). Methods for hybridizing and cloning nucleic acid sequences are well known in the art (see, for example, Molecular Cloning, A Laboratory Manual 2nd Edition, Volumes 1-3, Sambrook et al. (Eds) Cold Spring Harbor Laboratory Press (1989) and cited therein references). At least two classes of SAR transduction signal mutants have been isolated that constitutively express the SAR genes. One class is designated "Isd" (Isd-lesion simulating disease) mutants, which are also referred to as "cim class 1" mutants. See WO 94/16077. Isd mutants (cim class l) create spontaneous lesions on leaves, accumulate elevated SA concentrations, high levels of PR-1, PR-2, and PR-5 mRNA, and are resistant to fungal and bacterial pathogens (Dietrich et al., 1994; Weymann et al., 1995). The second class has been designated as "cim" mutants (cim = constitutive immunity), which are also referred to as "cim class II" mutants. See WO 94/16077. The cim mutants have all the features of the Isd mutants except for spontaneous damage. Thus, the cim mutants appear phenotypically normal.

When plants that constitutively express SAR genes are selected, they can be used in breeding programs to incorporate constitutive expression of SAR genes and pathogen resistance into plant lines. The progeny for further crosses are selected based on the expression of the SAR genes and disease resistance as well as for other characteristics important to production and quality according to methods well known to those skilled in the art of plant breeding. For example, since Isd mutants show lesion formation and necrosis, cim mutants with their normal phenotypes are preferred for use in such breeding programs and the method of the present invention, although Isd mutants could be used if desired.

C. Transformation of plants with SAR genes

A third way to obtain immunomodulated plants is by transforming the plants with the SAR gene, preferably with a functional form of the NIM1 gene.

1. Recombinant expression of the wild-type NIM1 gene

Recombinant overexpression of wild-type NIM1 (SEQ ID NO: 1) results in transgenic plants with a disease resistant phenotype. See concurrent US Patent Application Serial Number 08 / 880,179 incorporated herein by reference. Increased levels of the active NIM1 protein have the same disease resistance effect as chemical induction with inducing chemicals such as BTH, INA and SA. Preferably the expression of the NIM1 gene is at a level that is at least twice the expression level of the NIM2 gene in wild-type plants and is more preferably at least ten times the expression level of the wild-type. The section below entitled "Recombinant DNA Technology" lists protocols that may be used for recombinant expression

The wild-type NIM1 gene in transgenic plants at levels higher than in the wild-type. Alternatively, plants can be transformed with the wild-type NPR1 gene to produce disease resistant plants as described in Cao et al. (1997).

2. Recombinant expression of an altered form of the NIM1 gene

Immunomodulated plants for use in the method of the present invention can also be created by recombinant expression of an altered form of the NIM1 gene, whereby altering the NIM1 gene takes advantage of both the finding that the SAR pathway in plants shows functional analogies to the NF-κΒ / ΙκΒ regulatory pattern in mammals and flies. and to discover that the NIM1 gene product is structural homologue of the mammalian signal transduction factor IkB of the α subclass. See co-reported PCT "Methods of using the NIM1 gene to confer disease resistance in plants" incorporated herein by reference.

The sequence of the NIM1 gene (SEQ ID NO: 1) was used in the BLAST searches and matches were identified based on the homology of the highly conserved domain in the NIM1 gene sequence to the ankyrin domains found in a number of proteins such as spectrins, ankyrins, NF-kB and IkB (Michaely and Bennett , Trends Cell Biol. 2, 127-129 (1992)). A pairwise eye comparison was performed between the NIM1 protein (SEQ ID NO: 2) and 70 known ankyrin-containing proteins and found striking similarities to members of the α class of the transcription regulator class (Bauerle and Baltimore 1996; Baldwin 1996). As shown in figure 1, the NIM1 protein (SEQ ID NO: 2) has significant homology to the ^ α proteins from mouse, rat and pig (SEQ ID NO: 3, 4 and 5, respectively). NIM1 contains several important ^ α structural domains throughout the length of the protein, including ankyrin domains (shown by the dashed lines in Figure 1), 2 N-terminal serines (amino acids 55 and 59 of NIM1), and a pair of lysines (amino acids 99 and 100 of NIM1) and the acidic C-terminus. Generally, NIM1 and ^ α share 30% residue identity and have conservative substitutions on 50% of the residues. Thus, there is homology between ^ α and NIM1 among proteins, and an overall similarity of 80%.

One of the modes of action of ^ α in signal transduction is by binding to the cytosolic transcription factor NF-kB and preventing it from entering the nucleus and altering the transcription of target genes (Bauerle and Baltimore, 1996; Baldwin, 1996). The target NFkB genes regulate (activate or inhibit) several cellular processes, including cell death responses against viruses and microorganisms (Bauerle and Baltimore, 1996). When the signal transduction pathway is activated, ^ a is phosphorylated at two serine residues (amino acids 32 and 36 of murine ^ a). This programs ubiquitination on double lysines (murine ^ α amino acids 21 and 22). After ubiquitination, the NF-kB / IkB complex is routed to the proteosome, where αα is degraded and NF-κB is released into the nucleus.

Phosphorylated serine residues important in the function of ^ α are conserved on NIM1 within a large contiguous block of the conserved sequence of amino acids 35 to 84 (Figure 1). In contrast to the ^ α where the double lysine is located about 15 amino acids towards the N-terminus of the protein, in NIM1 the double lysine is located 40 amino acids towards the C-terminus. Moreover, there is a high degree of homology between NIM1 and ^ α in the serine / threonine rich C-terminal domain which has been shown to be important in the basal rate (Sun et al., Mol. Cell. Biol. 16, 1058-1065 ( 1996)). According to the present invention, based on the analysis of structural homology and in the presence of elements known to be important for the function of ^ a, NIM1 is expected to act as ^ a with analogous effects on plant gene regulation.

When plants containing the wild-type NIM1 gene are treated with inducing chemicals, they are expected to have more NIM1 gene product (IkB homolog) or less phosphorylation of the NIM1 gene product (IkB homolog). According to this model, the result is that the plant NF-kB homolog is maintained outside the nucleus and allows the expression of SAR genes and immune responses. In nim1 mutant plants, the NF-kB homolog is free to go to the nucleus and reprimand resistance and SAR gene expression.

According to this concept, animal cells treated with salicylic acid show increased stability and amount of IkB and a decrease in active NF-KB in the nucleus (Kopp and Ghosh, 1994); IkB mutations are known that are known to act as superrepressors or negative dominant factors (Britta-Mareen Traenckner et al., EMBO 14: 2876-2883 (1995); Brown et al., Science 267: 1485-1488 (1996); Brockman et al., Molecular and Cellular Biology 15: 2809-2818 (1995); Wang et al., Science 274: 784-787 (1996)). These mutant forms of IkB bind to NF-kB but are not phosphorylated or ubiquitinated so they are not degraded. NF-kB remains bound to IkB and cannot migrate to the nucleus.

PL 191 355 B1

In view of the above, altered forms of NIM1 that act as dominant negative regulators of the SAR signal transduction pathway can be obtained. Plants transformed with these dominant negative forms of NIM1 have the opposite phenotype to nim1 mutant plants in that plants transformed with altered forms of NIM1 exhibit constitutive SAR gene expression and thus a ClM phenotype, i.e., transgenic plants are immunomodulated. Due to the position that the NIM1 gene occupies in the SAR signal transduction pathway, a number of changes in this gene, in addition to those specifically disclosed herein, are expected to result in constitutive expression of the SAR genes and thus the CIM phenotype. The section below entitled "Recombinant DNA technology" lists protocols that can be used for the recombinant expression of altered forms of the NIM1 gene in transgenic plants at levels higher than that of the wild-type. Several altered forms of the NIM1 gene that act as dominant-negative regulators of the SAR signal transduction pathway are described below.

a. Change of serine residues 55 and 59 to alanine residues

Phosphorylation of the serine residues in human α α is required for stimulus-activated degradation of α, thereby activating NF-κB. Mutagenesis of human ^ α serine (S32 and S36) residues to alanine residues inhibits stimulus-induced phosphorylation of ^ α mediated degradation by proteosome (Traenckner et al., 1995; Brown et al., 1996; Brockman et al., 1995 ; Wang et al. 1996). This altered form of ^ α can act as a dominant negative form by trapping NF-kB in the cytoplasm, thus blocking downstream signal transduction events. Based on the amino acid comparison between NIM1 and IkB shown in Figure 1, serines 55 (S55) and 59 (S59) in NIM1 (SEQ ID NO: 2) are homologous to S32 and S36 in human ^ α. 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, the NIM1 gene is altered such that the encoded protein has alanines instead of serines at amino acid positions corresponding to positions 55 and 59 of the Arabidopsis NIM1 amino acid sequence (SEQ IDNO: 2).

b. N-terminus deletion

Deletion of amino acids 1-36 (Brockman et al. 1995; Sun et al. 1996) or 1-72 (Sun et al. 1998) of human ^ α, which includes ubiquitinated residues K21 and K22 as well as the phosphorylation sites S32 and S36 results in dominant negative phenotype of ^ α in transfected cultures of human cells. The N-terminal deletion of the first 125 amino acids of the NIM1 gene product removes eight lysine residues that could serve as ubiquitination sites as well as the putative phosphorylation sites S55 and S59 discussed above. Thus, in a preferred embodiment, the NIM1 gene is altered such that the encoded product is missing about the first 125 amino acids compared to the native Arabidopsis NIM1 amino acid sequence (SEQ ID NO: 2).

c. C-terminus deletion

The deletion of the amino acids 261-317 of human ^ α can result in increased endogenous stability by blocking the constitutive phosphorylation of serine and threonine residues at the C-terminus. This altered form of ^ α is expected to function as a dominant negative form. The serine and threonine rich region is present at amino acids 522-593 at the C-terminus of NIM-1. Thus, in a preferred embodiment, the NIM-1 gene is altered such that the encoded product is missing approximately its C-terminal portion, including amino acids 522-593, as compared to the native Arabidopsis NIM1 amino acid sequence (SEQ ID NO: 2).

d. N-terminal deletion-C-terminal deletion hybrid and ankyrin domain deletion

Altered forms of the NIM1 gene product may also be generated by deletions of C-terminal and N-terminal sections of deletions or hybrids. In yet another embodiment of the present invention, constructs comprising the ankyrin domains of the NIM1 gene are provided.

3. Recombinant expression of other SAR genes

Immunomodulated plants for use in the method of the present invention can be produced by recombinant expression of various SAR genes such as those described in Ward et al. (1991). See, for example, US Patent No. 5,614,395, which describes disease resistant plants created by overexpression of one or more PR protein genes. While it refers to recombinant expression in particular forms of the NIM1 gene, the section below named "Recombinant DNA Technology" presents protocols that can also be used to recombinantly express other SAR genes such as PR protein genes in transgenic plants at levels higher than wild-type.

Recombinant DNA technology

Wild or altered form of the NIM1 gene conferring disease resistance to plants by increasing SAR expression can be incorporated into plant cells using conventional recombinant DNA technology. This generally involves inserting the DNA molecules that encode the chosen one

The NIM1 form described above into an expression system in which the DNA molecule is heterologous (ie, is not normally present) using standard cloning techniques known in the art. The vector contains the necessary elements for transcription and translation of inserted protein-coding sequences. A large number of expression 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 the lambda λgt1, λgt10, and Charon 4 vector systems; plasmid vectors such as pBI121, pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101, pCDNAII and other similar systems. The components of the expression system can also be modified to increase expression. For example, truncated sequences, nucleotide substitutions, or other modifications may be used. The expression systems described herein can be used to transform essentially any crop plant under suitable conditions. The transformed cells can be regenerated into whole plants such that the selected form of the NIM1 gene activates SAR in transgenic plants.

A. Construction of plant expression cassettes

The sequences of the genes to be expressed in transgenic plants are first assembled into expression cassettes after the appropriate plant-expressible promoter. The expression cassette may also include any other sequences required or selected for expression of the transgene. Such sequences include, but are not limited to, transcriptional terminators, external sequences to enhance expression such as introns, essential sequences, sequences intended to target the gene product to specific organelles and cell compartments. These expression cassettes can then be easily transferred into the plant transformation vectors described above. The following is a description of the various components common to expression cassettes.

1. Promoters

The choice of promoter used in the expression cassettes will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. The promoters selected will express transgenes in specific cell types (such as leaf epidermis cells, mesophyll cells, root cortex cells) and selection will reflect the desired location for gene product accumulation. Alternatively, the selected promoter may drive expression of the gene under various induction conditions. Promoters vary in strength, ie the ability to promote transcription. Depending on the host system used, any suitable promoter may be used. Non-limiting examples of promoters that can be used in expression cassettes are given below.

a. Constitutive expression, CaMV 35S promoter

The construction of the plasmid pCGN1761 is described in published patent application EP 0392 225 (example 23), which is hereby incorporated by reference. pCGN1761 contains the "dual" 35S CaMV promoter and the tml transcription terminator with a unique EcoRI site between the promoter and terminator and has a pUC-type backbone. A derivative of pCGN1761 is constructed that has a modified polylinker that includes NotI and XhoI sites in addition to an existing EcoRI site. The derivative is referred to as pCGN1761ENX. pCGN1761ENX is useful in the cloning of cDNA or gene sequences (including microorganism ORF sequences) in its polylinker for expression under the control of the 35S promoter in transgenic plants. The entire 35S promoter-tml terminator gene sequence so constructed can be excised with HindIII, SphI, SalI and Xbal from the 5 'side of the promoter and Xbal, BamHI and BglII 3' with respect to the terminator for transfer to transformation vectors such as described below. Moreover, the 35S dual promoter fragment can be removed by a 5 'cleavage from HindIII, SphI, SalI, Xbal or Pstl, and a 3' cleavage at any of the polylinker restriction sites (EcoRI, NotI or Xhol) to be replaced by a different promoter. If desired, modifications can be made around the cloning sites by introducing sequences that can enhance translation. This is especially useful when overexpression is desired. For example, pCGN1761ENX can be modified by optimizing a translation initiation site as described in Example 37 of US Patent 5,639,949 incorporated herein by reference.

b. Expression under a chemically / pathogen regulated promoter

The dual 35S promoter in pCGN1761ENX can be replaced by any other selected promoter which will give sufficiently high expression levels. As an example, one of the chemically regulated promoters described in US Patent No. 5,614,395 can replace the dual 35S promoter. The selected promoter is preferably excised from its source using restriction enzymes, but may alternatively be amplified by PCR using primers that carry the appropriate restriction end sites. If PCR amplification is undertaken, the promoter should be in again

It was sequenced to check for amplification errors after cloning the amplified promoter in the target vector. The chemical / pathogen regulated tobacco promoter PR-1a is excised from plasmid pCIB1004 (for construction see example 21 in EP 0 332 104, incorporated herein by reference) and transferred to pCGN1761ENX (Uknes et al., 1992). pCIB1004 is cleaved with HindIII and the resulting fragment is gel purified and cloned into pCHN1761ENX in which the double 35S promoter has been removed. This is done by digestion with XhoI and blunting with T4 polymerase followed by cleavage with HindIII and isolation of the larger fragment containing the vector-terminator into which the pCIB1004 promoter fragment is cloned. This generates a pCGN1 76 1 ENX derivative with the PR-1a promoter and the tml terminator and between them a polylinker with unique EcoRI and NotI sites. The selected coding sequence can be inserted into this vector, and the fusion products (i.e., promoter-gene-teminator) can then be transferred into any chosen transformation vector, including those described below. Various chemical regulators may be used to induce the expression of a selected coding sequence in plants transformed by the method of the present invention, including the benzothidiazole, isonicotinic acid, and salicylic acid compounds disclosed in US Patent Nos. 5,523,311 and 5,614,395.

c. Constitutive expression, actin promoter

It is known that several isoforms of actin are expressed in most cell types and, consequently, the actin promoter is a good choice for constitutive promoter. In particular, the promoter from the Act1 rice gene has been cloned and characterized (McEIroy et al. Plant Cell 2: 163-171 (1990)). The 1.3 kb fragment of the promoter was found to contain all regulatory elements required for expression in rice protoplasts. Moreover, numerous expression vectors based on the Act1 promoter have been constructed specifically for use in monocots (McEIroy et al. Mol. Gen. Genet. 231: 150-160 (1991)). They integrate the Act1 intronl, the Adh1 5 'flanking sequences and the Adh1 intron 1 (from the maize alcohol dehydrogenase gene), and sequenced from the 35S CaMV promoter. The vectors showing the highest expression were fusions of 35S and the Act1 intron or the 5 'flanking sequence of Act1 and the Act1 intron. Sequence optimization around the initiator ATG (GUS reporter gene) also enhances expression. The promoter expression cassettes described by McEIroy et al. (Mol. Gen. Genet. 231: 150-160 (1991)) can be easily modified for gene expression and are particularly suitable for use in monocot hosts. For example, promoter-containing fragments are removed from the McEIroy constructs and used to replace the double 35S promoter in pCGN1761ENX which is then available for insertion of specific gene sequences. The fusion genes thus constructed may then be transferred into appropriate transformation vectors. In a separate report, it was found that the rice Act1 promoter along with its first intron also drives high expression of cultured barley cells (Chibbar et al. Plant CellRep. 12: 506-509 (1993)).

d. Constitutive expression, ubiquitin promoter

Ubiquitin is another gene product known to accumulate in many cell types and its promoter has been cloned from several species for use in transgenic plants (e.g., sunflower - Binet et al. Plant Science 79: 87-94 (1991) and maize - Christiansen et al. Plant Molec. Biol. 12: 619-632 (1989)). The maize ubiquitin promoter was developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in EP 0 342 926 (to Lubrizol), which is incorporated herein by reference. Taylor et al. (Plant Cell Rep. 12: 491-495 (1993)) describe a vector (pAHC25) which contains the maize ubiquitin promoter and first intron and its high activity in cell suspensions of many monocots upon introduction by microprojectile bombardment. The ubiquitin promoter is suitable for gene expression in transgenic plants, in particular monocots. Suitable vectors are derived from pAHC25 or any of the transformation vectors described in this application, modified by introducing an appropriate ubiquitin promoter and / or intron sequences.

e. Root specific expression

Another pattern of expression is expression in the roots. A suitable root promoter is described by deFramond (FEBS 290: 103-106 (1991)) and also in published patent application EP 0452 269, which is hereby incorporated by reference. This promoter is transferred into an appropriate vector such as pCGN1761ENX to insert the selected gene and then transfer the entire promoter-gene-terminator expression cassette into the transformation vector of interest.

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f. Injury Inducible Promoters

Wound-induced promoters may also be suitable for gene expression. A number of 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), Rohreiner and Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al., Plant Molec. Biol. 22: 129-142 (1993), Warner et al. Plant J. 3: 191-201 (1993)) and all are suitable for use with with the present invention. Logemann et al. Describe the 5 'flanking sequences of the wun1 gene of the potato dicot. Xu et al. Show that the wound-inducible promoter from potato dicot (pin2) is active in monocotyledonous rice. Moreover, Rohrmeier and Lehle describe the cloning of maize Wip1 cDNA which is wound-induced and which can be used to isolate an appropriate promoter using standard techniques. Similarly, Firek and Warner et al. Described a wound-induced gene from the monocotyledonous Asparagus officinalis that is expressed at local invasion sites by injury and pathogens. Using cloning techniques well known in the art, these promoters can be transferred into suitable vectors, linked to genes related to the present invention, and used to express these genes at sites where plants are injured.

g. Preferred expression in the core

Patent application WO 93/07278, which is incorporated herein by reference, describes the isolation of the maize trpA gene that is preferentially expressed in core cells. The gene and promoter sequence extending down to -1726 bp from the start of transcription are shown. Using standard molecular biology techniques, this promoter or portions thereof can be transferred into a vector such as pCGN1761 where it can replace the 35S promoter and can be used to drive expression of the foreign gene in a core-preferred manner. In fact, fragments containing a core-preferred promoter or parts thereof can be transferred into any vector and modified for use in transgenic plants.

h. Leaf-specific expression

The maize gene encoding phosphoenol carboxylase (PEPC) has been described by Hudspeth and Grula (Plant Molec. Biol 12: 579-589 (1989)). Using standard molecular biology techniques, the promoter of this gene can be used to drive expression in a leaf-specific manner in transgenic plants.

2. Transcription terminators

A variety of transcription terminators are available for use in expression cassettes. They are responsible for the termination of transcription behind the transgene and its correct polyadenylation. Suitable transcription terminators are those known to function in plants and include the 35S CaMV terminator, the tml terminator, the nopaline synthase terminator, and the pea rbcS E9 terminator. They can be used in both monocotyledons and dicots.

3. Sequences for enhancing or regulating expression

Numerous sequences have been found that enhance the expression of genes within the transcriptional unit, and these sequences can be used with the genes of the present invention to increase their expression in transgenic plants.

Various intron sequences have been found to enhance expression in particular in monocots. For example, the introns of the maize Adh1 gene have been found to significantly enhance expression of the wild-type gene under its corresponding promoter when introduced into maize cells. Intron 1 has been found to be particularly effective for enhancing expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al., Genes Develop. 1: 1183-1200 (1997)). In the same experimental setup, the intron from the maize bronze1 gene had a similar effect on enhancing expression. Intron sequences have been routinely incorporated into plant transformation vectors, typically within the untranslated leader.

It is also known that a number of viral-derived untranslated leader sequences also enhance expression, and are particularly effective in dicotyledonous cells. Specifically, leader sequences from tobacco mosaic virus (TMV, sequence "W"), maize chlorotic spot virus (MCMV), and alfalfa mosaic virus (AMV) have been shown to be effective in enhancing expression (eg, Gallie et al. Nucleic Acids Res. 15: 8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990)).

4. Addressing the gene product within the cell

It is known that there are different mechanisms for targeting gene products in plants and the sequences controlling the action of these mechanisms have been characterized in some detail. For example, the targeting of gene products to chloroplasts is controlled by a signal sequence found on

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The N-terminus of various proteins, which is cleaved on import into chloroplasts to yield a mature protein (e.g., Cornai 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 gene products into the chloroplast (van den Broeck et al. Nature 313: 358-363 (1985)). DNA encoding the appropriate signal sequences can be isolated from the 5 'ends of the cDNAs encoding RUBISCO protein, CAB protein, EPSP synthase enzyme, GS2 protein, and many other proteins known to be located in chloroplasts. See also the section entitled "Expression targeting chloroplasts" in Example 37 of US Patent No. 5,639,949.

Other gene products are localized to other organelles such as the mitochondria and the peroxisome (e.g., Unger et al. Plant Molec. Biol. 13: 411-418 (1989)). The cDNAs encoding these products can be manipulated to ensure that heterologous gene products are targeted to these organelles. Examples of such sequences are the nucleus-encoded ATPase and mitochondria-specific isoforms of aspartate aminotransferase. Addressing to cell protein bodies is described by Rogers et al. (Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).

In addition, the sequences that target the gene products to other cell compartments have been characterized. The N-terminal sequences are responsible for targeting the ER, apoplast, and extracellular secretion from aleurone cells (Koehler and Ho, Plant Cell 2: 769-783 (1990)). In addition, N-terminal sequences along with C-terminal sequences are responsible for the targeting of gene products (Shinsh et al. Plant Molec. Biol. 14: 357-368 (1990)).

By fusing the appropriate targeting sequences described above to the transgene sequences of interest, it is possible to target the transgene product to any organ or cell compartment. For example, for targeting chloroplasts, the chloroplast signal sequence from the RUBISCO gene, the CAB gene, the ESPS synthase gene, or the GS2 gene is fused in a consistent phase to the N-terminal ATG of the transgene. The selected signal sequence should span a known cleavage site and the constructed fusion should include possible post-cleavage amino acids that are required for cleavage. In some cases, this requirement may be met by adding a small number of amino acids between the cleavage site and the ATG of the transgene, or alternatively replacing certain amino acids within the transgene sequence. Fusions engineered for chloroplast import can be tested for the efficiency of chloroplast uptake by in vitro translation of in vitro transcribed constructs followed by in vitro uptake by chloroplasts using the techniques described by Bartlett et al. In Edelmann et al. (Red) 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 cellular targeting mechanisms can be used not only with their respective promoters but also together with heterologous promoters so as to achieve a specific targeting target in cells under the transcriptional regulation of a promoter that has an expression pattern different from the promoter from which the targeting signal is derived.

B. Construction of plant transformation vectors

Numerous transformation vectors available for transforming plants are known to those of ordinary skill in the art of plant transformation, and the genes essential to the invention may be used in conjunction with any such vectors. Vector selection will depend on the preferred transformation technique and the target species to be transformed. For some target species, different antibiotic or herbicide selection markers may be advantageous. Selectable markers routinely used in transformation include the nptII gene, which confers resistance to kanamycin and related antibiotics (Messing and Vierra, Gene 19: 259-268 (1982); Bevan et al., Nature 304: 184-187 (1983)), the gene barium 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)) hph gene, which confers resistance to the antibiotic hygromycin (Blochinger and Diggelman, Mol. Cell Biol 4: 2929-2931), and the dhfr gene which confers resistance to methotrexate (Bourouis et al., EMBO J. 2 (7) 1099-1104 (1983) ) and the EPSPS gene that confers glyphosate resistance (US Patent Nos. 4,940,935 and 5,186,642).

1. Vectors suitable for transformation of Agrobacterium

A variety of transformation vectors are available using Agrobacterium tumefaciens. They typically contain vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984). The constructs of two vectors suitable for Agrobacterium transformation are described below.

PL 191 355 B1

a. pCIB200 and pCIB2001

The binary vectors pCIB200 and pCIB2001 are used to construct recombinant vectors for use with Agrobacterium and can be constructed as follows. pTJS75kan was created by digesting pTJS75 Narl (Schmidhauser and Helinski, J. Bacteriol. 164: 446-455 (1985)) allowing excision of the tetracycline resistance gene followed by insertion of an AccI fragment from pUC4K bearing NPTIl (Messing and Vierra, Gene 19: 259) -268 (1982), Bevan et al., Nature 304: 184-187 (1983); McBride et al., Plant Molecular Biology 14: 266-276 (1990)). The Xhol linkers were ligated into the EcoRV fragment of pCIB7 which contains the left and right TDNA border sequences, the plant selection marker nos / nptII hybrid gene and the pUC polylinker (Rothstein et al., Gene 53: 151-161 (1987)) and the Xhol digested fragment was cloned into the digested Sall pTJS75kan to create a pCIB2000 (see also EP-A-332 104, example 19). pCIB2000 contains the following unique restriction sites in the polylinker: EcoRI, SstI, KpnI, BglII, Xbal and SalI. pCIB2001 is a derivative of pCIB2000 that is made by inserting additional restriction sites into the polylinker. Unique restriction sites in the pCIB2001 polylinker are EcoRI, Sstl, Kpnl, Bglll, Xbal, SalI, Mlul, Bcll, Avrll, Apal, Hpal and Stul. pCIB2001 in addition to having these unique restriction sites also has kanamycin selection in bacteria and plants, T-DNA right and left border sequences for Agrobacterium mediated transformation, RK2 derived trfA function for mobilization between E. coli and other hosts and OriT and OriV functions as well with RK2. The pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals.

b. pCIB10 and derivatives thereof for selection on hygromycin

The binary vector pCIB10 contains the kanamycin resistance gene for plant selection, T-DNA right and left border sequences, and has sequences from the broad host range plasmid pRK252, which allows it to replicate in both E. coli and Agrobacterium. Its construction is described in Rothstein et al., Gene 53: 153-161 (1987). Various derivatives of pCIB have been constructed which incorporate the gene for hygromycin B phosphotransferase described by Gritz et al., Gene 25: 179-188 (1983). These derivatives allow the selection of transgenic plant cells on only hygromycin (pCIB743) or hygromycin and kanamycin (pCIB715, pCIB717).

2. Vectors suitable for transformation without Agrobacterium

Transformation without the use of Agrobacterium circumvents the requirement for T-DNA sequences in the selected transformation vector and consequently vectors lacking these sequences may be used in addition to vectors such as those described above that contain T-DNA sequences. Transformation techniques which do not rely on Agrobacterium include transformation by particle bombardment, protoplast uptake (e.g., PEG and electroporation), and microinjection. The choice of the vector is highly dependent on the preferred selection for the transformed species. Typical vectors suitable for non-Agrobacterium transformation are described below.

a. pCIB3064 pCIB3064 is a pUC derived vector suitable for direct gene transfer techniques in combination with the herbicide basta (or phosphinothricin). The plasmid pCIB246 contains the 35S CaMV promoter functionally fused to the E. coli GUS gene and the 35S CaMV transcription terminator and is described in published PCT application WO 93/07278. The 35S promoter of this vector contains two ATG sequences 5 'from the start site. These sites are mutated using standard PCR techniques to remove ATG and generate Sspl and Pvull restriction sites. The new restriction sites are 96 and 37 bp distant from the unique Sq11 site and 101 and 42 bp distant from the current start site. The resulting derivative of pCIB246 is referred to as pCIB3025. The GUS gene is then excised from pCIB3025 by digesting with SalI and SacI, the ends are blunted and religated to obtain the plasmid pCIB3060. Plasmid pJIT82 is obtained from John Innes Center, Norwich, and a 400 bp Smal fragment containing the bar gene from Streptomyces viridiochormogenes is excised and inserted into the Hpal site of pCIB3060 (Thompson et al., EMBO J. 6: 2519-2523 (1987)). This results in pCIB3064, which contains the bar gene under the control of the 35S CaMV promoter and terminator for herbicide selection, the ampicillin resistance gene (for E. coli selection), and a polylinker with unique Sphl, PstI, HindIII and BamHI sites. 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 that uses the E. coli dihydrofolate reductase gene as a selectable marker conferring resistance to methotrexate. PCR was used to amplify the 35S promoter (approx. 800 bp), intron 6 from the maize Adh1 gene (approx. 550 bp) and 18 bp of the GUS leader untranslated sequence from pSOG10. A 250 bp fragment encoding the E. coli dihydrofolate reductase type II gene

The PL 191 355 B1 was also amplified by PCR and these two PCR fragments were ligated to the Sacl-Pstl fragment from pB1221 (Clontech) which includes the vector backbone pUC19 and the nopaline synthase terminator. Combining these fragments generated pSOG19 which contains the 35S promoter fused to the intron 6 sequence, the GUS leader, the DHFR gene and the nopaline synthase terminator. Replacing the GUS leader in pSOG19 with the leader sequence from maize chlorotic spot virus (MCMV) generated the vector pSOG35. pSOG19 and pSOG35 have the pUC gene for ampicillin resistance and have HindIII, SphI, and EcoRI sites available for the cloning of foreign sequences.

C. Transformation

After the sequence of interest is cloned into an expression system, it is transformed into a plant cell. Methods for transforming and regenerating plants are well known in the art. Ti plasmid vectors were used to introduce foreign DNA as well as direct DNA uptake, liposomes, electroporation, microinjection, and microprojectiles. Additionally, bacteria of the genus Agrobacterium can be used to transform plant cells. Representative techniques for transforming both dicotyledonous and monocotyledonous plants are described below.

1. Transformation of dicots

Dicotyledon transformation techniques are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium. The non-Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be achieved by PEG-mediated uptake or electroporation, delivery by microprojectile bombardment, or microinjection. Examples of these techniques are described by Paszkowski et al., EMBO J. 3: 2717-2222 (1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1987), Reich et al., Biotechnology 4: 1001-1004 (1986) and Klein et al. Nature 327: 70-73 (1987). In any event, transformed cells are regenerated into whole plants using standard techniques known in the art.

The Agrobacterium-mediated technique is the preferred dicotyledonous transformation technique due to its high transformation efficiency and wide application in a wide variety of species. Agrobacterium transformation typically involves the transfer of a binary vector carrying the foreign DNA of interest (e.g., pCIB200 or pCIB2001) to the appropriate Agrobacterium strain, which may depend on the set of vir genes carried by the Agrobacterium host strain either on the co-resident Ti plasmid or on the chromosome (e.g., strain CIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell5: 159-169 (1993)) The transfer of a recombinant binary vector into Agrobacterium is achieved by a three-parent crossing procedure using E. coli carrying the recombinant binary vector, an E. coli helper carrying a plasmid such as pRK2013 which is capable of mobilizing a recombinant binary vector into Agrobacterium by transforming DNA (Hoefgen and Willmizer, Nucl. Acids Res. 16: 9877 (1988)).

Transformation of a target plant species by recombinant Agrobacterium generally involves the co-cultivation of Agrobacterium with plant explants and follows protocols well known in the art. The transformed tissue is regenerated on selection medium containing an antibiotic or herbicide resistance marker present between the T-DNA boundaries of the binary plasmid.

Another approach to transforming plant cells with a gene involves shooting inactive or biologically active molecules into plant tissues and cells. This technique is disclosed in US Patent Nos. 4,945,050, 5,036,006 and 5,100,792 to Sanford et al. Generally, this procedure involves shooting inactive or biologically active molecules into cells under conditions effective to penetrate the outer surface of the cell and to incorporate into it. When passive molecules are used, the vector may be introduced into the cell by coating the molecules with a vector containing the desired gene. Alternatively, the target cell may be surrounded by the vector such that the vector is carried into the cell following the molecule. Biologically active molecules (e.g., dried yeast cells, dried bacteria or bacteriophage, each carrying the DNA to be introduced) can also be inserted into the plant tissue.

2. Transformation of monocots

Transformation of most monocotyledons has also now become routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and bombardment with callus tissue particles. Transformations can be undertaken with a single DNA species or with multiple DNA species (i.e., co-transformation) and both of these techniques are suitable for use with the present invention. Cotransformation may have the advantage of avoiding the construction of a complete vector and generating transgenic plants with unconjugated loci for the gene of interest and the selectable marker, which allows the removal of the selection marker in subsequent

From one generation to the next, as long as it is deemed desirable. However, a disadvantage of using cotransformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al. Biotechnology 4: 1093-1096 (1986)).

European patent applications EP 0 292 435, EP 0 392 225 and WO 93/07278 describe techniques for the preparation of callus and protoplasts from the inbred elite maize line, transformation of protoplasts using PEG or electroporation, and regeneration of maize plants from transformed protoplasts. Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and Fromm et al. (Biotechnology 8: 833-839 (1990)) describe techniques for transforming A188-derived maize line by particle bombardment. Moreover, WO 93/07278 and Koziel et al. (Biotechnology 11: 194-200 (1993)) describe techniques for transforming elite maize inbred lines by particle bombardment. This technique uses 1.5-2.5 mm long immature corn embryos cut from a corn cob 14-15 days after pollination and a PDS-1000He Biolistics device for bombing.

Transformation of rice can also be undertaken by direct gene transfer techniques using 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 transformed using particle bombardment (Christou et al. Biotechnology 8: 957-962 (1991)).

Patent application EP 0 332 581 describes techniques for transforming and regenerating Pooidae protoplasts. These techniques allow the transformation of Dactylis and wheat. Moreover, the transformation of wheat was described by Vasil et al. (Biotechnology 10: 667-674 (1992) using particle bombardment into C-cells of long-lasting regenerative callus, and also by Vasil et al. (Biotechnology 11: 1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102: 667-674 (1992) using particle bombardment of immature embryos or callus derived from immature embryos. However, a preferred technique for transforming wheat involves transforming wheat by bombarding immature embryo particles and includes a high sucrose or high maltose step prior to Prior to bombardment, any number of embryos (0.75-1 mm long) are seeded on MS medium with 3% sucrose (Murashige and Skoog, Physiologia Plantarum 15: 474-497 (1962)) and 3 mg / ml 2, 4-D for somatic embryo induction that occurs in the dark On the selected bombardment day, the embryos are removed from the induction medium and placed in an osmoticum (i.e. to the desired concentration with sucrose or maltose, typically 15%). Embryos are allowed to plasmolize for 2-3 hours and then bombarded. Typical are 20 embryos on the target plate, although this is not critical. The appropriate gene carrying plasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer size gold particles using standard procedures. Each plate is shot with the DuPont Biolistics helium device using a pressure ^® interrupting about 1000 psi and using a standard mesh size of 80. After bombardment, the embryos are placed back into the dark to regenerate for about 24 hours (still on osmoticum). After 24 hours the embryos are removed from the osmoticum and placed back on the regeneration medium (MS + 1 mg / liter NAA, 5 mg / liter GA), also containing the appropriate selection factor (10 mg / l basta for pCIB3064 and 2 mg / l methotrexate for pSOG35). After about 1 month, developed shoots are transferred to larger sterile containers known as "GA7" which contain half concentration MS, 2% sucrose and the same concentration of selection agent.

The transformation of monocots using Agrobacterium has recently been described. See WO 94/00977 and US Patent 5,591,616, both incorporated herein by reference.

Breeding

Immunomodulated plants obtained by transformation with the SAR gene, such as the form of the NIM1 gene, can be any of a wide variety of plant species, including monocotyledons and dicotyledons, however, immunomodulated plants used in the method of the invention are preferably selected from the list of agriculturally important target plants given above. Expression of the selected form of the NIM1 gene in combination with other characteristics important for production and quality can be incorporated into plant lines through breeding. Breeding approaches and techniques are known in the art. See, for example, Welsh J.R. Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, NY (1981); Crop Breeding, Wood

D.R. (ed.) American Society of Agronomy, Madison, Wisconsin (1983); Mayo O. The Theory of Plant Breeding 2nd 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 Breeding, Walter de Gruyter and Co, Berlin (1986).

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The genetic properties introduced into the transgenic seeds and plants mentioned above are transmitted by sexual reproduction or vegetative growth, and can therefore be maintained and propagated in progeny plants. Generally, said maintenance and multiplication employs known agricultural methods designed to suit specific purposes such as growing, sowing, and harvesting. Since the growing crop is susceptible to attack and damage from insects or infections, steps are taken to control plant diseases, insects, nematodes and other unfavorable conditions to improve yield. These include mechanical steps such as soil cultivation or removal of contaminated plants as well as the application of agrochemicals such as fungicides, gametocides, nematicides, growth regulators, maturation promoters and insecticides.

Utilization of the beneficial genetic properties of transgenic plants and seeds can also be carried out by plant breeding with the aim of developing plants with improved properties such as pest, herbicide or stress tolerance, improved nutritional value, improved yield, or better structure resulting in less deposition loss or disintegrating. The various breeding steps are determined by well-defined intervention such as selecting lines for crossing, controlling pollination of the parent lines, or selecting suitable progeny plants. Depending on the desired properties, different breeding steps are applied. Suitable techniques are well known in the art and include, but are not limited to, hybridization, inbreeding, backcrossing, multi-lineage, variety mixing, interspecies hybridization, aneuploid techniques, etc. Cross-pollination of a male sterile plant with pollen from a different line ensures that the male sterile genome but the female fertile plant will obtain the homogeneous properties of both parent lines.So the transgenic seeds and plants can be used to grow improved plant lines that, for example, enhance the effectiveness of conventional methods such as herbicide or pesticide treatments or allow such methods to be absent 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 products of better quality than products that have not been able to tolerate comparable adverse developmental conditions.

In seed production, the necessary characteristics are the quality and uniformity of the seeds, while the quality and uniformity of the seeds harvested by the farmer are not important. Since it is difficult to keep a crop free from other crop plants and weeds to control seed borne diseases and to obtain seeds with good germination, seed producers have developed fairly extensive and well-developed seed production practices, they are experienced in the art of growing, conditioning, and selling pure seeds. Thus, it is common practice for a farmer to purchase certified seeds that meet specific quality standards, rather than using seeds harvested from his own crops. The propagation material for use as seed is conventionally treated with a protective coating containing herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, and mixtures thereof. Commonly used protective coatings include compounds such as captan, carboxin, thiram (TMTD®), metalaxyl (Apron®) and pirimpos-methyl (Actellic®). If desired, these compounds are formulated together with future carriers, surfactants or application adjuvants customary in the art of formulation to provide protection against damage by bacteria, fungi or animal pests. Protective coatings can be applied by impregnating the propagation material with a liquid formula or by coating with a combined wet or dry formula. Other methods of application are also possible, such as treatment directed at the buds or the fruit.

The seeds may be provided in a bag, container or vessel composed of a suitable packaging material, bag or package that can be closed to contain the seed. The bag, container or vessel may be specified for short term or long term storage, or both, of the seed. Examples of suitable packaging material include paper such as thick paper, rigid or flexible plastic or other polymeric material, glass or metal. Preferably the bag, container or vessel is comprised of a plurality of layers of packaging materials, of the same or different types. In one embodiment, the bag, container, or vessel is provided so as to preclude or limit the ingress of moisture into the vessel. In one example, the bag, container or vessel is sealed, such as by heat, to prevent ingress of water or moisture. In another embodiment, the water-absorbent materials are disposed between or next to the layers of packaging materials. In yet another embodiment, the bag, container or vessel, or the packaging material of which it is assembled is treated to reduce, suppress, or prevent disease, contamination.

Or other adverse effects of storage or transportation of seeds. An example of such treatment is sterilization by, for example, chemicals or exposure to radiation. Encompassed by the present invention is a commercial bag containing seeds of a transgenic plant containing a form of the NIM1 gene or a NIM1 protein that is expressed at higher levels in said transformed plant than in a wild-type plant, together with a suitable carrier, along with label instructions for how to use them in conferring resistance. with a wide range of plants.

Application of the microbicide to immunomodulated plants

As described herein, the plant protection method of the invention comprises two steps: firstly activating the SAR pathway to deliver an immunomodulated plant and secondly providing a microbicide to such immunomodulated plants to obtain a synergistically increased disease resistance.

A. Conventional microbicides

According to the method of the present invention, any commercial or conventional microbicide can be applied to the immunodulated plants obtained by any of the three routes described above. Examples of suitable microbicides include, but are not limited to, the following fungicides: 4- [3- (4-chlorophenyl) -3- (3,4-dimethoxyphenyl) acryloyl] morpholine ("dimethomorph") (reference: C. Tomlin ( editor: The Pesticide Manual, 10th edition, Farnhan, UK, 1994, 351-352);

5-methyl-1,2,4-triazolo [3,4-b] [1,3] benzothiazole ("tricyclazole"), (reference: C. Tomlin (editor): The Pesticide Manual, 10th edition, Farnhan, UK , 1994, 1017-1018); 3-allyloxy-1,2-benzothiazole-1,1-dioxide ("probonazole") (reference: C. Tomlin (editor): The Pesticide Manual, 10th edition, Farnhan, UK, 1994, 831-832); α- [2- (4-chlorophenyl) ethyl] -α (1,1-dimethylethyl) -1H-1,2,4-triazole-1-ethanol ("tebuconazole"), (reference: EP-A-40 345 ); 1 [- [3- (2-chlorophenyl) -2- (4-fluorophenyl) oxiran-2-yl) methyl] 1H-1,2,4-triazole (epoxiconazole), (reference: EP-A-196 038) ; μ- (4-chlorophenyl) -μ- (1-cyclopropylethyl-1H-1,2,4-triazole-1-ethanol ("cyclopronazole") (reference: US-4,664,696); 5- (4-chlorobenzyl) -2-2-dimethyl-1- (1H-1,2,4-triazol-1-ylmethyl) cyclopentanol, ("metconazole") (reference EP-A-267 778); 2- (2,4-dichlorophenyl) -3 (1H-1,2,4-triazol-1-yl) propyl-1,1,2,2-tetrafluoroethylether, ("tetraconazole") (reference: EP-A 234 242); methyl- (E) - 2- [2- [6- (2-Cyanophenoxy) pyrimidin-4-yloxy] phenyl] -3-methoxyacrylate, ("ICI 5504" "azoxystrobin") (reference EP-A-382 375); methyl 2 E 4 - methoxyimino 1 -pha-o-tolyloxy-o-tolyl-ctane ("BAS 490 F" "Methyl Kresoxime") (reference: EPA 400 417); 2- (2-phenoxyphenyl) - (E) -2-methoxymino-N -methylacetamide (reference EP-A-398 692); [2- (2,5-dimethylphenoxymethyl) -phenyl] - (E) -2-methoxymino-N-methylacetamide, (reference: EP-A-398 692); ( 1R, 3S / 1S, 3R) 2,2-Dichloro-N - [(R) -1 (4-chlorophenyl) ethyl] -1-ethyl-3-methylcyclopropanecarboxamide, ("KTU3616") (reference: EP-A- 341 475); ethylene bis (dithiocarbamate) zinc manganese polymer complex ("manko zeb ") (reference US 2,974,156); 1- [2- (2,4-dichlorophenyl) -4-propyl-1,3-dioxolan-2-yl [methyl] -1H-1,2,4-triazole ("propiconazole"), (reference GB-1522657 ); 1- [2- [2-chloro-4- (4-chlorophenoxy) phenyl] -4-methyl-1,3-dioxolan-2-yl-methyl) -1H-1,2,4-triazole ("diphenoconazole" ), (reference: GB-209860); 1- [2- (2,4-dichlorophenyl) pentyl-1H-1,2,4-triazole ("penconazole") (reference GB-1589852); cis-4- [3- (4-tert-butylphenyl) -2-methylpropyl] -2,6-dimethylmorpholine ("fenpropimorph") (reference: DE 275212 \ 35); 1- [3- (4-tert-butylphenyl) -2-methylpropyl-piperidine ("fenpropidine") (DE reference: 2752135); 4-cyclopropyl-6-methyl-N-phenyl-2-pyrimidinamide ("cyprodinil") (reference: EP-A-310550); (RS) -N- (2,6-dimethylphenyl-N- (methoxyacetyl) -alanine ("metalaxyl") methyl ester (reference: GB-1500581); (R) -N- (2,6-dimethylphenyl) methyl ester N- (methoxyacetyl) alanine ("R-metalaxyl"), (reference: GB-1500581); 1,2,5,6-tetrahydro-4H-pyrrolo [3,2,1,1] quinoline-4-one ( "Pyrochilone") (reference GB-1394373); ethyl hydride phosphonate ("fosetyl") (reference: C. Tomlin (editor): The Pesticide Manual, 10th edition, Farnhan, UK, 1994, 530-532); and copper hydroxide (reference: C. Tomlin (editor): The Pesticide Manual, 10th edition, Farnhan, UK, 1994, 229-230).

The selected microbicide is preferably applied to the immunomodulated plants to be protected in the form of a composition with further carriers, surfactants or other application-promoting adjuvants customary in formulation technology. Suitable carriers and adjuvants can be solid or liquid, and are substances customary in formulation technology; e.g. natural or regenerated minerals, solvents, dispersants, wetting agents, binders, thickeners, binders or fertilizers.

A preferred method of applying the microbicide composition is by application to parts of the plant that are above ground, especially to the leaves (foliar application). The frequency and rate of application depend on the biological and climatic living conditions of the pathogen. However, the microbicide can also penetrate plants via the roots through the ground or through water (systemic action) if the plant site is impregnated with a liquid formula (e.g. in rice cultivation) or if the microbicide is incorporated in solid form into the soil, e.g. in the form of granules (soil application). To treat

The microbicide seed can also be applied to the seed (coating) either by impregnating the rhizomes or grains with a liquid microbicide formula, or by coating them with an already combined wet or dry formula. In addition, in special cases, other methods of application to plants are possible, for example, treatments directed at the buds or the fruit.

The microbicide can be used unmodified or preferably together with adjuvants conventionally used in formulation technology and is therefore formulated in a known manner, e.g. into emulsifiable concentrates, coating pastes, spraying or diluting solutions, dilute emulsions, wettable powders, soluble powders, dusts, granules, or by encapsulation into e.g. polymeric substances. As with the nature of the compositions, the methods of application, such as spraying, atomizing, dusting, scattering, coating, or pouring, are selected in accord with the objectives and prevailing conditions. Preferred application concentrations of the microbicide are normally from 50 g to 2 kg a.i./ha, preferably from 100 g to 1000 g a.i./ha, especially from 150 g to 700 g a.i./ha. In the case of seed treatment, the application concentrations are from 0.5 g to 1000 g, preferably from 5 g to 100 g a.i. per 100 kg of seeds.

The formulations are prepared in a manner known per se, e.g. by homogeneously mixing and / or grinding the microbicide with fillers, e.g. solvents, solid carriers and, where appropriate, surfactants.

Suitable solvents are: aromatic hydrocarbons, preferably fractions containing 8 to 12 carbon atoms, e.g. mixtures of xylene and substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols and glycols and esters thereof, such as ethanol, ethylene glycol, monomethyl ethylene glycol or monomethyl ether, ketones such as cyclohexanone, highly polar solvents such as N-methyl-2-pyrrolidone, dimethyl sulfoxide, or dimethylformamide, and vegetable oils or epoxidized vegetable oils such as epoxidized coconut oil or soybean oil, or water.

The solid carriers used, for example, for dusts or dispersible powders are normally natural mineral fillers such as calcite, talcum, kaolin, motmorillonite or attapulgite. To improve the physical properties it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. Suitable granular adsorptive carriers are porous types, for example calcite or sand. Additionally, a great number of pregranular inorganic or organic materials can be used, for example, in particular dolomite or powdered plant residues.

Depending on the nature of the microbicide, suitable surfactants are nonionic, cationic and / or anionic surfactants having good emulsifying, dispersing and wetting properties. The term "surfactants" will also be understood to mean mixtures of surfactants.

Particularly preferred application-promoting adjuvants are also natural or synthetic phospholipids of the kephalin and lecithin series, e.g. phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and lyso-lecithin.

The agrochemical compositions generally comprise 0.1 to 99%, preferably 0.1 to 95%, active microbicidal agent 99.9 to 1%, preferably 99.9 to 5%, solid or liquid adjuvant and 0 to 25%, preferably 0, 1 to 25% surfactant.

While commercial products will preferably be formulated as concentrates, the end user will generally employ dilute formulations.

B. Microbicides activating plants

When applied to immunomodulated plants obtained via the second or third route described above (selective breeding or genetic engineering), the microbicide may alternatively be a chemical SAR inducer (plant activating microbicide) such as a benzothiadiazole compound, an isonicotinic acid compound, or a salicylic acid compound that is described in US Patent Nos. 5,523,311 and 5,614,395. Thus, the two methods of immunomodulation are used together. By the use of plant activating microbicides obtained by selective breeding or genetic engineering, "extraimmunomodulation" is produced and synergistic enhanced disease resistance is obtained.

As described below, the NIM1-overexpressing immunomodulated transgenic plants responded much faster and to much lower doses of BTH, as demonstrated by zPR-1 gene expression and resistance to P. parasitica, than wild-type plants. See example 35 and Northern blot analysis in figure 3. Synergistically enhanced disease resistance in NIM1 overexpressing plants can be obtained with as little as 10 μΜ BTH, a concentration normally insufficient for any

PL 191 355 B1 effectiveness. Normally phytotoxic or otherwise undesirable concentrations of SAR-inducing chemicals can be avoided by exploiting synergy. Additionally, the shift in time course of SAR activation that occurs when SAR-inducing chemicals are applied to already immunomodulated plants, such as plants overexpressing NIM1, can be exploited. Moreover, economic gains can be obtained from the reduced amount of chemicals required to provide a given level of plant protection.

C. Conventional microbicides in combination with plant activating microbicides

For even greater disease resistance, both a conventional microbicide and a plant activating microbicide can be applied to immunomodulated plants obtained either by selective breeding or genetic engineering. This results in an even higher level of synergistic disease resistance compared to the level of disease resistance obtained by immunomodulation alone, by immunomodulation plus only one type of microbicide, or by the simultaneous use of both types of microbicides (conventional and plant activating). See for example Table 35 in Example 19.

Disease resistance assessment

Assessment of disease resistance is performed by methods known in the art. See Uknes et al. (1993) Molecular Plant Microbe Interactions 6: 680-685; Gorlach et al., (1996) Plant Cell8: 629-643; Alexander et al., Proc. Natl. Acad. Sci. USA 90: 7327-7331 (1993). For example, several representative disease resistance assays are described below.

A. Determination of resistance to Phythophthora parasitica

The determination of resistance to Phytophthora parasitica, a black pedicel organism, is performed on six-week-old plants grown as described in Alexander et al., Proc. Natl. Acad. Sci. USA 90: 7327-7331 (1990). The plants are watered, allowed to drain well, and then inoculated by applying 10 ml of a sporangia suspension (300 sporangia / ml) to the soil. The inoculated plants are kept in a greenhouse maintained at 23-25 ° C during the day and 20-22 ° C at night. The wilt index is used for determination as follows: 0 = no symptoms; 1 = no symptoms; 1 = some amount of wilting with reduced turgor; 2 = obvious wilting symptoms but no rotting or stunting; 3 = clear wilting symptoms with stunting but no visible stem rot; 4 = severe wilting, with pronounced stem rot, some damage to the root system; 5 = like 4, but plants near death or dead and with severe reduction in root system. All determinations are made blindly on plants arranged randomly.

B. Pseudomonas syringae resistance assay

Pseudomonas syringae pv. tabaci strain # 551 is injected into the two lower leaves of several 6-7 week old plants at a concentration of 10 ⁶ or 3x10 ⁶ per ml of H 2 O. Six plants are scored at each time point. Plants infected with Pseudomonas tabaci are scored on a 5-point disease severity scale. 5 = 100% dead tissue, 0 = no symptoms. A T test (LSD) is performed on the scores each day and groupings are given after the mean disease score. Values followed by the same letter on a given evaluation date are not statistically significantly different.

C. Cercospora nicotiana resistance determination

A spore suspension of Cercospora nicotiana (ATCC # 18366) is sprayed until it is almost running down to the leaf surface. Plants are kept at 100% humidity for 5 days. After that, the plants are sprinkled with water 5-10 times a day. Six plants are scored at each time point. Cercospora nicotiana is assessed by the% leaf area showing symptoms. A T test (LSD) is performed on the scores each day and groupings are given after the mean disease score. Values followed by the same letter on that evaluation date are not statistically significantly different.

D. Determination of resistance to Peronospora parasitica

Peronospora parasitica resistance determination is performed on plants as described in Uknes et al. (1993). Plants are inoculated with the compatible P. parasitica isolate by spraying a spore suspension (approximately 5x10 ⁴ spores per milliliter). The inoculated plants are incubated under humid conditions at 17 ° C in a growth chamber with a 14 h / 10 h cycle. night. Plants are assessed 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 blue (Keogh et al., Trans. Br. Mycol. Soc. 74: 329-333 (1980)) for microscopic examination.

Brief description of the drawings

Figure 1 is a comparison of the NIM1 protein sequence with ΙκΒα from mouse, rat and pig. Vertical lines above the (l) sequence indicate amino acid identity between the NIM1 sequence and ΙκΒα (value

PL 191 355 B1 of the matrix is 1.5); colons (:) above the sequence show a similarity value> 0.5; single dots (.) above the sequence indicate a similarity value between <0.5 but> 0.0, and a value <0.0 indicates no similarity and no index above the sequence (See examples). The positions of the mammalian ΙκΒα ankyrin domains were identified according to de Martin et al., Gene 152, 252-255 (1995). Dots within the sequence indicate the gaps between the NIM1 and ΙκΒα proteins. The five ankyrin repeats in ΙκΒα are shown by the dashed lines below the sequence. Amino acids are numbered relative to the NIM1 protein with gaps inserted where appropriate. Plus (+) signs are placed above the sequence every 10 amino acids.

Figure 2 is the amino acid sequence composed of the regions of the NIM1 protein (the numbers correspond to the amino acid positions in SEQ ID NO: 2) and the rice EST protein products (SEQ ID NO: 17-24).

Figure 3 shows the results of the Northern analysis showing the expression time curve of the wild-type PR-1 gene and overexpression lines after treatment with water or BTH. RNA was prepared from treated plants and analyzed as described in the examples. "Ws" is the wild-type Ws Arabidopsis ecotype. "3A", "5B", "6E" and "7C" are single NIM1 overexpressing plant lines produced according to Example 21. "BTH" is water treatment, "10 BTH" is 10 μΜ BTH treatment, "100 BTH" is 100 treatment μΜ BTH, "0" is day zero control, "1", "3" and "5" are samples on days 1,3 and 5.

Brief Description of the Sequences in the Sequence Listing SEQ ID NO: 1 is a genomic sequence comprising the coding region of the Arabidopsis wild-type gene.

SEQ ID NO: 2 is the amino acid sequence of the wild-type NIM1 Arabidopsis thaliana protein encoded by the coding region of SEQ IDNO: 1.

SEQ ID NO: 3 is the amino acid sequence of the mouse iKBa in figure 1.

SEQ ID NO: 4 is the rat IKBa amino acid sequence of Figure 1.

SEQ ID NO: 5 is the porcine IKBa amino acid sequence of Figure 1.

SEQ ID NO: 6 is the cDNA sequence of the Arabidopsis thaliana N I M 1 gene.

SEQ ID NOS: 7 and 8 are respectively the coding sequence of the cDNA and the coded amino acid sequence of the dominant negative form of the NIM1 protein having alanine residues instead of serine residues at positions 55 and 59.

SEQ ID NOS: 9 and 10 are respectively the coding sequence of the cDNA and the coded amino acid sequence of the dominant negative form of the NIM1 protein having an N-terminal deletion.

SEQ ID NOS: 11 and 12 are the coding sequence of the cDNA and the coded amino acid sequence, respectively, of the dominant negative form of the NIM1 protein having a C-terminal deletion.

SEQ ID NOS: 13 and 14 are respectively the coding sequence for the cDNA and the coding sequence for the amino acid of the dominant negative form of the NIM1 protein having both the N-terminal and the C-terminal deletions.

SEQ ID NOS: 15 and 16 are the coding sequence of the cDNA and the coded amino acid sequence of the NIM1 ankyrin domain, respectively.

SEQ ID NO: 17 is the amino acid sequence 33-155 of Rice-1 in Figure 2.

SEQ ID NO: 18 is the amino acid sequence 215-328 of Rice-1 in Figure 2.

SEQ ID NO: 19 is the amino acid sequence 33-155 of Rice-2 in Figure 2.

SEQ ID NO: 20 is the amino acid sequence 208-288 of Rice-2 in Figure 2.

SEQ ID NO: 21 is the amino acid sequence 33-155 of Rice-3 in Figure 2.

SEQ ID NO: 22 is the amino acid sequence 208-288 Rice-3 in Figure 2.

SEQ ID NO: 23 is the amino acid sequence 33-155 of Rice-4 in Figure 2.

SEQ ID NO: 24 is the amino acid sequence 215-271 of Rice-4 in Figure 2.

SEQ ID NOS: 25 to 32 are oligonucleotide primers.

DEFINITIONS

The following definitions will aid the understanding of the present invention

Plant cell: the structural and physiological unit of plants, composed of the protoplast and the cell wall. The term "plant cell" refers to any cell that is either part of or derived from a plant. Some examples of plant cells include the differentiated cells that are part of a living plant; differentiated cells in culture; undifferentiated cells in culture; 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. Every tissue of a plant in a plant or culture is included. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any groups of plant cells organized into structural and / or functional units. Use of this term

PL 191 355 B1 with or without a specific type of tissue as recited above or otherwise covered by this definition is not intended to exclude any other type of plant tissue.

Protoplast: a plant cell without a cell wall

Daughter plant: A future generation plant of sexual or asexual origin that includes, but is not limited to, progeny.

Transgenic plant: a plant that has stably integrated recombinant DNA in its genome.

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

Recombinant DNA technology. A technology that produces recombinant DNA in vitro and transfers recombinant DNA into cells in which it can be expressed or expanded (see Concise Dictionary of Biomedicine and Molecular Biology, Juo Ed., CRC Press, Boca Raton (1996)), for example transferring the DNA into protoplast (s) or cell (s) in various forms, including but not limited to (1) naked DNA in circular, linear, or super-helical form; (2) DNA contained in nucleosomes or chromosomes or nuclei or parts thereof; (3) DNA complexed or linked to other molecules, (4) DNA encapsulated in liposomes, spheroplasts, cells or protoplasts, or (5) DNA transferred from organisms other than the host organism (e.g., Agrobacterium tumefaciens). These and various other methods of introducing recombinant DNA into cells are known in the art and can be used to produce the transgenic cells or transgenic plants of the present invention.

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 used to increase peroxidase activity in a monocotyledonous plant. Specifically, regulatory regions (e.g., promoters) may be introduced into the plant genome to increase endogenous peroxidase expression.

Also included in the recombinant DNA technology is the insertion of a peroxidase coding sequence lacking the selected expression signals into a monocotyledonous plant and the determination of the transgenic monocotyledon for increased peroxidase expression due to endogenous control sequences in the monocotyledonous plant. This would increase the number of copies of the peroxidase coding sequences within the plant.

The original insertion of the recombinant DNA into the genome of the R ⁰ plant is not defined as achieved by traditional plant breeding methods but by technical methods as described herein. Following the original insertion, transgenic descendants can be propagated using essentially traditional breeding methods.

Hybrid gene: A DNA molecule containing at least two heterologous parts e.g. parts derived from preexisting DNA sequences that are not linked in their preexisting states as these sequences have preferably been generated using recombinant DNA technology.

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

Coding sequence: A DNA molecule that, when transcribed and translated, produces a polypeptide or protein.

Gene: a distinct region of the chromosome containing a regulatory DNA sequence that is responsible for expression control, ie, transcription and translation, and a coding sequence that is transcribed and translated to produce a distinct polypeptide or protein.

acd: plant with accelerated cell death

AFLP: length polymorphism of amplified fragments avrRpt2: Rpt2 avirulence gene, isolated from Pseudomonas syringae BAC: bacterial artificial chromosome

BTH: benzo [1,2,3] thiadiazole-7-carbothioic acid, S-methyl ester

CIM: constitutive resistance phenotype (SAR is constitutively activated) cim: mutant plant with constitutive resistance cM: centimorgans cprl: mutant plant constitutively expressing PR genes

Col-O: Arabidopsisecotype Columbia

Ecs: combinations of enzymes

PL 191 355 B1

Emwa: Compatible Peronospora parasitica isolate in the Ws-O Arabidopsis ecotype

EMS: ethyl methyl sulfate

INA: 2,6 dichloroisonicotinic acid

Ler: Arabidopsis ecotype Landsberg erecta

Isd: mutant plant with nahG disease simulation damage: Pseudomonas putida salicylate hydroxylase, which converts salicylic acid to catechol

NahG: Arabisopsis line transformed with the nahG gene ndr: mutant plant with non-breed specific disease resistance nim: mutant plant with non-inducible resistance

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

Nim1: mutant NIM1 allele conferring disease resistance also refers to mutant Arabidopsis thaliana plants having a mutated nim1 NIM1 allele

Noco: Peronospora parasitica isolate compatible on the Col-O Arabidopsis ecotype

ORF: - open reading frame

PCs: primer combinations

PR: related to pathogenesis

SA: salicylic acid

SAR: Acquired Systemic Resistance

SAR-on: immunomodulated plants in which SAR is activated, typically exhibiting greater than wild-type SAR gene expression and having a disease resistant phenotype

SSLP: Simple sequence length polymorphism

UDS: universal disease susceptibility phenotype

Wela: Peronospora parasitica isolate compatible with the Weiningen Arabidopsis ecotype

Ws-O: Issilewskija Arabidopsis ecotype

WT: wild type

YAC: yeast artificial chromosome

Examples

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

Standard recombinant DNA and molecular cloning techniques are well known in the art and are described by Sambrook et al., Molecular Cloning, ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and T.J. Silhavy M.L. Bernam and L.W. Enquist Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and Ausubel F.M. et al., Current Protocols in Molecular Biology, edited by Greene Publishing Associates and WileyInterscience (1987).

1. Synergistic effects of disease resistance achieved by coordinated application to plants of a chemical inducer of acquired systemic resistance with a conventional microbicide

In this set of examples, SAR was induced in plants by the application of a chemical SAR inducer such as benzothiodiazole. In addition, conventional microbicides were applied. The plants were then attacked by various pathogenic pathogens. The combination of both methods of pathogen control (including chemical + microbicide) resulted in a greater than additive, i.e. synergistic, disease resistance. This was defined as the synergy factor (SF), i.e. the ratio of observed (O) effect to expected (E).

The expected effect (E) reported for a combination of active ingredients can be described by the so-called Colby formula and can be calculated as follows (Colby, SR "Calculating synergistic and antagonistic responses of herbicide combinations". Weeds15, pages 20-22 (1967) ).

ppm = milligrams of active ingredient (= a.i.) per liter of spray mixture

X =% of the effect caused by active ingredient I at an application concentration of p ppm of active ingredient

Y =% of the effect caused by active ingredient II at an application concentration of q ppm of active ingredient

E = expected effect of active ingredients l + II at application concentration p + q ppm of active ingredient (additive effect)

X + Y

Colby's formula is: E = X + Y - '

100

PL 191 355 B1

Example 1: Action against Erysiphe graminis on barley

Residual protective action: Barley plants about 8 cm high were sprayed to the point of formation of droplets with an aqueous mixture (maximum 0.02% of active ingredient) and sprinkled 3 to 4 days later with conidia of the fungus. Infected plants were grown in a greenhouse at 22 °. The fungus infestation was generally determined 10 days after infection.

Table a 1

Action against Erysiphe graminis on barley component I: benzothiodiazole-7-carboxylic acid component II: metoconazole

Test No. mg of active ingredient per liter (ppm) l: ll % activity SF (synergy factor) O / E class l class II O (watched) E (expected) 1 0.6 0 2 2 40 3 6 89 4 0.6 10 5 2 40 6 6 51 7 twenty 65 8 0.6 0.6 1: 1 37 10 3.7 9 0.6 2 1: 3 59 40 1.5 10 0.6 6 1:10 81 51 1.6 11 0.6 twenty 1:30 am 78 65 1.2 12 2 6 1: 3 78 71 1.1 13 2 twenty 1:10 98 79 1.2

Table a 2

Action against Erysiphe graminis on barley component I: benzothiodiazole-7-carboxylic acid component II: tetraconazole

Test No. mg of active ingredient per liter (ppm) l: ll % activity SF (synergy factor) O / E O (watched) E (expected) class l class II 1 0.6 14 2 2 27 3 0.6 45 4 2 63 5 0.6 0.6 1: 1 70 53 1.3 6 0.6 2 1: 3 82 68 1.2 7 2 0.6 3: 1 79 60 1.3

PL 191 355 B1

Table 3

Action against Erysiphe graminis on barley component I: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: metoconazole

Test No. mg of active ingredient per liter (ppm) l: ll % activity SF (synergy factor) O / E O (watched) E (expected) class l Skł. II 1 0.6 0 2 2 33 3 6 17 4 twenty 33 5 60 50 6 0.6 6 1:10 33 17 1.9 7 0.6 0.6 1:10 50 33 1.5 8 0.6 60 1: 100 83 50 1.7

Example 2: Action against Colleotrichium lagenarium on Cucumis sativus L.

After a cultivation period of 10 to 14 days, cucumber plants were sprayed with a mixture prepared from a wettable powdered formulation of the test compound. After 3 to 4 days, the plants are infected with a spore suspension _{of 5} (1.0 x 10 ⁵ spores / ml) of the fungus and incubated for 30 hours at high humidity and a temperature of 23 ° C. Incubation was then continued at normal humidity at 22 ° C to 23 ° C. The protective effect was assessed 7 to 10 days post infection based on the degree of fungal infection.

After a cultivation period of 10 to 14 days, cucumber plants were treated by applying to the soil a spray mixture prepared from a wettable powdered formulation of the test compound. After 3 to 4 days, the plants were infected with a spore suspension (1.0 x 10 ⁵ spores / ml) of the fungus and incubated for 30 hours under high humidity at 23 ° C. Incubation was then continued at normal humidity at 22 ° C. The protective effect was assessed 7 to 10 days post infection based on the degree of fungal infection.

Table 4

Action against Colleotrichium lagenarium on Cucumis sativa L. / listed component I: benzothiodiazole-7-carboxylic acid component II: azoxystrobin

Test No. mg of active ingredient per liter (ppm) l: ll % activity SF (synergy factor) O / E O (watched) E (expected) class l class II 1 0.06 0 2 0.2 5 3 2 22 4 0.06 5 5 0.2 9 6 0.6 12 7 6 17 8 0.06 0.06 1: 1 16 5 3.2 9 2 0.2 10: 1 65 29 2.2 10 2 0.6 3: 1 49 31 1.6 11 2 6 1: 3 44 35 1.3

PL 191 355 B1

Table a 5

Action against Colleotrichium lagenarium on Cucumis sativa L. / soil application, component l: benzothiodiazole-7-carboxylic acid, component II: azoxystrobin

Test No. mg of active ingredient per liter (ppm) l: ll % activity SF (synergy factor) O / E O (watched) E (expected) class l class II 1 0.006 0 2 0.02 40 3 0.06 49 4 0.2 91 5 0.2 0 6 0.6 9 7 2 28 8 6 66 9 0.006 0.2 1:30 am 11 0 _* 10 0.6 1: 100 thirty 9 3.3 11 2 1: 300 83 28 3.0 12 0.02 6 1: 300 97 80 1.2 13 0.06 6 1: 100 100 82 1.2

* the synergy factor SF cannot be calculated

Table a 6

Action against Colleotrichium lagenarium on Cucumis sativa L. / Listing Component I: Benzothiodiazole-7-carboxylic acid Component II: Kresoxime-methyl

Test No. mg of active ingredient per liter (ppm) l: ll % activity SF (synergy factor) O / E O (watched) E (expected) class l class II 1 0.2 3 2 0.6 51 3 2 0 4 twenty 41 5 0.2 2 1:10 15 3 5 6 0.2 twenty 1: 100 61 43 1.45

PL 191 355 B1

Table 7

Action against Colleotrichium lagenarium on Cucumis sativa L. / listed component I: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: azoxystrobin

Test No. mg of active ingredient per liter (ppm) l: ll % activity SF (synergy factor) O / E O (watched) E (expected) class l class II 1 0.06 16 2 0.2 22 3 6 60 4 2 18 5 6 75 6 0.06 2 1:30 am 43 31 1.4 7 0.2 2 1:10 57 36 1.6

Table 8

Action against Colleotrichium lagenarium on Cucumis sativa L. / soil application, component I: S-methyl ester of benzo [1,2,3] thiodiazole-7-carbothioic acid, component II: azoxystrobin

Test No. mg of active ingredient per liter (ppm) l: ll % activity SF (synergy factor) O / E O (watched) E (expected) class l class II 1 0.006 0 2 0.02 6 3 0.06 23 4 0.2 36 5 0.02 1 6 0.06 5 7 0.6 27 8 2 61 9 6 93 10 0.006 0.02 1: 3 26 1 26 11 0.006 0.6 1: 100 44 27 1.6 12 0.006 2 1: 300 84 61 1.4 13 0.02 0.02 1: 1 23 7 3.3 14 0.02 2 1: 100 77 64 1.2 15 0.06 0.02 3: 1 42 24 1.8 16 0.06 2 1:30 am 92 70 1.3 17 0.2 2 1:10 93 75 1.2

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Example 3: Action against Cercospora nicotianae on tobacco plants

Tobacco plants (6 weeks of age) were sprayed with a solution of the test compound formulation (concentration of maximum 0.02% active ingredient). Four days after spraying, the plants were inoculated with a suspension of Cercospora nicotianae sporangia (1,500,000 spores / ml) and kept at high humidity for 4 to 5 days and then incubated with the normal day and night sequence. Assessment of the symptoms in the test was based on the leaf surface infested with the fungus.

Table 9

Action against Cercospora nicotianae on tobacco plants component I: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: tebuconazole

Test No. mg of active ingredient per liter (ppm) l: ll % activity SF (synergy factor) O / E O (watched) E (expected) class l class II 1 0.2 0 2 2 17 3 6 55 4 twenty 78 5 2 0 6 6 0 7 0.2 2 1:10 87 0 _* 8 0.2 6 1:30 am 97 0 _* 9 2 2 1: 1 87 17 5.1 10 2 6 1: 3 94 17 5.5 11 6 2 3: 1 87 55 1.6 12 6 6 1: 1 90 55 1.6 13 twenty 2 10: 1 97 78 1.2 14 twenty 6 3: 1 97 78 1.2

Table 10

Action against Cercospora nicotianae on tobacco plants component I: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: cyproconazole

Test No. mg of active ingredient per liter (ppm) l: ll % activity SF (synergy factor) O / E O (watched) E (expected) class l class II 1 2 3 4 5 6 7 1 0.2 0 2 2 17 3 6 55 4 twenty 78 5 2 0

Table 10 continues

1 2 3 4 5 6 7 6 6 0 7 0.2 2 1:10 78 0 _* 8 0.2 6 1:30 am 84 0 _* 9 2 2 1: 1 90 17 5.3 10 2 6 1: 3 94 17 5.5 11 6 2 3: 1 87 55 1.6 12 6 6 1: 1 93 55 1.7 13 twenty 2 10: 1 100 78 1.3 14 twenty 6 3: 1 100 78 1.3

Table 11

Action against Cercospora nicotianae on tobacco plants component I: benzothiodiazole-7-carboxylic acid component II: fenpropimorph

Test No. mg of active ingredient per hectare l: ll % activity SF (synergy factor) O / E class l class II O (watched) E (expected) 0 - - 0 (control) 1 0.2 0 2 0.6 3 3 2 69 4 6 79 5 2 13 6 6 23 7 10 42 8 0.2 2 1:10 52 13 4 9 0.2 6 1:30 am 61 23 2.7 10 0.6 2 1: 3 71 16 4.4 11 6 6 1: 1 100 83 1.2

PL 191 355 B1

Table 12

Action against Cercospora nicotianae on tobacco plants Component I: Benzothiodiazole-7-carboxylic acid Component II: Diphenoconazole

Test No. mg of active ingredient per hectare l: ll % activity SF (synergy factor) O / E class l class II O (watched) E (expected) 0 - - 0 (control) 1 2 69 2 6 79 3 twenty 100 4 0.6 3 5 2 23 6 6 32 7 2 0.6 3: 1 90 70 1.3 8 6 0.6 10: 1 100 80 1.3

Example 4: Action against Pyricularia orizae on rice plants

The rice plants, about 2 weeks old, were placed together with the soil at the roots in a container filled with the spray mixture (maximum 0.006% active ingredient). 96 hours later, the rice plants were infected with a conidia suspension of the fungus. The fungus infestation was assessed after incubating the infected plants for 5 days at 95-100% relative humidity and about 24 ° C. Table 13

Action against Pyricularia orizae on rice plants component I: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: KTU 3616

Test No. mg cz active (pp class l the ratio of it per liter m) class II l: ll % acts O (watched) food E (expected) SF (synergy factor) O / E 1 6 16 2 0.02 0 3 0.06 28 4 0.2 47 5 0.6 79 6 2 83 7 6 91 8 6 0.02 300: 1 42 15 2.8 9 6 0.06 100: 1 76 39 1.9 10 6 0.2 30: 1 98 55 1.8 11 6 0.6 10: 1 98 82 1.2 12 6 2 3: 1 100 86 1.2 13 6 6 1: 1 98 92 1.1

PL 191 355 B1 ₂

In a plot of 12 m ² , rice plants were sprayed with a mixture prepared from a wettable powder of the active ingredient. Infection happened naturally. To evaluate the results, the area of fungus infected leaves was measured 44 days after spraying. The following results were obtained:

Table a 14

Action against Pyricularia orizae on rice plants in the open, component I: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: pyroquilone

Test No. mg of active ingredient per hectare l: ll % activity SF (synergy factor) O / E class l class II O (watched) E (expected) - - - 0 (control) 1 0.25 22 2 0.5 50 3 0.75 46 4 0.5 82 5 0.25 0.75 1: 3 80 58 1.4 6 0.5 0.75 1: 1.5 85 73 1.2

The rice plants, about 2 weeks old, were placed with the soil at the roots in a container filled with the spray mixture. The fungus infestation was assessed 36 days later. The degree of infection in the untreated plants corresponded to 0% of the activity.

Table a 15

Action against Pyricularia orizae on rice plants component I: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: tricyclazole

Test No. mg cz active (pp class l the ratio of it per liter m) class II l: ll % acts O (watched) food E (expected) SF (synergy factor) O / E 1 0.5 65 2 0.25 39 3 0.1 18 4 0.05 5 5 1 74 6 0.5 71 7 0.25 48 8 0.1 32 9 0.25 0.25 1: 1 75 68 1.1 10 0.1 0.25 1: 2.5 69 57 1.2 11 0.1 0.1 1: 1 61 44 1.4 12 0.05 1 1:20 80 75 1.1 13 0.05 0.25 1: 5 58 50 1.2

PL 191 355 B1

Example 5: Action against Colleotrichium sp. (Anthracnose) and Cercospora sp. (Leaf blotch) on chili peppers.

₂

Effect on crop yield: In an area of approximately 10 m ² (test site: Cikampek, Java, Indonesia), chili pepper plants were sprayed a total of seven times with an interval of approximately 7 days with 500-700 liters of spray mixture per hectare. Three days after the first spraying, the plants were infected artificially with the fungus.

Table 16

Action against Colleotrichium: determined on the basis of the assessment of infection on chili pepper fruits after the fifth spraying component l: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: mancozeb

Test No. mg of active ingredient per liter (ppm) l: ll % activity SF (synergy factor) O / E O (watched) E (expected) class l class II 1 2 5 100 55 12 3 5 100 1:20 77 59 1.3

Table 17

Action against Cercospora: determined on the basis of the assessment of infection on the leaves after the sixth spraying component I: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: mancozeb

Test No. mg of active ingredient per liter (ppm) l: ll % activity SF (synergy factor) O / E O (watched) E (expected) class l class II 1 2 5 100 76 8 3 5 100 1:20 87 78 1.1

Table 18

Effect on the crop yield: chili fruits were harvested after the sixth spraying component l: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: mancozeb

Test No. mg of active ingredient per liter (ppm) l: ll yield in kg per hectare SF (synergy factor) O / E O (watched) E (expected) class l class II 1 2 5 100 459 8 3 5 100 1:20 1400 ca 460 ca 3

P ric e d 6: Action against Puccinia recondita in wheat

Seven-day-old maize plants were sprayed until droplets were formed with a mixture prepared from the preparation of the active agent or a combination of active agents. After 4 days, the sprayed plants were infected with a conidia suspension of the fungus, the sprayed plants were then incubated for 2 days at a relative atmospheric humidity of 90-100% and 20 ° C. 10 days after infection, fungal infection was assessed.

PL 191 355 B1

Table a 19

Action against Puccinia recondita on wheat component I: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: propioconazole

Test No. mg of active ingredient per liter l: ll % activity SF (synergy factor) O / E O (watched) E (expected) class l class II 1 100 - 0 (control) 51 2 5 10 3 100 5 20: 1 79 56 1.4

Table a 20

Action against Puccinia recondita on wheat, component I: benzothiodiazole-7-carboxylic acid, component II: fenpropidin

Test No. kg of active ingredient per hectare l: ll % activity SF (synergy factor) O / E class l class II O (watched) E (expected) - - - 0 (control) 1 6 twenty 2 twenty 40 3 twenty 40 4 60 60 5 6 twenty 1: 3 73 52 1.4 6 6 twenty 1:10 75 68 1.1

Example 7: Action against Erysiphe graminis on wheat ₂

In field trials (10 m ² ), winter wheat in the growing phase was sprayed with a spray mixture prepared from a wettable active ingredient powder. Infection happened naturally. 10 days after infection, the degree of fungal infection was assessed. The following results were obtained:

Table 21

Action against Erysiphe graminis on wheat component I: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: propioconazole

Test No. g of active ingredient per hectare l: ll % activity SF (synergy factor) O / E O (watched) E (expected) class l class II - - - 0 (control) 1 5 29 2 50 2 3 100 31 4 5 50 1:10 49 32 1.5 5 5 100 1:20 59 51 1.2

PL 191 355 B1

Table 22

Action against Erysiphe graminis on wheat component I: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: cyprodinil

Test No. g of active ingredient per hectare l: ll % activity SF (synergy factor) O / E O (watched) E (expected) class l class II - - - 0 (control) 1 5 29 2 50 2 3 100 31 4 5 50 1:10 49 32 1.5 5 5 100 1:20 59 51 1.2

Example 8: Action against Mycosphaerella fijiensis on banana plants of banana plants, a plot of 300 m ^{2 was} sprayed intermittently for 17-19 days with a spray mixture prepared from a wettable active ingredient powder a total of six times. Infection happened naturally. The area of the leaves infected with the fungus was measured to evaluate the results. The following results were obtained:

Table 23

Action against Mycosphaerella fijiensis on banana trees component I: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: propioconazole

Test No. g of active ingredient per hectare l: ll % activity SF (synergy factor) O / E class l class II O (watched) E (expected) 1 50 - 0 (control) 19 2 50 26 3 50 50 1: 1 46 40 1.15

Example 9: Action against Alternaria solani on tomatoes

Tomato plants in a plot of 7 m ^{2 were} sprayed intermittently for 7 days with a spray mixture prepared from a wettable powder of the active ingredient nine times in total. The infection was occurring naturally and the King's surfaces contaminated with fungus were measured to evaluate the results. The following results were obtained:

PL 191 355 B1

Table 24

Action against Alternaria salted on tomatoes in the open, component I: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: cyprodinil

Test No. g of active ingredient per hectare l: ll % activity SF (synergy factor) O / E class l class II O (watched) E (expected) - - - 0 (control) 1 2.5 32 2 12.5 thirty 3 25 51 4 2.5 12.5 1: 5 79 53 1.5 5 2.5 25 1:10 80 67 1.2

Example 10: action against Phytophora infestans on tomatoes

Tomato plants of the cultivar "Roter Gnom" were sprayed until the formation of droplets with a mixture prepared from the preparation of the active agent or a combination of active agents. After 4 days, the sprayed plants were infected with a sporangia suspension of the fungus, and then incubated in a cabinet for 2 days at 18-20 ° C and a relative atmospheric humidity of 90-100%. 5 days after infection, fungal infection was assessed. The following results were obtained:

Table 25

Action against Phytophora infestans on tomatoes component I: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: metalaxyl

Test No. mg of active ingredient per liter l: ll % activity SF (synergy factor) O / E class l class II O (watched) E (expected) - - - 0 (control) 1 5 14 2 25 36 3 100 61 4 500 72 5 0.1 13 6 1 23 7 10 35 8 50 68 9 5 0.1 50: 1 50 25 2.0 10 5 1 5: 1 62 34 1.8 11 5 10 1: 2 87 44 2.0 12 5 50 1:10 84 73 1.2 13 25 50 1: 2 92 80 1.2 14 100 10 10: 1 85 75 1.1 15 100 50 2: 1 95 88 1.1 16 500 10 50: 1 97 82 1.2

PL 191 355 B1

Table a 26

Action against Phytophora infestans on tomatoes component l: benzothiodiazole-7-carboxylic acid component II: metalaxyl

Test No. mg of active ingredient per liter l: ll % activity SF (synergy factor) O / E class l class II O (watched) E (expected) - - - 0 (control) 1 0.1 0 2 0.5 9 3 1 22 4 5 45 5 1 13 6 10 33 7 50 63 8 100 83 9 0.1 1 1:10 36 13 2.8 10 0.5 1 1: 2 29 21 1.4 11 1 1 1: 1 57 32 1.8 12 1 10 1:10 79 48 1.6 13 5 1 5: 1 61 52 1.2

Example 11: Action against Pseudoperonospora cubensis on cucumbers

16-19 day old cucumber plants ("Wisconsin") were sprayed to form droplets with a mixture prepared from the formulation of the active agent or a combination of active agents. After 4 days, the sprayed plants were infected with a sporangia suspension of Pseudoperonospora cubensis (strain 365, Ciba; maximum 5000 per ml), the sprayed plants were then incubated for 1-2 days at 18-20 ° C and 70-190% relative atmospheric humidity. 10 days after infection, the degree of fungus infestation was assessed and compared with that of non-sprayed plants. The following results were obtained:

Table a 27

Action against Pseudoperonospora cubensis on cucumbers component I: benzothiodiazole-7-carboxylic acid component II: metalaxyl

Test No. mg of active ingredient per liter l: ll % activity SF (synergy factor) O / E O (watched) E (expected) class l class II 1 2 3 4 5 6 7 - - - 0 (control) 1 0.05 0 2 0.5 6 3 5 66 4 0.5 31

Table 27 continues

1 2 3 4 5 6 7 5 5 66 6 50 91 7 0.05 0.5 1:10 66 31 2.1 8 0.05 5 1: 100 83 66 1.3 9 0.5 0.5 1: 1 83 35 2.4 10 0.5 5 1:10 83 68 1.2

Example 12: Action against Peronospora tobacco on tobacco plants

Tobacco plants (6 weeks old) were sprayed with a solution of the test compound formulation. Four days after spraying, the plants were inoculated with a sporangia suspension of the fungus and kept at high humidity for 4 to 5 days and then incubated with the normal day-night sequence. Assessment of the symptoms in the test was based on the leaf surface infested with the fungus. The degree of contamination of untreated plants corresponds to 0% activity.

Table 28

Action against Peronospora tabacina on tobacco plants component I: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: dimethomorph

Test No. mg of active ingredient per liter (ppm) l: ll % activity SF (synergy factor) O / E O (watched) E (expected) class l class II 1 0.03 14 2 0.1 34 3 0.3 88 4 0.3 52 5 1 52 6 0.03 1 1:33 74 59 1.3 7 0.1 0.3 1: 3 92 68 1.4 8 0.1 1 1:10 95 68 1.4

Example 13: Action against Peronospora parasitica on Arabidopsis thaliana

Metalaxyl, fosetyl and copper hydroxide fungicides and the SAR activator S-methyl ester of benzo [1,2,3] thiodiazole-7-carbothioic acid (BTH), prepared at the concentration of the active ingredient (ai), respectively 25%, 80%, 70% and 25% with a wettable powder carrier, was applied as a thin mist to the leaves of three-week-old plants. The wettable powder itself was used as a control. Three days later, the plants were inoculated with a conidia suspension of Peronospora parasitica as described by Delaney et al. (1995). Ws plants were inoculated with the appropriate P. parasitica isolate Emwa (1-2 x 10 ⁵ spores / ml); Col plants were inoculated with P. parasitica isolate the corresponding NoCo2 (0,5-1x 10 ⁵ spores / ml). After incubation, the plants were covered to maintain high humidity and placed in a Percival growth chamber at 17 ° C with a 14 hour day / 10 hour night cycle (Uknes et al., 1993). Tissue was harvested 8 days after inoculation.

The progress of the fungal infection was followed for 12 days by viewing under a dissection microscope to observe the development of conidiophores (Delaney et al. (1994); Dietrich et al. (1994)). In order to observe the growth of the fungus in the leaf tissue, single-leaf lactophenyl blue staining was used. Fungal growth was quantified using a PCR-derived fungal rRNA probe according to White et al. (1990; PCR Protocols: A guide to methods and Application, 315-322) using

The primers were NS1 and NS2 and P. parasitica EmWa DNA as template. RNA was purified from frozen tissue by phenol / chloroform extraction after lithium chloride precipitation (Lagrimini et al: PNAS, 84: 7542-7546). Samples (7.5 µg) were separated by formaldehyde agarose gel electrophoresis and transferred to a nylon filter (Hybond N +, Amersham) as described by Ausbel et al. (1987). Hybridization and washing were performed according to Church and Gilbert (1984, PNAS, 81: 1991-1995). Relative amounts of transcript were determined using Phosphorlmager (Molecular Dynamics, Sunnyvale, CA) according to the manufacturer's instructions, sample loading was normalized by hybridizing the de-briquetted filter with constitutively expressed Arabidopsis b-tubulin cDNA. The degree of infection of the untreated plants corresponded to 0% fungal growth inhibition, the following results were obtained:

Table 29

Action against Peronospora parasilicaNoCo2 on Arabidopsis thaliana (Col-0) component I: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: metalaxyl

Test No. Ingredients % growth inhibition of the fungus synergy factor O / E BTH metalaxyl O (watched) E (expected) control - - 0 1 0.01 mM - 0 2 - 0.1 mg / l 0 3 0.01 mM 0.1 mg / l 40.7 0

Table 30

Action against Peronospora parasitica Emwa on Arabidopsis thaliana (Ws) component l: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: metalaxyl

Test No. Ingredients % growth inhibition of the fungus synergy factor O / E BTH metalaxyl O (watched) E (expected) control - - 0 1 0.01 mM twenty 2 0.003 mm 0 3 2.5 mg / l 74 4 0.5 mg / l 50 5 0.1 mg / l 50 6 0.01 mM 2.5 mg / l 100 90 1.1 7 0.01 mM 0.5 mg / l 95 70 1.4 8 0.01 mM 0.1 mg / l 88 70 1.3 9 0.003 mm 2.5 mg / l 100 75 1.3

PL 191 355 B1

T a b el a 31

Action against Peronospora parasitica Emwa on Arabidopsis thaliana (Ws) component l: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: fosetyl

Test No. Ingredients % growth inhibition of the fungus synergy factor O / E BTH fosetyl O (watched) E (expected) control - - 0 1 0.01 mM thirty 2 1.0 g / l 40 3 0.2 g / l 10 4 0.04 g / l 0 5 0.01 mM 0.1 g / l 100 70 1.4 6 0.01 mM 0.2 g / l 100 40 2.5 7 0.01 mM 0.04 g / l 95 thirty 3.2

T a b el a 32

Action against Peronospora parasitica Emwa on Arabidopsis thaliana (Ws) component l: benzo [1,2,3] thiodiazole-7-carbothioic acid S-methyl ester component II: copper hydroxide

Test No. Ingredients % growth inhibition of the fungus synergy factor O / E BTH Cu (OH) 2 O (watched) E (expected) control - - 0 1 0.01 mM - thirty 2 0.01 g / l 0 3 0.01 mM 0.01 g / l 85 thirty 2.8

As can be seen in Table 29, synergistic disease resistance effects were demonstrated in wild Arabidopsis Col-0 plants. No inhibition of fungal growth was observed when the plants were treated separately with both 0.01 mM BTH and 0.0001 g / L metalaxyl as these concentrations are normally insufficient to obtain an effect. However, when plants were treated with both of these compounds at these normally insufficient concentrations, 40.7% inhibition of fungal growth was observed, which is clearly a synergistic effect. Tables 30-32 show the synergistic disease resistance effects in wild Arabidopsis Ws plants. Only 20-30% inhibition of fungal growth was observed when treating Ws plants with 0.01 mM BTH. However, synergistic disease resistance was observed with the simultaneous treatment of plants with BTH and metalaxyl, fosetyl, or copper hydroxide. These combined antifungal effects reducing the effective concentrations of the fungicide and BTH required to control the pathogen allow a reduction in the chemical dose required to arrest fungal growth and thus reduce the frequency of chemical tolerance-related leaf damage.

II. Synergistic disease resistance effects obtained by the use of conventional antimicrobial agents and / or chemical inducers of acquired systemic resistance in constitutive immunity mutant (CIM) plants

In this group of examples, a high-throughput Northern hybridization assay was developed to identify plant mutants exhibiting high levels of PR-1 mRNA during normal growth, with the intention that these mutants also exhibit acquired systemic resistance. A number of mutants have been isolated using this assay and have been shown to accumulate not only PR-1 mRNA but also PR-2 and PR-5 (Lawton et al. (1993); Dietrich et al. (1994); and Weyman et al. (1995)).

PL 191 355 B1

These mutants also show elevated SA levels and are resistant to pathogen infection, confirming the usefulness of this strategy for the isolation of mutants in the SAR signaling pathway.

Two classes of mutants in the SAR signaling pathway have been isolated using this assay. One class is designated as Isd mutants (Isd = lesion simulating disease). This class of mutants is also referred to as "Class I cim" as described in WO 94/16077, the description of which is hereby incorporated by reference in its entirety. This class of Isd (also known as cim Class l) created spontaneous lesions on leaves, accumulated at an increased concentration of SA, showed high levels of PR-1, PR-2 and PR-5 mRNA and was resistant to fungal and bacterial pathogens 9 Dietrich et al. 1994; Weymann et al., 1995).

A second class, called cim (cim = constitutive jmmunity), is described below and exhibits all the features of Isd mutants with the exception of spontaneous lesions. This second class (cim) corresponds to the "Class II cim" mutants discussed in WO 94/16077. The cim3 plant mutant line described below belongs to this cim class (cim Class II) and corresponds to a predominant mutation which is wild in appearance and expresses stable, elevated levels of SA and mRNA SAR genes and has a broad spectrum of disease resistance.

Example 14: Isolation and characterization of cim mutants constitutively expressing SAR genes

1,100 individual mutagenized (EMS) Arabidopsis M2 plants were grown in Aracon trays (Lehie Seeds, Round Rock, TX) in groups of approximately 100. Plants were grown as described in Uknes et al., 1993 supra, taking particular care to avoid excessive hydration and infection with pathogens. Briefly, Metro Mix 360 was saturated with water and autoclaved three times for 70 minutes in 10 liter aliquots. The blend was thoroughly mixed between each autoclaving. Seeds were surface sterilized in 20% Clorox for 5 minutes and rinsed with seven changes of sterile water before sowing. The sown seeds were vernalized for 3-4 days and then grown in chambers on a 9 hour day and a 15 hour night at 22 ° C. When the plants were three to four weeks old, one or two leaves weighing 50 to 100 mg were harvested and total RNA was isolated using a rapid RNA mini-preparation method (Verwoerd et al. (1989) Nuc. Acid Res., 17, 2362). PR-1 gene expression was analyzed by Northern hybridization (Lagrimini et al. (1987) Proc. Natl. Acad. Sci. USA 84, 7542-7546; Watd et al., 1991). Each group of plants also contained controls of untreated A. thaliana Col-0 plants and those treated for two days with INA (0.25 mg / ml). All plants were grown as described by Weymann et al. (1995).

Eighty potential mutants displaying increased levels of PR-1 mRNA were identified. After examining the offspring, five were selected for further characterization. Potential cim mutants showed increased expression of SAR genes in the absence of pathogen or inducing activity. Examination of the progeny of potential cim mutants confirmed that constitutive expression of PR-1 is hereditary. Among the cim mutants, two were further characterized: cim2 and cim3 showing the highest and most stable expression of PR-1.

The recessive hairless trait was used as a marker in test crosses with Columbia plants. Col-g1 flower buds were de-stemmed prior to pollen discharge and then immediately, and the next day, mutant pollen was administered. F1 plants were grown in soil, and the disenchanted offspring were identified by the presence of the hairs.

After crossing cim2 and cim3 with the Col-0 or La-er ecotype, a significant proportion of F1 plants exhibited high SAR gene expression was identified, suggesting that these traits are dominant. In the case of cim2, some, but not all, F1 plants constitutively expressed SAR genes. Such a result would be expected if the cim2 mutation was dominant and occurred in the parent in a heterozygous system. Further genetic studies of the cim2 mutation showed a continuous variable segregation in the F2 generation, consistent with the hypothesis of incomplete penetration.

The cim3 mutation showed a 1: 1 distribution in the F1 generation, after which two F1 individuals showing high levels of PR-1 mRNA were self-bred to form the F2 population. The distribution in F2 examined by determining the accumulation of PR-1 mRNA gave 93 F2 plants with high PR-1 mRNA levels and 25 F2 plants without significant accumulation of R-1 mRNA, resulting in a ratio of 3.7: 1 (c ² = 1.77; 0.5>P> 0.1), which is consistent with the hypothesis that cim3 is a dominant mutation in one gene. Further crosses confirmed that cim3 is inherited as a dominant mutation.

For cim3, the starting M2 plant identified in the test and the M3 population were normal in appearance. However, after inbred cim3 plants, some of the best expressing lines

PL 191 355 B1 showed low fertility. Plants with normal appearance and fertility and high PR-1 expression were obtained after test cross-g1.

The pre-identified cim3 plants had a stunted appearance with thin distorted leaves. However, F2 plants crossed with the Col-g1 ecotype retained a high level of SAR gene expression and were not distinguishable from wild plants. This suggested that the dwarf and distorted leaf phenotype was caused by an independent mutation unrelated to the constitutive expression of the SAR genes. The cim3 mutant phenotype was also observed when the plants were grown under sterile conditions, confirming that the accumulation of PR-1 mRNA was not caused by the pathogen.

Example 15: SAR gene expression

In addition to PR-1, two other SAR genes, PR-2 and PR-5, are also highly expressed in cim3 mutants. The levels of SAR gene expression were variable among the offspring but were always more than 10 times higher than the untreated control and were close to the levels obtained after resistance-inducing treatment with INA (0.25 mg / ml) of wild plants.

Example 16: Analysis of salicylic acid

Endogenous SA concentration has been shown to increase with pathogen-induced necrosis in Arabidopsis (Uknes et al., 1993 supra). Salicylic acid and its glucose conjugate were analyzed as described by Uknes et al., 1993, leaf tissue was harvested from 10 cim3 plants and 10 controls at 4 weeks of age. Leaves from individual plants were harvested and the level of the PR-1 gene was determined. SA levels were measured in plants expressing PR-1. The concentration of free SA in the cim3 mutant was 3 to 4 times higher than in uninfected wild Arabidopsis plants (233 ± 35 and 69 ± 8 ng / g wet weight, respectively). The level of SA glucose conjugate (SAG) was 13.1 times higher in cim3 than in uninfected wild-type Arabidopsis (4519 ± 473 and 344 ± 58 ng / g fresh weight, respectively). These elevated SA and SAG concentrations are comparable to those obtained in pathogen infected tissues or in cpr mutants.

Example 17: Disease resistance

The cim3 mutant was tested for resistance to Peronospora parasitica, the causative agent of downy mildew disease in Arabidopsis. Thirty cim3 plants (confirmed by PR-1 expression study) and thirty controls (Columbia ecotype), each approximately 4 weeks old, were inoculated with P. parasitica as described by Uknes et al., 1992 supra. Seven days later, the plants were examined for sporulation and stained with trypan blue to reveal fungal structures as described by Keogh et al. (1980) Trans. Br. Mycol. Soc. 74, 329-333 and Koch and Slusarenko (1990) Plant Cell 2, 437-445. Wild plants (Col-0) show growth of hyphae, conidia and oospores, whereas wild plants treated with INA (0.25 mg / ml) and cim3 plants show no fungal growth. Immunity conferred by the cim3 mutation appears mostly as a small group of dead cells at the site of pathogen infection. This type of resistance resembles that seen in Isd mutants (Dietrich et al., 1984, supra; Weymann et al., 1995, supra) or SAR-induced wild plants (Uknes et al., 1992, supra). Occasionally, intermediate resistance phenotypes were observed, including post-hyphae necrosis in cim3 plants. Such necrosis resembles that seen in wild plants treated with low doses of SA or INA (Uknes et al., 1992, supra, Uknes et al., 1993, supra). However, sporulation was never observed in the cim3 plants, whereas sporulation did occur in all the control plants. No spontaneous lesions were observed on uninfected cim3 leaves after staining with trypan blue.

In addition to resistance to the fungal pathogen P. parasitica, cim3 plants were also resistant to infection with the bacterial pathogen Pseudomonas syringae DC3000. Six-week old wild plants (± INA treatment) and cim3 were inoculated with a suspension of P. syringae DC3000 and disease progression was observed by measuring the growth of bacteria extracted from the infected leaves with time progression. The bacterial titer difference between Col-0, Col-0 + INA and cim3 on day 0 or day 2 was not statistically significant. However, by day four, a 31-fold reduction in bacterial growth was observed in the cim3 plants compared with the wild-type (P <0.003; Sokal and Rohlf, 1981). Plants were also inspected visually for disease symptoms. Leaves from wild plants showed severe chlorosis, with disease symptoms spreading clearly beyond the initial injection zone. In contrast, INA treated wild plants or cim3 plants were practically free from disease symptoms.

In this example, Pseudomonas syringae cultures derived from DC3000 tomatoes were grown in King B medium (agar or liquid plates) with rifampicin (50 µg / ml) at 28 ° C (Walen et al. (1991) Plant Cell 3, 49-59). The overnight culture was diluted and resuspended in 10 mM MgCl 2 to a density of 2-5 x 10 ⁵ cells per ml and injected into Arabidopsis leaves. Injections were made by forming a small one

Use a 28 gauge needle in the middle of the leaf and inject about 250 µl of the diluted bacterial suspension with a 1 cm ³ syringe. At different time points, 10 random samples were taken containing 3 random leaflets taken with a corkbob # 1 from 10 plants of each group. 3 leaf clippings were placed in an Eppendorf tube with 300 µl of 10 mM MgCl ₂ and homogenized with a pestle. The resulting bacterial suspension was appropriately diluted and plated on King B medium with rifampicin (50 µg / ml) and cultured for 4 days at 28 ° C. Bacterial colonies were counted and the data were processed statistically by Student's t test (Sokal and Rohlf (1981), Biometry, ^2nd ed. New York: WHFreeman and Company).

Also in this example, 2,6-dichloroisonicotinic acid (INA) was suspended in sterile distilled water at a concentration of 25% of the active ingredient formulated as a wettable powder (0.25 mg / ml, 325 µΜ Kessmann et al. (1994) Annu. Rev. Phytopatol 32,439-59). All plants were sprayed with water or INA solution until drops were formed.

Example 18: Role of SA in SAR gene expression and disease resistance

To investigate the relationship between SA, SAR gene expression and resistance in the cim3 mutant, crosses with Arabidopsis plants expressing the salicylate hydroxylase (nahG) gene were performed (Delaney et al., 1994). These "NahG plants" were obtained by transforming the nahG gene under the control of the 35S promoter into Arabidopsis using Agrobacterium mediated transformation. See Huang, H., Ma, H. (1992) Plant Mol. Biol. Rep. 10, 372-383, incorporated herein by reference; and Delaney et al. (1994) Science 266, 1247-1250, incorporated herein by reference. ArabidopsisCol-nahG carries the dominant kanamycin resistance gene alongside the dominant nahG gene, so Col-nahG was used as pollen donor. F1 seeds were rehydrated in water for 30 minutes and then surface sterilized in 10% Clorox, 0.05% Tween 20 for five minutes and rinsed thoroughly in sterile water. Seeds were sown on a germination medium (GM, Murashige and Skoog medium containing 10 g / L of sucrose buffered with 0.5 g / L of 2 (N-morpholino) ethanesulfonic acid, pH 5.7 with KOH) containing 25 mg / mL of kanamycin for selection F1 plants. See Valvekens et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5536-5540. Kanamycin-resistant F1 plants were transferred to soil after 18 days. The presence of the nahG gene and PR-1 expression were confirmed in all experiments by Northern hybridization analysis. Since both the cim3 and nahG phenotypes are dominant, the epistasis between the two genes could be analyzed in F1 plants. Seventy F1 plants from the cim3 X nahG cross were analyzed for expression of the PR-1 and nahG genes. In mRNA expression analyzes by Northern hybridization, the presence of the nahG gene correlated with suppression of SAR gene expression. The presence of the cim3 mutation in each F1 individual was confirmed by determining the level of PR-1 mRNA in the obtained F2 segregants.

To determine whether the cim3 mutation is epistatic to nahG in terms of disease resistance, 5 plants 51 from the cim3 X nahG cross that were confirmed for nahG and PR-1 mRNA shoulder were self-crossed and 20-30 F2 seeds were seeded. NahG and PR-1 mRNA expression was analyzed in individuals from this F2 population, which were then exposed to P. parasitica (NoCo2) to test their susceptibility to disease. Disease resistance mediated by the cim3 mutation was eliminated by the presence of the nahG gene, showing that nahG is epistatic to cim3 in terms of SAR gene expression phenotypes and disease resistance.

Example 19: Synergistic disease resistance obtained by administration of microbicide and / or BTH to cim mutants

Three days before inoculation with the pathogen, a chemical inducer of systemic acquired resistance BTH (benzo [1,2,3] -thiodiazole-7-carbothioic acid S-methyl ester) formulated as 25% active ingredient (ai) with a wettable powder carrier (Metraux et al., 1991); and / or a metalaxyl microbicide (CGA 48988) formulated as 25% ai, or a wettable carrier alone. Plants were inoculated with a conidial suspension (1.8 x 10 ⁵ spores / ml) of the relevant pathogen Peronospora parasitica NoCo2. Following infection, the plants were covered to maintain high humidity and placed in a Percival growth chamber at 17 ° C with a 14 hr cycle. day / 10 hrs night (Uknes et al., 1993). Tissue was harvested 8 days post infection.

Fungal growth was determined using the PCR fungal rRNA probe according to White et al. (1990; PCR Protocols: A guide to methods and Application, 315-322) using NS1 and NS2 primers and P. parasitica EmWa DNA as template. RNA was purified from frozen tissue by phenol / chloroform extraction after lithium chloride precipitation (Lagrimini et al: PNAS, 84: 7542-7546). Samples (7.5 µg) were separated by formaldehyde agarose gel electrophoresis and transferred to a nylon filter (Hybond N +, Amersham) as described by Ausbel et al. (1987). Hybridization and washing were performed according to Church and Gilbert (1984, PNAS, 81: 1991-1995). Relative

Transcript amounts were determined using a Phosphorlmager (Molecular Dynamics, Sunnyvale, CA) according to the manufacturer's instructions, sample loading was normalized by hybridizing the de-briquetted filter to constitutively expressing Arabidopsis b-tubulin cDNA. The degree of infection of the untreated plants corresponded to 0% inhibition of the growth of the fungus.

Administration of metalaxyl alone, BTH "plant activator" alone, or both metalaxyl and BTH to the cim3 mutants described above produced an over-additive or synergistic disease resistance effect. This effect was designated as the synergy factor (SF), which is the ratio of the observed (O) to the expected (E) effect. The following results were obtained:

Table 33

Action against Peronospora parasitica on Arabidopsis component l: cim3 mutation component II: metalaxyl

Test No. Ingredients % growth inhibition of the fungus synergy factor O / E cim3 metalaxyl O (watched) E (expected) control Tue - 0 1 cim3 - 12.5 2 Tue 12.5 mg / l 52.7 3 Tue 2.5 mg / l 0 4 Tue 0.1 mg / l 0 5 Tue 0.02 mg / l ND 6 cim3 12.5 mg / l ND ND ND 7 cim3 2.5 mg / l 82.2 12.5 6.6 8 cim3 0.1 mg / l 57.8 12.5 4.6 9 cim3 0.02 mg / l 55.6 ND ND

wt = wild Col-0 ND = not determined

Table 34

Action against Peronospora parasitica on Arabidopsis component l: mutation cim3 component II: BTH

Test No. Ingredients % growth inhibition of the fungus synergy factor O / E cim3 BTH O (watched) E (expected) control Tue - 0 1 cim3 - 12.5 2 Tue 0.1 mm 85.7 3 Tue 0.03 mM 20.8 4 Tue 0.01 mM 0 5 cim3 0.1 mm ND 98.2 ND 6 cim3 0.03 mM 73.1 33.3 2.2 7 cim3 0.01 mM 16.6 12.5 1.3

wt = wild Col-0 ND = not determined

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Table a 35

Action against Peronospora parasitica on Arabidopsis component l: mutation cim3 component II: BTHi metalaxyl (M)

Test No. Ingredients % growth inhibition of the fungus synergy factor O / E cim3 BTH + M O (watched) E (expected) control Tue - 0 1 cim3 - 12.5 2 Tue BTH 0.01 mM + M 0.5 mg / l 100 3 Tue BTH 0.01 mM + M 0.1 mg / l 40.7 4 Tue BTH 0.01 mM + M 0.2 mg / l ND 5 cim3 BTH 0.01 mM + M 0.5 mg / l ND 100 ND 6 cim3 BTH 0.01 mM + M 0.1 mg / l 100 53.2 1.9 7 cim3 BTH 0.01 mM + M 0.02 mg / l 77.7 ND ND

wt = wild Col-0 ND = not determined

As can be seen in the tables above, synergistic disease resistance effects have been demonstrated in cim3 plants using metalaxyl alone, using BTH alone, and using a combination of BTH and metalaxyl. For example, a 12.5% inhibition of fungal growth was observed in untreated cim3 plants compared to the untreated wild-type plant, indicating that constitutive expression of the SAR genes in the cim3 mutant correlates with disease resistance. However, as shown in Table 30, the use of metalaxyl at a concentration of 0.0001 g / L (normally insufficient for effective activity) in an immunomodulated (SAR-constitutive) cim3 plant resulted in an increase in growth inhibition to 57.8%. A synergy factor of 4.6 calculated from these data clearly shows the synergistic effect obtained by treating an immunomodulated plant with a microbicide.

The data presented in Table 31 show that synergy is also obtained by administering a chemical inducer of systemic acquired resistance such as BTH to an immunomodulated (SAR-constitutive) cim3 plant. For example, in wild-type plants, a concentration of 0.03 mM BTH is normally insufficient for effective disease resistance, yielding only 20.8% fungal growth inhibition. However, in cim3 plants, this normally insufficient BTH concentration resulted in 73.1% fungal growth inhibition, almost as much as the inhibition obtained with 0.1 mM BTH, the recommended effective concentration. The synergy factor of 2.2 calculated from the data in Table 31 clearly shows the synergistic effect obtained by applying BTH to a plant immunomodulated by other agents.

The disease resistance effects were even more pronounced when using both BTH and metalaxyl on the cim3 plant. As shown in Example 13 (Table 29), no inhibition of fungal growth is observed in wild plants when either 0.01 mM BTH or 0.0001 g / L metalaxyl is administered separately because these concentrations are normally insufficient for efficient operation. However, applying both of these compounds to a plant at these normally insufficient concentrations results in an observed fungal growth inhibition of 40.7%, which is a synergistic effect in wild plants. In the cim3 plants, the simultaneous administration of 0.01 mM BTH and 0.0001 g / L metalaxyl resulted in 100% growth inhibition of the fungus, clearly demonstrating even further synergistic activity.

Thus, the combined use of immunomodulated cim plants and low, normally ineffective concentrations of chemicals to achieve disease resistance provides benefits that should be apparent to those skilled in the agricultural science. The use of normally toxic or otherwise undesirable concentrations of chemicals can be avoided by taking advantage of the synergies demonstrated here. Additionally, economic benefits may be obtained from the reduced amounts of chemicals required to provide a given level of plant protection.

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III. Synergistic effects of disease resistance obtained by the use of conventional microbicides and / or chemical inducers of systemic acquired resistance on transgenic plants containing forms of the NIM1 gene

The NIM1 gene is a key component of the systemic acquired resistance (SAR) pathway in plants (Ryals et al. 1996). The NIM1 gene is associated with SAR activation by chemical and biological inducers and, in combination with such inducers, is required for SAR acquisition and SAR gene expression. The location of the NIM1 gene was determined by molecular biology analysis of the genome of mutant plants carrying the nim1 mutation, which makes the plants extremely sensitive to a variety of pathogens and prevents them from responding to pathogens and chemical SAR inducers. The wild Arabidopsis NIM1 gene was mapped and sequenced (SEQ ID NO: 1). The wild-type NIM1 gene product (SEQ ID NO: 2) is involved in the signal transduction cascade leading to SAR and gene-for-gene resistance (Ryals et al., 1997). Overexpression of the recombinant wild-type form of NIM1 results in immunomodulated plants with a constitutive immunity phenotype (CIM) and thus confers disease resistance to transgenic plants. Elevated levels of the active NIM1 protein produce the same disease resistance effect as chemical induction using chemical inducers such as BTH, INA and SA. See co-filed U.S. Patent Application No. 08 / 880,179, incorporated herein by reference.

In addition, the NIM1 gene product has been shown to be structural homologue of a mammalian IkB signal transduction factor of subclass a (Ryals et al., 1997). IkB mutations acting as superrepressors or dominant negative mutations in the NFkB / IkB regulatory pathway have been described. Certain altered forms of NIM1 are the dominant negative regulators of the SAR transduction pathway. These altered forms of NIM1 impart to the plants transformed therewith a phenotype opposite to that of the nim1 mutant, that is, the immunomodulated plants transformed with altered forms of NIM1 display constitutive expression of the SAR genes and the CIM phenotype. See the co-filed PCT application "Methods of using the NIM1 gene to confer disease resistance in plants" incorporated herein by reference.

Example 20: Transformation of plants with cosmid clones containing the wild-allele of the NIM1 gene

Cosmid D7 (deposited in the ATCC collection on September 25, 1996 as ATCC 97736) was obtained from a clone spanning the NIM1 gene region and thus contains the wild-type allele of the NIM1 gene (SEQ ID NO: 1). Cosmid E1 was also obtained from a clone spanning the NIM1 gene region and thus also contains the wild-type allele of the NIM1 gene (SEQ ID NO: 1). Cosmids D7 and E1 were introduced into Agrobacterium tumefaciensAGL-1 by conjugation transfer in a tri-parent cross with the HB101 helper strain (pRH2.013) as described in US Patent Application No. 08 / 880,179. These cosmids were used to transform a kanamycin sensitive nim1 mutant Arabidopsis line using vacuum infiltration (Mindrinos et al., 1994, Cell78, 1089-1099). Seeds from infiltrated plants were harvested and germinated on GM agar plates containing 50 mg / ml kanamycin as selection agent. The surviving seedlings were transferred to the soil approximately two weeks after sowing.

Plants transferred to the soil were grown in a phytotron about one week after transfer. The plants were completely covered with 300 mM INA fog using a chromister. After two days, leaves were collected for RNA extraction and PR-1 expression analysis. The plants were then sprayed with Peronospora parasitica (EmWa isolate) and grown under high humidity conditions in a growth chamber at 19 ° C by day and 17 ° C by night with an 8 hour cycle. lights / 16 hours darkness. Eight to ten days after fungus infection, plants were examined and scored positive or negative for fungal growth. Ws and nim1 plants were treated in the same way as controls in each experiment.

Total RNA was extracted from harvested tissue using LiCl / phenol buffer (Verwoerd et al., 1989, Nucl. Acids Res. 2362). RNA samples were run on formaldehyde agarose gels and transferred to GeneScreen Plus filters (DuPont). The filters were hybridized with cDNA probe labeled PR-1 ³² P. The resulting filter was exposed to film to determine which transformants were able to induce PR-1 expression after INA treatment.

To determine whether any of the transformants D7 and E1 overexpressed NIM1 due to the insertion site (positional) effect, primary transformants containing the D7 or E1 cosmids were self-crossed and T2 seeds were harvested. Seeds from one E1 line and 95 D7 lines were sown on soil and grown as described above. When T2 plants had produced at least 4 correct leaves, one leaf from each plant was harvested. RNA was isolated from this tissue and tested for PR-1 and NIM1 expression. Plants were then inoculated with P. parasitica (EmWa) and analyzed for fungal growth 10 days after infection, a number of transformants showed reduced growth compared to normal, four out of nothing, specifically lines D7-2, D7-4, D7-89 and E1-1 showed no visible growth

PL 191 355 B1 of the fungus. Plants showing higher than normal expression of NIM1 and PR-1 and showing resistance to the fungus indicate that overexpression of NIM1 carries disease resistance.

Example 21: Overproduction of NIM1 under the control of the native promoter

Plants constitutively expressing the NIM1 gene were generated by transforming wild-type Ws plants with a genomic BamHI-HindIII fragment (SEQ ID NO: 1 - bases 1249-5655) containing a 1.4 kb 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 seeds were harvested and plated on GM agar containing 50 mg / ml kanamycin. Surviving seedlings were transferred to soil and tested as previously described for resistance to the Emwa Peronospora parasitica isolate. Selected plants were self-crossed and selected for two consecutive generations to generate homozygous lines. Seeds from several of these lines were sown into soil and 15-18 plants from each line were grown for three weeks and tested again for Emwa resistance without prior treatment with inducing chemicals. Approximately 24 hours, 48 hours, and five days after treatment with the fungus, tissue for each line was harvested, pooled, and frozen. The plants remained in the growth room for up to five days after starting cultivation, when they were tested for Emwa resistance.

RNA was obtained from all samples collected and analyzed as previously described (Delaney et al., 1995). The filter was hybridized with the probe for the Arabidposis PR-1 gene (Uknes et al., 1992). Five of the 13 transgenic lines analyzed showed early induction of PR-1 gene expression. For these lines, PR-1 mRNA was visible 24 or 48 hours after exposure to the fungus. These five lines also showed no apparent fungal growth. Leaves were stained with lactophenol blue as written (Dietrich et al. 1994) to confirm the absence of mycelium in the leaves. Expression of the PR-1 gene was not induced in the other eight lines for 48 hours and these plants showed no resistance to Emwa.

A set of resistant lines were also tested for increased resistance to the bacterial pathogen Pseudomonas syringae DC3000 to establish the spectrum of apparent resistance as previously described by Uknes et al. (1993). Experiments were performed essentially as described by Lawton et al. (1996). Bacterial growth was slower in those lines that also showed constitutive Emwa resistance. This shows that plants overproducing the NIM1 gene under their native promoter have constitutive pathogen resistance.

To perform additional characterization of the CIM phenotype in these lines, free and glucose-bound salicylic acid was measured in uninfected plants and leaves were stained with lactophenol blue to examine the presence of microscopic defects. Resistant plants were sexed with SAR mutants such as NahG and ndr1 to determine how these dominant negative NIM1 mutations might affect salicylic acid-dependent feedback.

Example 22: Overproduction of NIM1 under the control of 35S full-length NIM1 cDNA (SEQ ID NO: 6) was cloned into the EcoRI site of pGCN1761 ENX (Comai et al. (1990) Plant. Mol. Biol. 15, 373-381). From the resulting plasmid, an XbaI fragment containing the boosted CaMV 35S promoter, the NIM1 cDNA in the appropriate orientation for transcription, and the 3 'tml 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 seeds were harvested and plated on GM agar containing 50 mg / ml kanamycin. Surviving seedlings were transferred to soil and tested as previously described. Selected plants were self-crossed and selected for two consecutive generations to generate homozygous lines. Nine of the 58 lines tested were found to be resistant to treatment with Emwa without prior chemical treatment. Thus, overproduction of the NIM1 cDNA also resulted in the emergence of disease resistant plants.

Example 23: NIM1 is an iKBa homologue

Multiple sequences were aligned with the protein products of NIM1 and IkB, whereby the NIM1 gene product was found to match the iKBa homolog (Figure 1). Sequence homology searches were performed using BLAST (Altschul et al., J. Mol. Biol. 215, 403-410 (1990). Multiple sequence alignment was performed using Clustal V (Higgins et al., CABIOS 5,151-153 (1989) ) which is part of the Lasergene Biocomputing Software from DNASTAR (Madison, WI). The sequences subjected to alignment were NIM1 (SEQ ID NO: 2), mouse iKBa (SEQ ID NO: 3, GenBank Accession No. 1022734), rat iKBa (SEQ ID NO: 2). ID: 4, GenBank Accession No.: 57674 and Χ63594; Tewari et al., Nucleic Acids Res. 20, 607 (1992)) and porcine iKBa (SEQ

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ID NO: 5, GenBank Accession No .: 221968; de Martin et al., EMBO J. 12, 2773-2779 (1993); GenBank Accession No.: 517193, de Martin et al., Gene 152, 253-255 (1995)). For the sequences subjected to alignment, the parameters used in the Clustal analysis were used, with a penalty of 10 for creating a gap and a penalty of 10 for extending the gap. Evolutionary distance was calculated using the PAM250 matrix (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, MO, Dayhoff, ed. (National Biomedical Research Foundation, Washington, DC) pp. 345-358 (1978)). Residual similarity was calculated using a modified Dayhoff matrix (Schwartz and Dayhoff, "A model of evolutionary change in proteins." In Atlas of Protein Sequence and Structure, MO Dayhoff, ed. (National Biomedical Research Foundation, Washington, DC) pp. 353- 358 (1979); Gribskov and Burgess, Nucleic Acids Res. 14, 6745-6763 (1986)).

Homology searches indicate that NIM1 is similar to the ankyrin domains of several proteins including: ankyrin, NF-κΒ, and IkB. The greatest overall homology is to IkB and related molecules (Figure 1). NIM1 contains 2 serines at amino acid positions 55 and 59; serine at position 59 is in the context of (D / ExxxxS) and the position (N-terminal) corresponds to its role in phosphorylation dependent, inducible ubiquitin-mediated degradation. All of these IKBa have these N-terminal serines and are required for the inactivation of IkB and the 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 common feature of IkB and NF-kB molecules. The C-ends of IkB may be dissimilar. NIM1 has some homology to the QL-rich region (amino acids 491-499) found at the C-terminus of some IkBs.

Example 24: Generation of altered forms of NIM1 - replacement of serine residues 55 and 59 with alanine residues

Phosphorylation of serine residues in human IKBa is required for stimulus-activated IKBa degradation, thus activating NF-kB. Mutagenesis of serine residues (S32-S36) in human IKBa to alanine residues inhibits stimulus-induced phosphorylation and thus blocks degradation mediated by the IKBa proteosome (E. Britta-Mareen Traenckner et al., EMBO J. 14: 2876-2883 (1995) ; Brown et al., Science 267: 1485-1488 (1996); Brockman et al., Molecular and Cellular Biology 15: 2809-2818 (1995); Wang et al., Science 274: 784-787 (1996)).

This altered form of IKBa acts as a dominant negative form by trapping NF-KB in the cytoplasm, thereby blocking further signaling. Based on sequence comparison between NIM1 and IKB, serines 55 (S55) and 59 (S59) in NIM1 are homologous to S32 and S36 in human IKBa. To construct dominant negative forms of NIM1, the serines at amino acid positions 55 and 59 were mutagenized to alanine residues. This can be done by any method known to those skilled in the art such as, for example, using the QuikChange Site Directed Mutagenesis Kit (# 200518: Strategene).

Using the full-length NIM1 cDNA (SEQ ID NO: 6), including the 42 bp 5 'untranslated (UTR) sequence and the 187 bp 3'UTR, a mutagenized construct can be generated according to the manufacturer's instructions using the following primers ( (SEQ ID NO: 6, items 192-226): 5'CAA CAG CTT CGA AGC CGT CTT TGA CGC GCC GGA TG-3 '(SEQ ID NO: 25) and 5'CAT CCG GCG CGT CAA AGA CGG CTT CGA AGC TGT TG-3 '(SEQ ID NO: 26), where the underlined bases indicate mutations. The strategy is as follows: The NIM1 cDNA, cloned in the pSE936 vector (Elledge et al., Proc. Nat. Acad. Sci. USA 88: 1731-1735 (1991)) is denatured and primers containing the altered bases attached. DNA polymerase (Pfu) extends the primers by replacing the missing strand, resulting in circular strands with a cut. The DNA is digested with the restriction endonuclease DpnI, which cuts only the methylated sites (non-mutagenized template strand). The remaining circular DNA is transformed into E. coli strain XL1-Blue. Plasmids are extracted from the resulting colonies and sequenced to confirm the presence of the mutant bases and to verify that no other mutations have occurred.

The mutagenized NIM1 cDNA is digested with EcoRI restriction endonuclease and cloned into pCGN1761 under the transcriptional control of the 35S double cauliflower mosaic virus promoter. A transformation cassette containing the 35S promoter, NIM1 cDNA and tml terminator is released from plasmid pCGN1761 by partial digestion with XbaI restriction enzyme and is ligated into the XbaI site of dephosphorylated pCIB200. SEQ ID NOS: 7 and 8 show the DNA coding sequence and the coded amino acid sequence, respectively, of such an altered form of the NIM1 gene.

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Example 25: 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.) In human IKBa, which includes K21, K22, S32, and S36, results in a dominant negative IKBa phenotype in culture transfected human cells. The N-terminal deletion of the first approximately 125 amino acids of the encoded NIM1 cDNA product removes eight lysine residues that may serve as potential ubiquitinylation sites and also removes putative phosphorylation sites S55 and S59 (see Example 2). Such an altered gene construct can 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 form of NIM1 can be prepared in which the DNA encoding the first 125 amino acids is deleted. A 1612 bp PCR product is produced using the following primers (SEQ ID NO: 6: 418 to 2011): 5'-gg aat tca-ATG GAT TCG GTT GTG ACT GTT TTG-3 '(SEQ ID NO: 27) and 5'-gga att cTA CAA ATC TGT ATA CCA TTG G-3 '(SEQ ID NO: 28), in which the synthetic start codon is underlined (ATG) and the EcoRI linker sequence is in lower case. The fragment amplification uses a reaction mixture containing 0.1 to 100 ng of template DNA, 10 mM Tris pH 8.3 / 50 mM KCl / 2 mM MgCl2 / 0.001% gelatin / 0.25 mM each dNTP / 0.2 mM each primer and 1 unit rTth DNA polymerase in a final volume of 50 ml and a Perkin Elmer Cetus 9600 PCR machine. The PCR conditions are as follows: 94 ° C 3 min: 35 x (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 digestion with the restriction enzyme EcoRI and ligated into the EcoRI site of dephosphorylated pCGN1761, under the transcriptional control of the double 35S promoter. The construct is sequenced to confirm the presence of the synthetic ATG start codon and to verify that no other mutations have occurred. The transformation cassette containing the 35S promoter, the modified NIM1 cDNA and the tml terminator is released from plasmid pCGN1761 by partial digestion with the restriction enzyme XbaI and is ligated into the XbaI site of dephosphorylated pCIB200. SEQ ID NOs: 9 and 10 show the DNA coding sequence and the encoded amino acid sequence, respectively, of an altered form of the NIM1 gene having an N-terminal amino acid deletion.

Example 26: Generation of altered forms of NIM1 - C-terminal deletion

The deletion of amino acids 261-317 in human IKBa is believed to result in increased internal stability by blocking the constitutive phosphorylation of serine and threonine residues at the C-terminus. A serine-threonine-rich region is present in the C-terminus 522-593 region of NIM1. The C-terminal coding region of the NIM1 gene can be modified by deleting the nucleotide sequences that code for amino acids 522-593. Using the method of Ho et al. (1989), PCR removes the C-terminal coding region and the 3'UTR of the NIM1 cDNA (SEQ ID NO: 6: 1606-2011) generating a 1623 bp fragment using the following primers: 5 ' -cggaattcGATCTCTTTAATTTGTGAATTT C-3 '(SEQ ID NO: 29) and 5'-ggaattcTCAACAGTT CATAATCTGGTCG-3' (SEQ ID NO: 30) where the synthetic stop codon is underlined (TGA on the complementary strand) and the EcoRI linker sequence is written lowercase. The components of the PCR reaction are as described previously and the cycle parameters are as follows: 94 ° C 3 min: 35 x (94 ° C 30 sec: 52 ° C 30 sec: 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 digestion with the restriction enzyme EcoRI and ligated into the EcoRI site of dephosphorylated pCGN1761 under the transcriptional control of the double 35S promoter. The construct is sequenced to confirm the presence of the synthetic stop codon in the frame and to verify that no other mutations have occurred during the PCR reaction. The transformation cassette containing the 35S promoter, modified NIM1 cDNA and the tml terminator is released from plasmid pCGN1761 by partial digestion with XbaI restriction enzyme and is ligated into the XbaI site of dephosphorylated DCIB200. SEQ ID NOS: 11 and 12 show the DNA coding sequence and the coded amino acid sequence, respectively, of an altered form of the NIM1 gene having a C-terminal amino acid deletion.

Example 27: Generation of altered forms of NIM1 - hybrid with N-terminal / C-terminal deletion

The N-terminal and C-terminal deleted form of NIM1 is generated using a single KpnI restriction site at position 819 (SEQ ID NO: 6). The N-terminal deleted form (Example 25) is digested with the restriction enzymes EcoRI / KpnI and the 415 bp fragment corresponding to the modified N-terminus is obtained by gel electrophoresis. Analogously, the C-terminal deleted form (Example 26) is digested with the restriction enzymes EcoRI / KpnI and the 790 bp fragment corresponding to the modified N-terminus is obtained by gel electrophoresis. Fragments

The PL 191 355 B1 is ligated at 15 ° C, digested with EcoRI to eliminate EcoRI concatamers and cloned into the EcoRI site of dephosphorylated pCGN1761. The N / C-terminal deletion form of NIM1 is under transcriptional control of the double 35S promoter. Similarly, a hybrid form of NIM1 is prepared that contains mutagenized putative phosphorylation sites (example 24) attached to the C-terminal deletion (example 26). The constructs are sequenced to confirm the correct in-frame start and stop codons and to check that no other mutations have occurred. Appropriate transformation cassettes containing the 35S promoter, NIM1 hybrids and tml terminator are released from plasmid pCGN1761 by partial digestion with XbaI restriction enzyme and ligated into the XbaI site of dephosphorylated pCIB200. SEQ ID NOS: 13 and 14 show the DNA coding sequence and the encoded amino acid sequence, respectively, of an altered form of the NIM1 gene having an N-terminal and C-terminal amino acid deletion.

Example 28: Generation of altered forms of NIM1 - ankyrin domains

NIM1 shows homology to the ankyrin motifs around amino acids 103-362. Using the method of Ho et al. (1989), the DNA sequence encoding putative ankyrin domains (SEQ ID NO: 1: 3093-3951) is amplified by PCR (conditions: 94 ° C 3 min: 35 x (94 ° C 30 sec. .: 62 ° C 30 sec: 72 ° C 2 min.): 72 ° C 10 min.) With NIM1 cDNA (SEQ ID NO: 6: 349-1128), using the following primers 5'-ggaattcaATGGACTCCAACAACACCGCCGC-3 ' (SEQ ID NO: 31) and 5'-ggaattcTCAACCTTCCAA-AGTTGCTTCTGATG-3 '(SEQ ID NO: 32). The resulting product is digested with the restriction enzyme EcoRI and inserted into the EcoRI site of dephosphorylated pCGN1761, under the transcriptional control of the double 35S promoter. The construct is sequenced to confirm the presence of the synthetic start codon (ATG), the stop codon (TGA) in the frame and to verify that no other mutations have occurred during the PCR reaction. A transformation cassette containing the 35S promoter, ankyrin domains and the tml terminator is released from plasmid pCGN1761 by partial digestion with the restriction enzyme XbaI and is ligated into the XbaI site of dephosphorylated pCIB200. SEQ ID NOS: 15 and 16 show the coding DNA sequence and encoded amino acid sequence of the NIM1 ankyrin domain, respectively.

Example 29: Construction of hybrid genes

To increase the likelihood of appropriate spatial and temporal expression of altered forms of NIM1, a HindIII / BamHI fragment of 4407 bp (SEQ ID NO: 1: bases 1249-5655) and / or an EcoRV / BamHI fragment of 5655 bp (SEQ ID NO: 1) : bases 1-5655) containing the promoter and the NIM1 gene are used to generate the altered forms of NIM1 of Examples 24-28 above. Although the construction steps may vary, the concept is comparable to the examples previously described herein. Strong overproduction of altered characters can be potentially lethal. Thus, the altered forms of the NIM1 gene described in Examples 24-28 are placed under the control of promoters other than the endogenous NIM1 promoter, including, but not limited to, the nos promoter or the Rubisco small subunit promoter. Analogously, altered forms of the NIM1 gene are expressed under the control of the pathogen-responsive PR-1 promoter (US Patent No. 5,614,395). Such expression allows for strong expression of altered forms of the NIM1 gene only during pathogen attack or other SAR induction conditions. In addition, disease resistance may be seen in transformants expressing altered forms of the NIM1 gene under the control of the PR-1 promoter when exposed to concentrations of SAR activating compounds (i.e., BTH or INA) that do not normally activate SAR, thereby activating feedback. (Weymann et al. (1995) Plant Cell 7: 2013.

Example 30: Transformation of altered forms of the NIM1 gene into Arabidopsis thaliana

The produced constructs (examples 24-29) were introduced into Agrobacterium tumefaciens by electroporation into the GV3101 strain. These constructs are used to transform Arabidopsis Col-0 and Ws-0 ecotypes by vacuum infiltration (Mindrinos et al., Cell 78, 1089-1099 (1994)) or standard root transformation. Seeds from these plants are harvested and allowed to germinate on agar plates with kanamycin (or other suitable antibiotic) as selection agent. Only seedlings that are transformed can neutralize the selection factor and survive. Seedlings that survive selection are transferred to soil and tested for the CIM (constitutive resistance) phenotype. Plants are assessed for any observable differences from wild-type plants.

Example 31: Testing the CIM phenotype in plants transformed with the NIM1 gene or with altered forms of the NIM1 gene

A leaf is harvested from each primary transformant, RNA is isolated (Verwoerd et al., 1989, Nuc Acid Res, 2362), and tested for constitutive expression of PR-1 by analyzing RNA filters.

PL 191 355 B1 (Uknes et al., 1992). A conidia suspension of 5-10 x 10 ⁴ spores / ml is prepared from two corresponding isolates of P. parasitica, Emwa and Noco (i.e. these fungal strains cause wild-type disease of Ws-0 and Col-0, respectively) and the transformants are sprayed an appropriate isolate, depending on the ecotype of the transformant. The inoculated plants are incubated under high humidity conditions for 7 days. Plants are checked for disease on day 7 and single leaves are collected for analysis of RNA filters using a probe that allows measurement of fungal infection.

Transformants that exhibit a CIM phenotype are taken to generate the T1 generation and homozygous plants are identified. Transformants are subjected to the battery of disease resistance tests described below. The Noco and Emwa fungal infection is repeated and the leaves stained with lactophenol blue to identify the presence of mycelial hyphae as described in Dietrich et al. (1994). Transformants are infected with the bacterial pathogen Pseudomonas syringae DC3000 to establish the spectrum of apparent resistance as previously described by Uknes et al. (1993). Both free and glucose-bound salicylic acid are measured in uninfected plants and leaves are stained with lactophenol blue to test for microscopic cavities. Resistant plants are sexually bred with SAR mutants such as NahG (US Patent No. 5,614,395) and ndr1 to determine how these dominant negative NIM1 mutations may affect SA-dependent feedback.

Example 32: Isolation of NIM1 homologs

NIM1 homologs can be obtained that hybridize under moderately stringent conditions either to the entire Arabidopsis NIM1 gene or, more preferably, to an Arabidopsis-derived oligonucleotide probe that contains a contiguous portion of its coding sequence of at least 10 nucleotides in length. The factors that influence the stability of the hybrids determine the stringency of hybridization. One of these factors is the melting point 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, Part One: Molecular Hybridization Technology; page 8 ff. The preferred hybridization temperature is about 25 ° C below the calculated melting point Tm, preferably in the range 12-15 ° C below the calculated melting point Tm, and for oligonucleotides, about 5-10 ° C below the calculated melting point Tm.

Using the NIM1 cDNA (SEQ ID NO: 6) as a probe, NIM1 Arabidopsis homologs are identified by screening genomic or cDNA libraries from various crops, such as, but not limited to, those listed below in Example 33. Standard techniques used for this purpose include screening seeded DNA libraries (plaques or colonies, see, e.g., Sambrook et al., Molecular Cloning, Ed., Cold Spring Harbor Laboratory Press. (1989)) PCR amplification using oligonucleotide primers (see, e.g., Innis et al., PCR Protocols, a Guide to Methods and Applications eds., Academic Press (1990)). The identified homologues are inserted by genetic engineering into the expression vectors discussed herein and retransformed into the crop plants listed below. Transformants are assessed for increased disease resistance using appropriate pathogens on the test crops.

NIM1 homologues from the cucumber, tomato, tobacco, maize and barley genomes were detected by analyzing the DNA filters. Genomic DNA was isolated from cucumber, tomato, tobacco, corn and barley, digested with BamHI, HindIII, Xbal or SalI restriction enzymes, electrophoretically separated on 0.8% agarose gels, and capillary transferred to nylon filters. After binding the DNA to the support by UV, the filter was hybridized under low stringency conditions [(1% BSA; 520 mM NaPO4) pH 7.2; 7% lauric sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride) at 55 ° C for 18-24 h.] With the radiolabeled ³² P NIM1 cDNA from Arabidopsis thaliana. After hybridization, the filters were washed under low stringency conditions [6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55 ° C; 1XSSC is 0.15 M NaCl, 15 mM Na-citrate (pH 7.0)] and exposed to X-ray film to visualize bands that correspond to NIM1.

In addition, identified expressed sequence tags (ESTs) showing similarity to the NIM1 gene can be used to isolate homologues. For example, several expressed sequence tags (ESTs) from rice showing similarity to the NIM1 gene have been identified. Multiple sequence alignments were 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 software * (1228 South Park Street, Madison Wisconsin, 53715) Lasergene Biocomputing Software for Macintosh (1994). Certain regions of the NIM1 protein share amino acid sequence homology to 4 different cDNA protein products

PL 191 355 B1 from rice. Homology was identified using the NIM1 sequence in GenBank searches. A comparison of the regions of NIM1 homology and the rice cDNA products are shown in Figure 2 (See also, SEQ ID NO: 2 and SEQ ID NO: 17-24). The NIM1 protein fragments share 36 to 48% amino acid sequence identity with the 4 rice products. These rice ESTs may be particularly useful for the isolation of NIM1 homologs from other monocots.

Homologs can also be obtained by PCR. In this method, comparisons are made between known homologues (e.g. from rice and Arabidopsis). Regions of high amino acid and DNA similarity or identity are then used to make PCR primers. Preferably, regions rich in M and W residues are preferably followed by regions rich in F, Y, C, H, Q, K, and E residues, as these amino acids are encoded by a limited number of codons. Once the appropriate region has been identified, primers for that region are made with different substitutions at the third position of the codon. The various substitutions at the third position can be narrowed down depending on the target species. For example, because maize is rich in GC, primers are designed that use G or C in the third position if possible. The PCR reaction is performed with cDNA or genomic DNA under a variety of standard conditions. If a band is visible, it is cloned and / or sequenced to determine if it is a NIM1 homologue.

Example 33: Expression of the NIM1 form in crops

Constructs that conferred the CIM phenotype in Col-0 or Ws-0 are transformed into crops for testing. Alternatively, altered native NIM1 genes, isolated from the crops in the previous example, are inserted back into the corresponding crops. Although the NIM1 gene can be inserted into any plant cell belonging to these extensive classes, it is particularly useful in cells of crops such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, beans, peas, etc. 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, blackcurrant, pineapple, avocado, papaya, mango, banana, soybeans, tobacco, tomato, sorghum and sugarcane. Transformants are assessed for increased disease resistance. In a preferred embodiment, the expression of the NIM1 gene is at a level that is at least twice the expression level of the native NIM1 gene in wild-type plants, and preferably ten times the expression level of the wild-type.

Example 34: Synergistic Disease Resistance by Using Conventional Fungicide Against Transgenic Plants Overproducing NIM1

The plant lines used in this example (6E and 7C) were generated by transforming Arabidopsis thaliana wild-type (Ws ecotype) with the BamHI-HindIII genomic NIM1 fragment (SEQ ID NO: 1 - bases 1249-5655) as described above in Example 21. Fungicides metalaxyl, fosetyl and copper hydroxide as active ingredients in a composition with a wettable carrier powder of 25%, 80% and 70%, respectively, were applied as a fine mist to the leaves of three-week-old transgenic Ws plants constitutively expressing the NIM1 gene. The wettable powder itself was used as control. Three days later, the plants were inoculated with a conidia suspension of Emwa Peronospora parasitica isolate (1-2 x 10 ⁵ spores / ml) as described by Delaney et al. (1995). After inoculation, the plants were covered to maintain humidity and placed in a growth room at 17 ° C for a 14 hour cycle. day / 10 hrs night (Uknes et al., 1993). Tissues were harvested 8 days after inoculation.

The development of the fungus was followed for 12 days by observation under a preparative microscope for the development of conidiophores (Delaney et al. (1995), Dietrich et al. (1994)). Lactophenolrypan blue staining of individual leaves was performed in order to observe the growth of the fungus inside the leaf tissue. Fungal growth was quantified using the fungal rRNA probe obtained by PCR according to White et al. (1990; PCR Protokols: A guide to Methods and Application, 315-322) using NS1 and NS2 primers and P. parasitica Emwa DNA as templates. RNA was purified from frozen tissue by phenol / chloroform extraction followed by lithium chloride precipitation (Lagrimini et al. 1987: PNAS, 84: 7542-7546). Samples (7.5 µg) were electrophoretically separated on formaldehyde agarose gels and transferred to nylon filters (Hybond-N +, Amersham) as described by Ausubel et al. (1987). Hybridizations and washes were according to Church and Gilbert (1984, PNAS, 81: 1991-1995). Relative amounts of transcript were determined using a Phosphor Imager (Molecular Dynamics, Sunnyvale, CA) according to the manufacturer's instructions. The loading of samples was normalized by re-hybridizing the filters after removal

From labeled DNA, from the constitutively expressed Arabidopsis b-tubulin cDNA. Infection of untreated plants corresponds to 0% inhibition of fungal growth.

Application of metalaxyl, fosetyl, or copper hydroxide to NIM1 overproducing plant lines had more than additive, ie synergistic, effects on disease resistance. This effect was defined as the synergy factor (SF), which is the ratio of the effect observed (O) to the effect expected (E). The following results were obtained:

Table 36

Action against Peronospora parasitica in Arabidopsis component l: overproduction of NIM1 (line 6E) component II: metalaxyl

Test No. Ingredients % growth inhibition of the fungus synergy factor O / E NIM1 metalaxyl O (watched) E (expected) control Tue - 0 1 NIM1 - 10 2 Tue 0.0125 g / l 59 3 Tue 0.0012 g / l 27 4 NIM1 0.0125 g / l 76 69 1.1 5 NIM1 0.0012 g / l 56 37 1.5

wt = wild type WS

Table 37

Action against Peronospora parasitica in Arabidopsis component l: overproduction of NIM1 (line 6E) component II: fosetyl

Test No. Ingredients % growth inhibition of the fungus synergy factor O / E NIM1 fosetyl O (watched) E (expected) control Tue - 0 1 NIM1 - 10 2 Tue 5.0 g / l 7 3 Tue 0.5 g / l 2 4 Tue 0.05 g / l 0 5 NIM1 5.0 g / l 93 17 5.5 6 NIM1 0.5 g / l 83 12 6.9 7 NIM1 0.05 g / l 42 10 4.2

wt = wild type WS

PL 191 355 B1

Table 38

Action against Peronospora parasitica in Arabidopsis component l: overproduction of NIM1 (line 7C) component II: fosetyl

Test No. Ingredients % growth inhibition of the fungus synergy factor O / E NIM1 fosetyl O (watched) E (expected) control Tue - 0 1 NIM1 - 14 2 Tue 5.0 g / l 7 3 Tue 0.5 g / l 2 4 NIM1 5.0 g / l 80 21 3.8 5 NIM1 0.5 g / l 56 16 3.5

wt = wild type WS

Table a 39

Action against Peronospora parasitica in Arabidopsis component l: overproduction of NIM1 (line 6E) component II: copper hydroxide

Test No. Ingredients % growth inhibition of the fungus synergy factor O / E NIM1 Cu (OH) 2 O (watched) E (expected) control Tue - 0 1 NIM1 - 10 2 Tue 2.0 g / l 0 3 Tue 0.2 g / l 0 4 Tue 0.02 g / l 0 5 NIM1 2.0 g / l 66 10 6.6 6 NIM1 0.2 g / l 14 10 1.4 7 NIM1 0.02 g / l twenty 10 2.0

wt = wild type WS

Table a 40

Action against Peronospora parasitica in Arabidopsis component l: overproduction of NIM1 (line 6E) component II: copper hydroxide

Test No. Ingredients % growth inhibition of the fungus synergy factor O / E NIM1 Cu (OH) 2 O (watched) E (expected) control Tue - 0 1 NIM1 - 14 2 Tue 2.0 g / l 0 3 Tue 0.2 g / l 0 4 Tue 0.02 g / l 0 5 NIM1 2.0 g / l 77 14 5.5 6 NIM1 0.2 g / l 51 14 3.6 7 NIM1 0.02 g / l 55 14 3.9

wt = wild type WS

PL 191 355 B1

As can be seen from the tables above, a synergistic effect on disease resistance is shown in NIM1 overproducing plants by the use of metalaxyl, fosetyl or copper hydroxide. For example, an untreated NIM1 plant (line 6E) shows 10% inhibition of fungal growth relative to an untreated wild-type plant; this shows that constitutive expression of the SAR gene in this NIM1 overproducer correlates with disease resistance. However, as shown above in Table 37, when using fosetyl at a dose of 5.0 g / L (concentration typically insufficient for efficacy) to an immunomodulated NIM1 overproducing plant (SAR on), the observed level of fungal growth inhibition increased to 93%. A synergism factor of 5.5 calculated from these data clearly shows the synergistic effect obtained by applying a fungicidal agent to an immunomodulated plant (SAR-on). In another example, an untreated NIM1 plant (line 7C) showed a 14% inhibition of fungal growth relative to the untreated wild-type plant, showing that constitutive expression of the SAR gene in this NIM1 overproducer correlates with disease resistance. However, as shown above in Table 40, when using copper hydroxide at a concentration of 2.0 g / L (a concentration typically insufficient for efficacy) against an immunomodulated NIM1 overproducing plant (SAR-on), the observed level of fungal growth inhibition was increased to 77%. A synergism factor of 5.5 calculated from these data clearly shows the synergistic effect obtained by using a fungicide applied to an immunomodulated plant (SAR-on).

Thus, the use of a combination of an immunomodulated NIM1 overproducing plant with low, normally ineffective concentrations of fungicide to achieve disease resistance provides benefits that will be apparent to those skilled in the art of agriculture. The use of a normally toxic or otherwise undesirable concentration of fungicide can be avoided by taking advantage of the synergy demonstrated here. Moreover, savings can be achieved as a result of the reduced amount of fungicides required to achieve a given level of plant protection.

Example 35: Synergistic disease resistance obtained by using a chemical SAR inducer against transgenic plants overproducing NIM1

Transgenic plants containing the NIM1 genomic DNA fragment under their own promoter (Example 21) were also analyzed for response to different BTH concentrations relative to the wild-type lineage. Seeds from each line were sown and grown as previously described. Approximately three weeks after planting, leaf samples were collected from each line (day 0 control) and the remaining leaves were treated with H ₂ O, 10 µM BTH or 100 µM BTH. Additional samples were collected on days 1, 3 and 5 after treatment. After sampling on day 3, a group of plants from each line was picked and treated with an Emwa Peronospora parasitica isolate as described above. RNA was obtained from the harvested tissue and Northern analysis was performed using the Arabidopsis PR-1 gene probe. Plants were tested for fungal resistance 8 days after infection.

The results of the Northern analysis for Ws and the four NIM1 overproducing lines (3A, 5B, 6E and 7C) are shown in Fig. 3. The expression of the PR-1 gene in the wild-type Ws line is barely detectable after exposure to a low dose of 10 μl BHT (BHT concentration). 100-300 μM is normally required for efficacy). Ws plants from this action are still susceptible to the fungal pathogen P. parasitica (Emwa). However, in all lines overproducing NIM1, the response to PR-1 gene expression was much stronger after low-level BHT treatment. Moreover, all NIM-1 overproducing lines treated with 10 µl BTH showed almost complete resistance to P. parasitica. Leaves stained with lactophenol blue to identify the presence of hyphae ((Dietrich et al. (1994) confirmed no fungal growth in lines overproducing NIM1 relative to the wild type. Thus, immunomodulated plants have the ability to respond more quickly and to much lower doses of BTH, such as shown by expression of the PR-1 gene and resistance to P. parasitica than wild-type plants These data show that synergistic disease resistance can be obtained by applying a systemic acquired resistance chemical inducer, such as BTH, to an immunomodulated plant (SAR-on) , such as a plant that overproduces NIM1.

Thus, the use of a combination of an immunomodulated NIM1 overproducing plant with low, normally ineffective concentrations of SAR-inducing chemicals such as BTH to achieve disease resistance provides benefits that will be apparent to those skilled in the art of agriculture. The use of normally toxic or otherwise undesirable concentrations of SAR-inducing chemicals can be avoided by using the demonstrated one

This is a combination of synergism. In addition, savings can be achieved as a result of the reduced amount of SAR-inducing chemicals required to achieve a given level of plant protection.

Sequence list (1) GENERAL INFORMATION:

(i) APPLICANTS:

(A) NAME: Novartis AG (B) STREET: Schwarzwaldallee 215 (C) CITY: Basel (E) COUNTRY: Switzerland (F) ZIP CODE (ZIP): 4002 (G) PHONE: +41 61 69 11 11 (H) TELEFAX: +41 61 696 79 76 (v) COMPUTER WRITING:

(A) ENVIRONMENT: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patent Release # 1.0, Version # 1.30 (ii) TITLE OF THE INVENTION: Plant protection method (iii) NUMBER OF SEQUENCES: 32 (2) INFORMATION FOR SEQ. ID NO: 1:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 5,655 base pairs (B) TYPE: Nucleic acid (C) STRAND: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTISENSIVE: NO ( ix) FEATURE:

(A) NAME / KEY: exon (B) ITEM: 2787..3347 (D) OTHER INFORMATION: / product = "1st exon NIM1

PL 191 355 B1 (ix> FEATURE:

(A) NAME / KEY: exon (B) ITEM: 3427..4162 (D) OTHER INFORMATION: / product = "2nd exon NIM1" (ix) FEATURE:

(A) NAME / KEY: exon (B) ITEM: 4271..4474 (D) OTHER INFORMATION: / product = "3rd exon NIM1" (ix) FEATURE:

(A) NAME / KEY: exon (B) ITEM: 4586..4866 (D) OTHER INFORMATION: / product = "4th exon NIM1" (ix) FEATURE:

(A) NAME / KEY: CDS (B) POSITION: connects (2787, .3347, 3427..4162, 4271..4474, 4586..4866) (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 1:

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

PL 191 355 B1

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 TATATAAAGT ACTCTGTTTA 1680

PL 191 355 B1

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

GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC GTC GCT ACC GAT AAC ACC Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val Ala Thr Asp Asn Thr

2861

PL 191 355 B1

GAC TCC TCT Cheese ATT GTT TAT Tyr CTG GCC GCC GAA CAA GTA CTC ACC GGA CCT 2909 Asp Cheese How much Val thirty Leu Ala Ala Glu 35 Gin Val Leu Thr Gly 40 Pro GAT GTA TCT GCT CTG CAA TTG CTC TCC AAC AGC TTC GAA TCC GTC TTT 2957 Asp Val Cheese Ala Leu Gin Leu Leu Cheese Asn Cheese Phe Glu Cheese Val Phe 45 50 55 GAC TCG CCG GAT GAT TTC TAC AGC GAC GCT AAG CTT GTT CTC TCC GAC 3005 Asp Cheese Pro Asp Asp Phe Tyr Cheese Asp Ala Lys Leu Val Leu Cheese Asp SO 65 70 GGC CGG GAA GTT TCT TTC CAC CGG TGC GTT TTG TCA GCG AGA AGC TCT 3053 Gly Arg Glu Val Cheese Phe His Arg Cys Val Leu Cheese Ala Arg Cheese Cheese 75 80 85 TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAG GAG AAA GAC TCC AAC 3101 Phe Phe Lys Cheese Ala Leu Ala Ala Ala Lys Lys Glu Lys Asp Cheese 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 How much 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 Cheese Val Val Thr Val Leu Ala Tyr Val Tyr Cheese 125 130 135 AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT GAA TGC GCA GAC GAG 3245 Cheese Arg Val Arg Pro Pro Pro Lys Gly Val Cheese 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 Underworld 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 How much Phe Lys How much Pro Glu Leu How much Thr Leu 170 175 180 185

TAT CAG GTAAAACACC ATCTGCATTA AGCTATGGTT ACACATTCAT GAATATGTTC 3397

Tyr Gin

PL 191 355 B1

TTACTTGAGT ACTTGTATTT GTATTTCAG AGG CAC TTA TTG GAC GTT GTA GAC 3450 Arg His Leu 190 Leu Asp Val Val Asp 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 200 205 Lys Leu Ala Asn 210 How much 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 215 220 Cys Lys Glu Ile 225 How much GTC AAG TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA 3594 val Lys Asn Val Asp Met Val Cheese Leu Glu Cheese 230 235 Lys Leu Pro cheese 240 Glu GAG CTT GTT AAA GAG ATA ATT GAT AGA CGT AAA GAG CTT GGT TTG GAG 3642 Glu Leu 245 Val Lys Glu Ile Ile Asp Arg Arg Lys 250 Glu 255 Leu Gly Leu Glu GTA CCT AAA GTA AAG AAA CAT GTC TCG AAT GTA CAT AAG GCA CTT GAC 3690 Val Pro 260 Lys Val Lys Lys His Val Ser Asn Val 265 270 His Lys Ala Leu ASp 275 TCG GAT GAT ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG GAT CAC ACC 3738 Asp cheese Asp Ile Glu Leu Val Lys Leu Leu Leu 280 285 Lys Glu Asp His 290 Thr 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 295 300 Val Ala Tyr Cys 305 Asn GTG AAG ACC GCA ACA GAT CTT TTA AAA CTT GAT CTT GCC GAT GTC AAC 3034 Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp 310 315 Leu Ala Asp Val 320 Asn CAT AGG AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG 3882 His Arg 325 Asn Pro Arg Gly Tyr Thr Val Leu His 330 Val 335 Ala Ala Met Arg AAG GAG CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA 3930

PL 191 355 B1

Lys 340 Glu Pro Gin Leu How much 345 Leu Cheese Leu Leu Glu 350 Lys Gly Ala Cheese Ala 355 TCA GAA GCA ACT TTG GAA GGT AGA ACC GCA CTC ATG ATC GCA AAA CAA 3978 Cheese Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Underworld How much Ala Lys Gin 360 365 370 GCC ACT ATG GCG GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT 4026 Ala Thr Underworld Ala Val Glu Cys Asn Asn How much Pro Glu Gin Cys Lys His 375 380 385 TCT CTC AAA GGC CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA 4074 Cheese Leu Lys Gly Arg Leu Cys Val Glu How much Leu Glu Gin Glu Asp Lys 390 395 400 CGA GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TTT GCA GTG GCG GCC 4122 Arg Glu Gin How much Pro Arg Asp Val Pro Pro Cheese 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 Underworld Thr Leu Leu Asp Leu Glu Asn Arg 420 425 430 GTATCTATCA AGTCTTATTT CTTATATGTT TGAATTAAAT TTATGTCCTC TCTA ' TTAGGA 4222 AACTGAGTGA ACTAATGATA ACTATTCTTT GTGTCGTCCA CTGTTTAG TT GCA CTT 4278

Val Ala Leu

435 GCT CAA CGT CTT TTT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC 4326 Ala Gin Arg Leu Phe Pro Thr Glu Ala Gin Ala Ala Underworld Glu How much Ala 440 445 450 GAA ATG AAG GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC 4374 Glu Underworld Lys Gly Thr Cys Glu Phe How much Val Thr Cheese 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 Cheese Pro Gly Val Lys How much Ala Pro 470 475 480 TTC AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA 4470 Phe Arg how much Leu Glu Glu His Gin Cheese Arg Leu Lys Ala Leu Cheese Lys

485 490 495

PL 191 355 B1

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 Asp CAG ATT ATG AAC TGT Cys GAG Glu GAC Asp TTG Leu ACT CAA CTG GCT TGC Cys GGA Gly 530 GAA Glu 4677 Gin How many Met Asn 520 Thr 525 Gin Leu Ala GAC GAC ACT GCT GAG AAA CGA CTA CAA AAG AAG CAA AGG TAC ATG GAA 4725 Asp Asp Thr Ala Glu Lys Arg Leu Gin Lys Lys Gin Arg Tyr Underworld Glu 535 540 545 ATA CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GAA TTA 4773 How much Gin Glu Thr Leu Lys Lys Ala Phe Cheese Glu Asp Asn Leu Glu Leu 550 555 560

GGA Gly AAT TCG TCC Cheese CTG Leu ACA GAT TCG Cheese ACT TCT TCC ACA TCG Cheese AAA Lys TCA Cheese ACC Thr 4821 Asn 565 Cheese Thr Asp 570 Thr Cheese Thr Cheese 575 GGT GGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT CGT CGG TGA 4866 Gly Gly Lys Arg Cheese Asn Arg Lys Leu Cheese 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

PL 191 355 B1

ttcttccttt AACCTTTTGT AACTCGAATT ACACAGCAAG TTAGTTTCAG GTCTAGAGAT 5286 AAGAGAACAC TGAGTGGGCG TGTAAGGTGC ATTCTCCTAG TCAGCTCCAT TGCATCCAAC 5346 ATTTGTGAAT GACACAAGTT AACAATCCTT TGCACCATTT CTGGGTGCAT ACATGGAAAC 5406 TTCTTCGATT GAAACTTCCC ACATGTGCAG GTGGGTTCGC 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: 2:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 594 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 2

Underworld 1 Asp Thr Thr How much 5 Asp Gly Phe Ala Asp 10 Cheese Tyr Glu How much Cheese 15 Cheese Thr Cheese Phe Val twenty Ala Thr Asp Asn Thr 25 Asp Cheese Cheese How much Val thirty Tyr Leu Ala Ala Glu 35 Gin Val Leu Thr Gly 40 Pro Asp Val Cheese Ala 45 Leu Gin Leu Leu Cheese 50 Asn Cheese Phe Glu Cheese 55 Val Phe Asp Cheese Pro 60 Asp Asp Phe Tyr Cheese Asp Ala Lys Leu Val Leu Cheese Asp Gly Arg Glu Val Cheese Phe His

PL 191 355 B1

Arg Cys Val Leu Ser Ala Arg Ser 85

Ala Ala Lys Lys Glu Lys Asp Ser 100

Glu Leu Lys Glu Ile Ala Lys Asp 115 120

Val Thr Val Leu Ala Tyr Val Tyr 130 135

Lys Gly Val Ser Glu Cys Ala Asp 145 150

Arg Pro Ala Val Asp Phe Met Leu 165

Phe Lys Ile Pro Glu Leu Ile Thr 180

Val Val Asp Lys Val Val Ile Glu 195 200

Ala Asn Ile Cys Gly Lys Ala Cys 210 215

Glu Ile Ile Val Lys Cheese Asn Val 225 230

Leu Pro Glu Glu Leu Val Lys Glu 245

Gly Leu Glu Val Pro Lys Val Lys 260

Ala Leu Asp Ser Asp Asp Ile Glu 275 280

Asp His Thr Asn Leu Asp Asp Ala 290 295

Phe Phe Lys cheese Ala Leu Ala cheese 90 95

Asn Asn Thr Ala Ala Val Lys Leu 105 110

Tyr Glu Val Gly Phe Asp Ser Val 125

Ser Ser Arg Val Arg Pro Pro Pro 140

Glu Asn Cys Cys His Val Ala Cys 155 160

Glu Val Leu Tyr Leu Ala Phe Ile 170 175

Leu Tyr Gin Arg His Leu Leu Asp 185 190

Asp Thr Leu Val Ile Leu Lys Leu 205

Met Lys Leu Leu Asp Arg Cys Lys 220

Asp Met Val Ser Leu Glu Lys Ser 235 240

How many How many Asp Arg Arg Lys Glu Leu 250 255

Lys His Val Ser Asn Val His Lys 265 270

Leu Val Lys Leu Leu Leu Lys Glu 285

Cys Ala Leu His Phe Ala Val Ala 300

PL 191 355 B1

Tyr Cys Asn Val 305 Lys Thr 310 Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala 315 320 Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala 325 330 335 Ala Underworld Arg Lys Glu Pro Gin Leu How much Leu Cheese Leu Leu Glu Lys Gly 340 345 350 Ala Cheese Ala Cheese Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Underworld How much 355 360 365 Ala Lys Gin Ala Thr Underworld Ala Val Glu Cys Asn Asn How much Pro Glu Gin 370 375 380 Cys Lys His Cheese Leu Lys dy Arg Leu Cys Val Glu How much Leu Glu Gin 385 390 395 400 Glu Asp Lys Arg Glu Gin How much Pro Arg Asp Val Pro Pro Cheese Phe Ala 405 410 415 Val Ala Ala Asp Glu Leu Lys Underworld Thr Leu Leu Asp Leu Glu Asn Arg 420 425 430 Val Ala Leu Ala Gin Arg Leu Phe Pro Thr Glu Ala Gin Ala Ala Underworld 435 440 445 Glu How much Ala Glu Underworld Lys Gly Thr Cys Glu Phe How much Val Thr Cheese Leu 450 455 460 Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Cheese Pro Gly Val Lys 465 470 475 480 How much Ala Pro Phe Arg How much Leu Glu Glu His Gin Cheese Arg Leu Lys Ala 485 490 495 Leu Cheese Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Cheese 500 505 510 Ala Val Leu Asp Gin How much Underworld Asn Cys Glu Asp Leu Thr Gin Leu Ala 515 520 525 Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gin Lys Lys Gin Arg 530 535 540

PL 191 355 B1

Tyr 545 Underworld Glu How much Gin Glu 550 Thr Leu Lys Lys Ala 555 Phe Cheese Glu Asp Asn 560 Leu Glu Leu Gly Asn 565 Cheese Cheese Leu Thr Asp 570 Cheese Thr Cheese Cheese Thr 575 Cheese Lys Cheese Thr Gly 560 Gly Lys Arg Cheese Asn 585 Arg Lys Leu Cheese His 590 Arg Arg

Arg * (2) INFORMATION FOR SEQ. ID NO: 3:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 314 amino acids (B) TYPE: amino acid (C) STRAND: unbound (D) TOPOLOGY: unbound (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 3

Underworld 1 Phe Gin Pro Ala 5 Gly His Gly Gin Asp 10 Trp Ala Underworld Glu Gly 15 Pro Arg Asp Gly Leu twenty Lys Lys Glu Arg Leu 2S Val Asp Asp Arg His thirty Asp Cheese Gly Leu Asp 35 Cheese Underworld Lys Asp Glu 40 Glu Tyr Glu Gin Underworld 45 Val Lys Glu Leu Arg 50 Glu How much Arg Leu Gin 55 Pro Gin Glu Ala Pro 60 Leu Ala Ala Glu Pro Trp Lys Gin Gin Leu Thr Glu Asp Gly Asp Cheese Phe Leu His Leu

70 75 80

PL 191 355 B1

PL 191 355 B1

Cys Val Phe Gly Gly Gin Arg Leu Thr Leu 305 310 (2) INFORMATION FOR SEQ. ID NO: 4:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 314 amino acids (B) TYPE: amino acid (C) STRAND: unbound (D) TOPOLOGY: unbound (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 4

Underworld 1 Phe Gin Pro Ala 5 Gly His Gly Gin Asp Trp Ala Met Glu Gly Pro 10 15 Arg Asp Gly Leu Lys Lys Glu Arg Leu Val Asp Asp Arg His Asp Cheese twenty 25 thirty Gly Leu Asp Cheese Underworld Lys Asp Glu Asp Tyr Glu Gin Underworld Val Lys Glu 35 40 45 Leu Arg Glu How much Arg Leu Gin Pro Gin Glu Ala Pro Leu Ala Ala Glu 50 55 60 Pro Trp Lys Gin Gin Leu Thr Glu Asp Gly Asp Cheese Phe Leu His Leu 65 70 75 80 Ala How much How much His Glu Glu Lys Thr Leu Thr Underworld Glu Val How much Gly Gin 85 90 95 Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gin Asn Asn Leu Gin Gin 100 105 110 Thr Pro Leu His Leu Ala Val How much Thr Asn Gin Pro Gly How much Ala Glu 115 120 125

PL 191 355 B1

Ala Leu 130 Leu Lys Ala Gly Cys Asp 135 Pro Glu Leu Arg 140 Asp Phe Arg Gly Asn Thr Pro Leu His Leu Ala Cys Glu Gin Gly Cys Leu Ala Cheese Val 145 150 155 160 Ala Val Leu Thr Gin Thr Cys Thr Pro Gin His Leu His Cheese Val Leu 165 170 175 Gin Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Cheese How much 180 185 190 His Gly Tyr Leu Gly How much val Glu His Leu Val Thr Leu Gly Ala Asp 195 200 205 Val Asn Ala Gin Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala 210 215 220 Val Asp Leu Gin Asn Pro Asp Leu Val Cheese Leu Leu Leu Lys Cys Gly 225 230 235 240 Ala Asp Val Asn Arg Val Thr Tyr Gin Gly Tyr Cheese Pro Tyr Gin Leu 245 250 255 Thr Trp Gly Arg Pro Cheese Thr Arg How much Gin Gin Gin Leu Gly Gin Leu 260 265 270 Thr Leu Glu Asn Leu Gin Thr Leu Pro Glu Cheese Glu Asp Glu Glu Cheese 275 280 285 Tyr Asp Thr Glu Cheese Glu Phe Thr Glu Asp Glu Leu Pro Tyr Asp Asp 290 295 300 Cys Val Phe Gly Gly Gin Arg Leu Thr Leu

305 310 (2) INFORMATION FOR SEQ. ID NO: 5:

(i) SEQUENCE CHARACTERISTICS (A) LENGTH: 314 amino acids (B) TYPE: amino acid

PL 191 355 B1 (C) STRAND: unbound (D) TOPOLOGY: unbound (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 5

Underworld 1 Phe Gin Pro Ala Glu 5 Pro Gly Gin Glu Trp Ala 10 Underworld Glu Gly 15 Pro Arg Asp Ala Leu Lys Lys Glu Arg Leu Leu Asp Asp Arg His Asp Cheese twenty 25 thirty Gly Leu Asp Cheese Underworld Lys Asp Glu Glu Tyr Glu Gin Underworld Val Lys Glu 35 40 45 Leu Arg Glu How much Arg Leu Glu Pro Gin Glu Ala Pro Arg Gly Ala Glu 50 55 60 Pro Trp Lys Gin Gin Leu Thr Glu Asp Gly Asp Cheese Phe Leu His Leu 65 70 75 80 Ala How much How much His Glu Glu Lys Ala Leu Thr Underworld Glu Val Val Arg Gin 85 90 95 Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gin Asn Asn Leu Gin Gin 100 105 110 Thr Pro Leu His Leu Ala Val How much Thr Asn Gin Pro Glu How much Ala Glu 115 120 125 Ala Leu Leu Glu Ala Gly Cys ASp Pro Glu Leu Arg Asp Phe Arg Gly 130 135 140 Asn Thr Pro Leu His Leu Ala Cys Glu Gin Gly Cys Leu Ala Cheese Val 145 150 155 160 Gly Val Leu Thr Gin Pro Arg Gly Thr Gin His Leu His Cheese How much Leu

165 170 175

PL 191 355 B1

Gin Ala Thr Asn 180 Tyr Asn Gly His Thr 185 Cys Leu His Leu Ala 190 Cheese How much His Gly Tzr Leu Gly How much Val Glu Leu Leu Val Cheese Leu Gly Ala Asp 195 200 205 Val Asn Ala Gin Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala 210 215 220 Val Asp Leu Gin Asn Pro Asp Leu Val Cheese Leu Leu Leu Lys Cys Gly 225 230 235 240 Ala Asp Val Asn Arg Val Thr Tyr Gin Gly Tyr Cheese Pro Tyr Gin Leu 245 250 255 Thr Trp Gly Arg Pro Cheese Thr Arg How much Gin Gin Gin Leu Gly Gin Leu 260 265 270 Thr Leu Glu Asn Leu Gin Underworld Leu Pro Glu Cheese Glu Asp Glu Glu Cheese 275 280 285 Tyr Asp Thr Glu Cheese Glu Phe Thr Glu Asp Glu Leu Pro Tyr Asp Asp 290 295 300 Cys Val Leu Gly Gly Gin Arg Leu Thr Leu

305 310 (2) INFORMATION FOR SEQ. ID NO: 6:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 2011 base pairs (B) TYPE: Nucleic acid (C) STRAND: Single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (vi) PRIMARY SOURCE:

(A) ORGANISM: Arabidopsis thaliana (ix) FEATURE:

(A) NAME / KEY: various features

PL 191 355 B1 (B) ITEM: 1..2011 (D) OTHER INFORMATION: / Note = "NIM1 cDNA sequence" (ix) FEATURE:

(A) NAME / KEY: CDS (B) ITEM: 43..1824 (D) OTHER INFORMATION: / product = "NIM1 protein (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 6:

GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC 54

Met Asp Thr Thr

ATT How much 5 GAT GGA TTC GCC Phe Ala GAT Asp 10 TCT TAT GAA ATC AGC AGC ACT Thr AGT Cheese TTC Phe GTC Val twenty 102 Asp Gly Cheese Tyr Glu How much Cheese 15 Cheese GCT ACC GAT AAC ACC GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA 150 Ala Thr Asp Asn Thr Asp Cheese Cheese How much Val Tyr Leu Ala Ala Glu Gin 25 thirty 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 Cheese Ala Leu Gin Leu Leu Cheese Asn Cheese 40 45 50 TTC GAA TCC GTC TTT GAC TCG CCG GAT GAT TTC TAC AGC GAC GCT AAG 246 Phe Glu Cheese Val Phe Asp Cheese Pro Asp Asp Phe Tyr Cheese 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 Cheese Asp Gly Arg Glu Val Cheese 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 Cheese Ala Arg Cheese Cheese Phe Phe Lys Cheese 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 Cheese Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu 105 110 115

PL 191 355 B1

ATT GCC AAG GAT TAC GAA GTC GGT TTC GAT TCG GTT GTG ACT GTT TTG 438 How much Ala Lys Asp 120 Tyr Glu Val Gly Phe 125 Asp Cheese Val Val Thr 130 Val Leu GCT TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT 486 Ala Tyr Val Tyr Cheese Cheese Arg Val Arg Pro Pro Pro Lys Gly Val Cheese 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 Underworld Leu Glu Val Leu Tyr Leu Ala Phe How much Phe Lys how much Pro 165 170 175 180 GAA TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA 630 Glu Leu How much Thr Leu Tyr Gin Arg His Leu Leu Asp Val val Asp Lys 185 190 195 GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT 678 Val Val How much Glu Asp Thr Leu Val How much Leu Lys Leu Ala Asn How much 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 Underworld Lys Leu Leu Asp Arg Cys Lys Glu How much How much Val 215 220 225 AAG TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA GAG 774 Lys Cheese Asn Val Asp Underworld Val Cheese Leu Glu Lys Cheese 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 how much how much 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 Cheese Asn Val His Lys Ala Leu Asp Cheese 265 270 275 GAT GAT ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG GAT CAC ACC AAT 918 Asp Asp How much Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn

280 285 290

PL 191 355 B1

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 Lys ACC Thr 310 GCA Ala ACA GAT Thr Asp CTT TTA AAA CTT GAT CTT GCC Ala 320 GAT Asp GTC AAC CAT His 1014 Leu Leu 315 Lys Leu Asp Leu Val Asn 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 Underworld Arg Lys 325 330 335 340 GAG CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA 1110 Glu Pro Gin Leu How much Leu Cheese Leu Leu Glu Lys Gly Ala Cheese Ala Cheese 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 Underworld How much Ala Lys Gin Ala 360 365 370 ACT ATG GCG GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT TCT 1206 Thr Underworld Ala Val Glu Cys Asn Asn How much Pro Glu Gin Cys Lys His Cheese 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 How much Leu Glu Gin Glu Asp Lys Arg 390 395 400 GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TTT GCA GTG GCG GCC GAT 1302 Glu Gin How much Pro Arg Asp Val Pro Pro Cheese 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 Underworld Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala 425 430 435 CAA CGT CTT TTT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC GAA 1398 Gin Arg Leu Phe Pro Thr Glu Ala Gin Ala Ala Underworld Glu How much Ala Glu 440 445 450 ATG AAG GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC CGT 1446 Underworld Lys Gly Thr Cys Glu Phe How much val Thr Cheese Leu Glu Pro Asp Arg

455 460 465

PL 191 355 B1

CTC Leu ACT Thr 470 GGT ACG AAG AGA Arg ACA Thr 475 TCA Cheese CCG GGT GTA AAG ATA GCA CCT Pro TTC Phe 1494 Gly Thr Lys Pro Gly Val Lys 480 How much Ala AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA ACC 1542 Arg How much Leu Glu Glu His Gin Cheese Arg Leu Lys Ala Leu Cheese 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 Cheese Ala Val Leu Asp 505 510 515 CAG ATT ATG AAC TGT GAG GAC TTG ACT CAA CTG GCT TGC GGA GAA GAC 1638 Gin How much Underworld Asn Cys Glu Asp Leu Thr Gin Leu Ala Cys Gly Glu Asp 520 525 530 GAC ACT GCT GAG AAA CGA CTA CAA AAG AAG CAA AGG TAC ATG GAA ATA 1686 Asp Thr Ala Glu Lys Arg Leu Gin Lys Lys Gin Arg Tyr Underworld Glu How much 535 540 545 CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GAA TTA GGA 1734 Gin Glu Thr Leu Lys Lys Ala Phe Cheese 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 Cheese Leu Thr Asp Cheese Thr Cheese Cheese Thr Cheese Lys Cheese Thr Gly 565 570 575 580 GGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT CGT CGG TGA 1824 Gly Lys Arg Cheese Asn Arg Lys Leu Cheese His Arg Arg Arg ★

585 590

GACTCTTGCC TCTTAGTGTA ATTTTTGCTG TACCATATAA TTCTGTTTTC ATGATGACTG 1884

TAACTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT

ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG

ATTTGTA

1944

2004

2011

(2) INFORMATION FOR SEQ. ID NO: 7:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 2011 base pairs (B) TYPE: Nucleic acid (C) STRAND: Single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) FEATURE:

(A) NAME / KEY: various features (B) ITEM: 43..1824 (D) OTHER INFORMATION: / product = "changed form of NIM1 / note =" serine residues at amino acid positions 55 and 59 in wild NIMI gene product have been changed for alanine residues (ix) FEATURE:

(A) NAME / KEY: various features (B) ITEM: 205..217 (D) OTHER INFORMATION: / note = "nucleotides 205 and 217 changed from T to G as compared to wild-type sequence" (xi) SEQUENCE DESCRIPTION: SEQUENCE . ID NO: 7:

GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC 54

Met Asp Thr Thr

ATT GAT GGA Gly TTC Phe GCC GAT TCT Cheese TAT GAA Tyr Glu ATC AGC AGC Cheese ACT AGT TTC Phe GTC Val twenty 102 How much 5 Asp Ala Asp 10 How much Cheese 15 Thr Cheese GCT ACC GAT AAC ACC GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA 150 Ala Thr Asp Asn Thr Asp Cheese Cheese how much Val Tyr Leu Ala Ala Glu Gin 25 thirty 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 Cheese Ala Leu Gin Leu Leu Cheese Asn Cheese 40 45 50

PL 191 355 B1

TTC Phe GAA Glu GCC GTC TTT GAC GCG CCG GAT GAT TTC TAC AGC GAC GCT AAG 246 Ala 55 Val Phe Asp Ala Pro Asp 50 Asp Phe Tyr Cheese 65 Asp Ala Lys CTT GTT CTC TCC GAC GGC CGG GAA GTT TCT TTC CAC CGG TGC GTT TTG 294 Leu Val Leu Cheese Asp Gly Arg Glu Val Cheese 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 Cheese Ala Arg Cheese Cheese Phe Phe Lys Cheese 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 Cheese 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 How much Ala Lys Asp Tyr Glu Val Gly Phe Asp Cheese 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 Cheese Cheese Arg Val Arg Pro Pro Pro Lys Gly val Cheese 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 Underworld Leu Glu Val Leu Tyr Leu Ala Phe How much Phe Lys How much Pro 165 170 175 180 GAA TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA 630 Glu Leu How much Thr Leu Tyr Gin Arg His Leu Leu Asp Val Val Asp Lys 185 190 195 GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT 678 Val Val How much Glu Asp Thr Leu Val How much Leu Lys Leu Ala Asn How much 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 Underworld Lys Leu Leu Asp Arg Cys Lys Glu How much How much Val

215 220 225

PL 191 355 B1

AAG TCT AAT GTA GAT ATG Underworld GTT AGT CTT GAA AAG TCA TTG CCG GAA GAG 774 Lys Asn cheese 230 Val Asp Val 235 Cheese Leu Glu Lys Cheese 240 Leu Pro Glu Glu CTT GTT AAA GAG ATA ATT GAT AGA CGT AAA GAG CTT GGT TTG GAG GTA 822 Leu Val Lys Glu How much How much 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 Cheese Asn Val His Lys Ala Leu Asp Cheese 265 270 275 GAT GAT ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG GAT CAC ACC AAT 918 Asp Asp How much 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 Underworld Arg Lys 325 330 335 340 GAG CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA 1110 Glu Pro Gin Leu How much Leu Cheese Leu Leu Glu Lys Gly Ala Cheese Ala Cheese 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 Underworld How much Ala Lys Gin Ala 360 365 370 ACT ATG GCG GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT TCT 1206 Thr Underworld Ala Val Glu Cys Asn Asn How much Pro Glu Gin Cys Lys His Cheese 375 380 385 CTC AAA GGC CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA CGA 1254

PL 191 355 B1

Leu Lys Gly Arg Leu Cys Val 395 Glu How much Leu Glu Gin 400 Glu Asp Lys Arg 390 GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TTT GCA GTG GCG GCC GAT 1302 Glu Gin How much Pro Arg Asp Val Pro Pro Cheese 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 Underworld Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala 425 430 435 CAA CGT CTT TTT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC GAA 1398 Gin Arg Leu Phe Pro Thr Glu Ala Gin Ala Ala Underworld Glu How much Ala Glu 440 445 450 ATG AAG GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC CGT 1446 Underworld Lys Gly Thr Cys Glu Phe How much Val Thr Cheese 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 Cheese Pro Gly Val Lys How much Ala Pro Phe 470 475 480 AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA ACC 1542 Arg How much Leu Glu Glu His Gin Cheese Arg Leu Lys Ala Leu Cheese 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 Cheese Ala Val Leu Asp 505 510 515 CAG ATT ATG AAC TGT GAG GAC TTG ACT CAA CTG GCT TGC GGA GAA GAC 1638 Gin How much Underworld Asn Cys Glu Asp Leu Thr Gin Leu Ala Cys Gly Glu Asp 520 525 530 GAC ACT GCT GAG AAA CGA CTA CAA AAG AAG CAA AGG TAC ATG GAA ATA 1686 Asp Thr Ala Glu Lys Arg Leu Gin Lys Lys Gin Arg Tyr Underworld Glu how much 535 540 545 CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GAA TTA GGA 1734 Gin Glu Thr Leu Lys Lys Ala Phe Cheese Glu Asp Asn Leu Glu Leu Gly

550 555 560

PL 191 355 B1

AAT Asn 565 TTG TCC CTG Leu ACA Thr GAT Asp 570 TCG Cheese ACT Thr TCT Cheese TCC Cheese ACA Thr 575 TCG AAA TCA ACC Thr GGT Gly 5Θ0 1782 Leu Cheese Cheese Lys Cheese GGA AAG AGG TCT AAC CGT AAA CTC tct CAT CGT CGT CGG TGA 1824 dy Lys Arg Cheese Asn Arg Lys Leu Cheese 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: 8:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 594 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 8

Underworld 1 Asp Thr Thr How much 5 Asp Gly Phe Ala Asp 10 Cheese Tyr Glu How much Cheese 15 Cheese Thr Cheese Phe Val twenty Ala Thr Asp Asn Thr 25 Asp Cheese Cheese How much Val thirty Tyr Leu Ala Ala Glu 35 Gin Val Leu Thr Gly 40 Pro Asp Val Cheese Ala 45 Leu Gin Leu Leu Cheese 50 Asn Cheese Phe Glu Ala 55 Val Phe Asp Ala Pro 60 Asp Asp Phe Tyr Cheese 65 Asp Ala Lys Leu Val 70 Leu Cheese Asp Gly Arg 75 Glu Val Cheese Phe His 80

PL 191 355 B1

Arg Cys Val Leu Ser 35 Ala Arg Ser Phe cheese 90 Phe Lys Cheese Ala Leu 95 Ala Ala Ala Lys Lys Glu Lys Asp Cheese Asn Asn Thr Ala Ala Val Lys Leu 100 105 110 Glu Leu Lys Glu How much Ala Lys Asp Tyr Glu Val Gly Phe Asp Cheese Val 115 120 125 Val Thr Val Leu Ala Tyr Val Tyr Cheese Cheese Arg Val Arg Pro Pro Pro 130 135 140 Lys Gly Val Cheese Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys 145 150 155 160 Arg Pro Ala Val Asp Phe Underworld Leu Glu Val Leu Tyr Leu Ala Phe How much 165 170 175 Phe Lys How much Pro Glu Leu How much Thr Leu Tyr Gin Arg His Leu Leu Asp 180 185 190 Val Val Asp Lys Val Val How much Glu Asp Thr Leu Val How much Leu Lys Leu 195 200 205 Ala Asn How much Cys Gly Lys Ala Cys Underworld Lys Leu Leu Asp Arg Cys Lys 210 215 220 Glu How much How much Val Lys Cheese Asn Val Asp Underworld Val Cheese Leu Glu Lys Cheese 225 230 235 240 Leu Pro Glu Glu Leu Val Lys Glu How much How much ASp Arg Arg Lys Glu Leu 245 250 255 Gly Leu Glu Val Pro Lys Val Lys Lys His Val Cheese Asn Val His Lys 260 265 270 Ala Leu Asp Cheese Asp Asp How much 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

PL 191 355 B1

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

Ala Met Arg Lys Glu Pro Gin Leu How much Leu 345 Cheese Leu Leu Glu 350 Lys Gly 340 Ala Cheese Ala Cheese Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Underworld How much 355 360 365 Ala Lys Gin Ala Thr Underworld Ala Val Glu Cys Asn Asn How much Pro Glu Gin 370 375 380 Cys Lys His Cheese Leu Lys Gly Arg Leu Cys Val Glu How much Leu Glu Gin 385 390 395 400 Glu Asp Lys Arg Glu Gin How much Pro Arg Asp Val Pro Pro Cheese Phe Ala 405 410 415 Val Ala Ala Asp Glu Leu Lys Underworld Thr Leu Leu Asp Leu Glu Asn Arg 420 425 430 Val Ala Leu Ala Gin Arg Leu Phe Pro Thr Glu Ala Gin Ala Ala Underworld 435 440 445 Glu How much Ala Glu Underworld Lys Gly Thr Cys Glu Phe How much Val Thr Cheese Leu 450 455 460 Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Cheese Pro Gly val Lys 465 470 475 480 How much Ala Pro Phe Arg How much Leu Glu Glu His Gin Cheese Arg Leu Lys Ala 485 490 495 Leu Cheese Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Cheese 500 505 510 Ala Val Leu Asp Gin How much Underworld Asn Cys Glu Asp Leu Thr Gin Leu Ala 515 520 525 Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gin Lys Lys Gin Arg 530 535 540

PL 191 355 B1

Tyr 545 Underworld Glu How many Gin Glu 550 Thr Leu Lys Lys Ais 555 Phe Cheese Glu Asp Asn 560 Leu Glu Leu Gly Asn Leu Cheese Leu Thr Asp Cheese Thr Cheese Cheese Thr Cheese 565 570 575 Lys Cheese Thr Gly Gly Lys Arg Cheese Asn Arg Lys Leu Cheese His Arg Arg

580 535 590

Arg (2) INFORMATION FOR SEQ. ID NO: 9:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 1597 base pairs (B) TYPE: Nucleic acid (C) STRAND: Single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) FEATURE:

(A) NAME / KEY: CDS (B) ITEM: 1..1410 (D) OTHER INFORMATION: / product = "changed form of NIM1" / note = "N-terminal deletion compared to wild NIM 1 sequence (xi) DESCRIPTION SEQUENCE: SEQ. ID NO: 9

ATG GAT TCG GTT GTG ACT GTT TTG GCT TAT GTT TAC AGC Cheese AGC Cheese AGA Arg 15 GTG Val 48 Underworld 1 Asp Cheese Val Val 5 Thr Val Leu Ala Tyr 10 Val Tyr AGA CCG CCG CCT AAA GGA GTT TCT GAA TGC GCA GAC GAG AAT TGC TGC 96 Arg Pro Pro Pro Lys Gly Val Cheese Glu Cys Ala Asp Glu Asn Cys Cys twenty 25 thirty CAC GTG GCT TGC CGG CCG GCG GTG GAT TTC ATG TTG GAG GTT CTC TAT 144

PL 191 355 B1

His Val Ala 35 Cys Arg Pro Ala Val 40 Asp Phe Underworld Leu Glu 45 Val Leu Tyr TTG GCT TTC ATC TTC AAG ATC CCT GAA TTA ATT ACT CTC TAT CAG AGG 192 Leu Ala 50 Phe How much Phe Lys How much 55 Pro Glu Leu How much Thr 60 Leu Tyr Gin Arg CAC TTA TTG GAC GTT GTA GAC AAA GTT GTT ATA GAG GAC ACA TTG GTT 240 His 65 Leu Leu Asp Val Val 70 Asp Lys Val Val How much 75 Glu Asp Thr Leu Val 80 ATA CTC AAG CTT GCT AAT ATA TGT GGT AAA GCT TGT ATG AAG CTA TTG 288 How much Leu Lys Leu Ala 85 Asn How much Cys Gly Lys 90 Ala Cys Underworld Lys Leu 95 Leu GAT AGA TGT AAA GAG ATT ATT GTC AAG TCT AAT GTA GAT ATG GTT AGT 336 Asp Arg Cys Lys 100 Glu How much How much Val Lys 105 Cheese Asn Val Asp Underworld 110 Val Cheese CTT GAA AAG TCA TTG CCG GAA GAG CTT GTT AAA GAG ATA ATT GAT AGA 384 Leu Glu Lys 115 Cheese Leu Pro Glu Glu 120 Leu Val Lys Glu How much 125 How much Asp Arg CGT AAA GAG CTT GGT TTG GAG GTA CCT AAA GTA AAG AAA CAT GTC TCG 432 Arg Lys 130 Glu Leu Gly Leu Glu 135 Val Pro Lys Val Lys 140 Lys His Val Cheese AAT GTA CAT AAG GCA CTT GAC TCG GAT GAT ATT GAG TTA GTC AAG TTG 480 Asn 145 Val His Lys Ala Leu 150 Asp Cheese Asp Asp How much 155 Glu Leu Val Lys Leu 160 CTT TTG AAA GAG GAT CAC ACC AAT CTA GAT GAT GCG TGT GCT CTT CAT 528 Leu Leu Lys Glu Asp 165 His Thr Asn Leu Asp 170 Asp Ala Cys Ala Leu 175 His TTC GCT GTT GCA TAT TGC AAT GTG AAG ACC GCA ACA GAT CTT TTA AAA 576 Phe Ala Val Ala 180 Tyr Cys Asn Val Lys 185 Thr Ala Thr Asp Leu 190 Leu Lys CTT GAT CTT GCC GAT GTC AAC CAT AGG AAT CCG AGG GGA TAT ACG GTG 624 Leu Asp Leu 195 Ala Asp Val Asn His 200 Arg Asn Pro Arg Gly 205 Tyr Thr Val

PL 191 355 B1

CTT Leu CAT His 210 GTT Val GCT Ala GCG Ala ATG Underworld CGG Arg 215 AAG Lys GAG Glu CCA CAA TTG ATA CTA TCT Cheese CTA Leu 672 Pro Gin Leu 220 How much Leu TTG GAA AAA GGT GCA AGT GCA TCA GAA GCA ACT TTG GAA GGT AGA ACC 720 Leu Glu Lys Gly Ala Cheese Ala Cheese 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 763 Ala Leu Underworld How much Ala Lys Gin Ala Thr Underworld 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 How much Pro Glu Gin Cys Lys His Cheese 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 How much Leu Glu Gin Glu Asp Lys Arg Glu Gin How much Pro Arg Asp Val Pro 275 260 285 CCC TCT TTT GCA GTG GCG GCC GAT GAA TTG AAG ATG ACG CTG CTC GAT 912 Pro Cheese Phe Ala Val Ala Ala Asp Glu Leu Lys Underworld Thr Leu Leu Asp 290 295 300 CTT GAA AAT AGA GTT GCA CTT GCT CAA CGT CTT TTT CCA ACG GAA GCA 960 Leu Glu Asn Arg Val Ala Leu Ala Gin Arg Leu Phe Pro Thr Glu Ala 305 310 315 320 CAA GCT GCA ATG GAG ATC GCC GAA ATG AAG GGA ACA TGT GAG TTC ATA 1008 Gin Ala Ala Underworld Glu How much Ala Glu Underworld Lys Gly Thr Cys Glu Phe How much 325 330 335 GTG ACT AGC CTC GAG CCT GAC CGT CTC ACT GGT ACG AAG AGA ACA TCA 1056 Val Thr Cheese Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Cheese 340 345 350 CCG GGT GTA AAG ATA GCA CCT TTC AGA ATC CTA GAA GAG CAT CAA AGT 1104 Pro Gly Val Lys How much Ala Pro Phe Arg How much Leu Glu Glu His Gin Cheese 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 Cheese Lys Thr Val Glu Leu Gly Lys Arg Phe Phe 370 375 380

PL 191 355 B1

CCG Pro 385 CGC TGT Arg Cys TCG GCA Ala cheese GTG CTC GAC CAG ATT ATG AAC TGT GAG GAC TTG 1200 Val 390 Leu Asp Gin How much Underworld 395 Asn Cys Glu Asp Leu 400 ACT CAA CTG GCT TGC GGA GAA GAC GAC ACT GCT GAG AAA CGA CTA CAA 1248 Thr Gin Leu Ala Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gin 405 410 415 AAG AAG CAA AGG TAC ATG GAA ATA CAA GAG ACA CTA AAG AAG GCC TTT 1296 Lys Lys Gin Arg Tyr Underworld Glu How much Gin Glu Thr Leu Lys Lys Ala Phe 420 425 430 AGT GAG GAC AAT TTG GAA TTA GGA AAT TTG TCC CTG ACA GAT TCG ACT 1344 Cheese Glu Asp Asn Leu Glu Leu Gly Asn Leu Cheese Leu Thr Asp Cheese Thr 435 440 445 TCT TCC ACA TCG AAA TCA ACC GGT GGA AAG AGG TCT AAC CGT AAA CTC 1392 Cheese Cheese Thr Cheese Lys Cheese Thr Gly Gly Lys Arg Cheese Asn Arg Lys Leu

450 455 460

TCT CAT CGT CGT CGG TGA GACTCTTGCC TCTTAGTGTA ATTTTTGCTG 1440

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: 10:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 470 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 10

PL 191 355 B1

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

Arg Pro Pro Pro Lys Gly Val Ser Glu Cys 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 Gin 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 Val Lys Ser Asn Val Asp Met Val Ser 100 105 110

Leu Glu Lys Cheese 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 Gin Leu Ile Leu Ser Leu 210 215 220

Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr

PL 191 355 B1

225 230 235 240 Ala Leu Underworld How much Ala Lys Gin Ala Thr Underworld Ala Val Glu cys Asn Asn 245 250 255 How much Pro Glu Gin Cys Lys His Cheese Leu Lys Gly Arg Leu Cys Val Glu 260 265 270 How much Leu Glu Gin Glu Asp Lys Arg Glu Gin How much Pro Arg Asp Val p 275 280 285 Pro Cheese Phe Ala Val Ala Ala Asp Glu Leu Lys Underworld Thr Leu Leu Asp 290 295 300 Leu Glu Asn Arg Val Ala Leu Ala Gin Arg Leu Phe Pro Thr Glu Ala 305 310 315 320 Gin Ala Ala Underworld Glu How much Ala Glu Underworld Lys Gly Thr Cys Glu Phe How much 325 330 335 Val Thr Cheese Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Cheese 340 345 350 Pro Gly Val Lys How much Ala Pro Phe Arg How much Leu Glu Glu His Gin Cheese 355 360 365 Arg Leu Lys Ala Leu Cheese Lys Thr Val Glu Leu Gly Lys Arg Phe Phe 370 375 380 Pro Arg Cys Cheese Ala Val Leu Asp Gin How much Underworld Asn Cys Glu Asp Leu 385 3 90 395 400 Thr Gin Leu Ala Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gin 405 410 415 Lys Lys Gin Arg Tyr Underworld Glu How much Gin Glu Thr Leu Lys Lys Ala Phe 420 425 430 Cheese Glu Asp Asn Leu Glu Leu Gly Asn Leu Cheese Leu Thr Asp Cheese Thr 435 440 445 Cheese Cheese Thr Cheese Lys Cheese Thr Gly Gly Lys Arg Cheese Asn Arg Lys Leu

450 455 460

PL 191 355 B1

His Arg Arg Arg *

465 470 (2) INFORMATION FOR SEQ. ID NO: 11:

(Ϊ) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 1608 base pairs (B) TYPE: Nucleic acid (C) STRAND: Single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) FEATURE:

(A) NAME / KEY: CDS (B) ITEM: 43..1608 (D) OTHER INFORMATION: / product = "changed form of NIM1" / note = "C-terminal deletion compared to wild NIM1 sequence (xi) SEQUENCE DESCRIPTION : SEQ. ID NO: 11:

GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC 54

Mec Asp Thr Thr

ATT GAT GGA TTC GCC GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC Phe cheese GTC Val twenty 102 How many Asp 5 Gly Phe Ala Asp 10 Cheese Tyr Glu How much Cheese 15 Cheese Thr GCT ACC GAT AAC ACC GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA 150 Ala Thr Asp Asn Thr Asp Cheese Cheese How much Val Tyr Leu Ala Ala Glu Gin 25 thirty 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 Cheese Ala Leu Gin Leu Leu Cheese ASn Cheese 40 4S 50 TTC GAA TCC GTC TTT GAC TCG CCG GAT GAT TTC TAC AGC GAC GCT AAG 246 Phe Glu Cheese Val Phe Asp Cheese Pro Asp Asp Phe Tyr Cheese Asp Ala Lys 55 60 65

PL 191 355 B1

CTT Leu GTT CTC TCC GAC GGC CGG GAA GTT TCT TTC CAC CGG TGC GTT TTG Leu 294 Val 70 Leu Asp Gly Cheese Arg 75 Glu Val Cheese Phe His 80 Arg Cys Val TCA GCG AGA AGC TCT TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAG 342 Cheese Ala Arg Cheese Cheese Phe Phe Lys Cheese 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 Cheese 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 How much Ala Lys Asp Tyr Glu Val Gly Phe Asp Cheese 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 Cheese Cheese Arg Val Arg Pro Pro Pro Lys Gly Val Cheese 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 Underworld Leu Glu val Leu Tyr Leu Ala Phe How much Phe Lys How much Pro 165 170 175 180 GAA TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA 630 Glu Leu How much Thr Leu Tyr Gin Arg His Leu Leu Asp Val Val Asp Lys 185 190 195 GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT 678 Val Val How much Glu Asp Thr Leu Val How much Leu Lys Leu Ala Asn How much 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 Underworld Lys Leu Leu Asp Arg Cys Lys Glu How much How much Val 215 220 225 AAG TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA GAG 774 Lys Cheese Asn Val Asp Underworld Val Cheese Leu Glu Lys Cheese Leu Pro Glu Glu

230 235 240

PL 191 355 B1

CTT Leu 245 GTT AAA GAG ATA ATT GAT AGA CGT AAA GAG CTT GGT TTG GAG Glu GTA Val 260 822 Val Lys Glu How much How many Asp 250 Arg Arg Lys Glu 255 Leu Gly Leu CCT AAA GTA AAG AAA CAT GTC TCG AAT GTA CAT AAG GCA CTT GAC TCG 870 Pro Lys Val Lys Lys His Val Cheese Asn Val His Lys Ala Leu Asp Cheese 265 270 275 GAT GAT ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG GAT CAC ACC AAT 918 Asp Asp How much 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 Underworld Arg Lys 325 330 335 340 GAG CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA 1110 Glu Pro Gin Leu How much Leu Cheese Leu Leu Glu Lys Gly Ala Cheese Ala Cheese 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 Underworld How much Ala Lys Gin Ala 360 365 370 ACT ATG GCG GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT TCT 1206 Thr Underworld Ala Val Glu Cys Asn Asn How much Pro Glu Gin Cys Lys His Cheese 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 How much Leu Glu Gin Glu Asp Lys Arg 390 395 400 GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TTT GCA GTG GCG GCC GAT 1302

PL 191 355 B1

Glu 405 Gin How much Pro Arg Asp 410 Val Pro Pro Cheese Phe Ala Val Ala Ala Asp 415 420 GAA TTG AAG ATG ACG CTG CTC GAT CTT GAA AAT AGA GTT GCA CTT GCT 1350 Glu Leu Lys Underworld Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala 425 430 435 CAA CGT CTT TTT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC GAA 1398 Gin Arg Leu Phe Pro Thr Glu Ala Gin Ala Ala Underworld Glu How much Ala Glu 440 445 450 ATG AAG GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC CGT 1446 Underworld Lys Gly Thr Cys Glu Phe How much Val Thr Cheese 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 Cheese Pro Gly Val Lys How much Ala Pro Phe 470 475 480 AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA ACC 1542 Arg How much Leu Glu Glu His Gin Cheese Arg Leu Lys Ala Leu Cheese 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 Cheese Ala Val Leu Asp 505 510 515 CAG ATT ATG AAC TGT TGA 1608 Gin How much Underworld Asn Cys * 520

(2) INFORMATION FOR SEQ. ID NO: 12:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 522 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 12

PL 191 355 B1

Met Asp Thr Thr Ile Asp Gly Phe 1 5

Thr Ser Phe Val Ala Thr Asp Asn 20

Ala Ala Glu Gin Val Leu Thr Gly 35 40

Leu Cheese Asn Cheese Phe Glu Cheese Val 50 55

Asp Ala Lys Leu Val Leu Cheese Cheese 65 70

Arg Cys Val Leu Ser Ala Arg Ser 85

Ala Ala Lys Lys Glu Lys Asp Ser 100

Glu Leu Lys Glu Ile Ala Lys Asp 115 120

Val Thr Val Leu Ala Tyr Val Tyr 130 135

Lys Gly Val Ser Glu Cys Ala Asp 145 150

Arg Pro Ala Val Asp Phe Met Leu 165

Phe Lys Ile Pro Glu Leu Ile Thr 180

Val Val Asp Lys Val Val Ile Glu 195 200

Ala Asn Ile Cys Gly Lys Ala Cys 210 215

Glu Ile Ile Val Lys Cheese Asn Val 225 230

Ala Asp Cheese Tyr Glu Ile Cheese Cheese 10 15

Thr Asp Cheese Ser Ile Val Tyr Leu 25 30

Pro Asp Val Ser Ala Leu Gin Leu 45

Phe Asp Ser Pro Asp Asp Phe Tyr 60

Asp Gly Arg Glu Val Ser Phe His 75 80

Phe Phe Lys cheese Ala Leu Ala cheese 90 95

Asn Asn Thr Ala Ala Val Lys Leu

105 110

Tyr Glu Val Gly Phe Asp Ser Val 125

Ser Ser Arg Val Arg Pro Pro Pro 140

Glu Asn Cys Cys His Val Ala Cys 155 160

Glu Val Leu Tyr Leu Ala Phe Ile 170 175

Leu Tyr Gin Arg His Leu Leu Asp

185 190

Asp Thr Leu Val Ile Leu Lys Leu 205

Met Lys Leu Leu Asp Arg Cys Lys 220

Asp Met Val Ser Leu Glu Lys Ser 235 240

PL 191 355 B1

Leu Pro Glu Glu Leu 245 Val Lys Glu How much How much 250 Asp Arg Arg Lys Glu 255 Leu Gly Leu Glu Val 260 Pro Lys Val Lys Lys 265 His Val Cheese Asn Val 270 His Lys Ala Leu Asp 275 Cheese Asp Asp How much Glu 280 Leu Val Lys Leu Leu 285 Leu Lys Glu Asp His 290 Thr Asn Leu Asp Asp 295 Ala Cys Ala Leu His 300 Phe Ala Val Ala Tyr 305 Cys Asn Val Lys Thr 310 Ala Thr Asp Leu Leu 315 Lys Leu ASp Leu Ala 320 Asp Val Asn His Arg 325 Asn Pro Arg Gly Tyr 330 Thr Val Leu His Val 335 Ala Ala Underworld Arg Lys 340 Glu Pro Gin Leu How much 345 Leu Cheese Leu Leu Glu 350 Lys Gly

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

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

Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gin

385 390 395 400

Glu Asp Lys Arg Glu Gin 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 Gin Arg Leu Phe Pro Thr Glu Ala Gin 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

PL 191 355 B1

Glu 465 Pro Asp Arg Leu Thr 470 Gly Thr Lys Arg Thr 475 Cheese Pro Gly Val Lys 480 How much Ala Pro Phe Arg 485 How much Leu Glu Glu His 490 Gin Cheese Arg Leu Lys 495 Ala Leu Cheese Lys Thr 500 Val Glu Leu Gly Lys 505 Arg Phe Phe Pro Arg 510 Cys Cheese Ala Val Leu Asp Gin How much Underworld Asn Cys ♦

515 520 (2) INFORMATION FOR SEQ. ID NO: 13:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 1194 base pairs (B) TYPE: Nucleic acid (C) STRAND: Single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) FEATURE:

(A) NAME / KEY: CDS (B) ITEM: 1..1194 (D) OTHER INFORMATION: / product = "NIM1 altered form Note =" N-terminal / C-terminal hybrid (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 13

ATG GAT TCG GTT GTG Val 5 ACT GTT TTG GCT TAT GTT TAC AGC AGC Cheese AGA Arg 15 GTG Val 48 Underworld 1 Asp Cheese Val Thr Val Leu Ala Tyr 10 Val Tyr Cheese AGA CCG CCG CCT AAA GGA GTT TCT GAA TGC GCA GAC GAG AAT TGC TGC 96 Arg Pro Pro Pro Lys Gly Val Cheese Glu Cys Ala Asp Glu Asn Cys Cys twenty 25 thirty 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 Underworld Leu Glu Val Leu Tyr 35 40 45

PL 191 355 B1

TTG Leu GCT TTC ATC TTC AAG ATC CCT GAA TTA ATT ACT CTC TAT CAG AGG Arg 192 Ala 50 Phe How much Phe Lys How much 55 Pro Glu Leu How much Thr 60 Leu Tyr Gin 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 How much 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 How much Leu Lys Leu Ala Asn How much cys Gly Lys Ala Cys Underworld 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 How much How much Val Lys Cheese Asn Val Asp Underworld Val Cheese 100 105 110 CTT GAA AAG TCA TTG CCG GAA GAG CTT GTT AAA GAG ATA ATT GAT AGA 384 Leu Glu Lys Cheese Leu Pro Glu Glu Leu val Lys Glu How much How much 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 Cheese 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 Cheese Asp Asp How much 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

PL 191 355 B1

Leu His Val Ala Ala Met Arg Lys Glu Pro Gin Leu 220 How much Leu Cheese Leu 210 215 TTG GAA AAA GGT GCA AGT GCA TCA GAA GCA ACT TTG GAA GGT AGA ACC 720 Leu Glu Lys Gly Ala Cheese Ala Cheese 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 Underworld How much Ala Lys Gin Ala Thr Underworld 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 How much Pro Glu Gin Cys Lys His Cheese 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 How much Leu Glu Gin Glu Asp Lys Arg Glu Gin How much 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 Cheese Phe Ala Val Ala Ala Asp Glu Leu Lys Underworld Thr Leu Leu Asp 290 295 300 CTT GAA AAT AGA GTT GCA CTT GCT CAA CGT CTT TTT CCA ACG GAA GCA 960 Leu Glu Asn Arg Val Ala Leu Ala Gin Arg Leu Phe Pro Thr Glu Ala 305 310 315 320 CAA GCT GCA ATG GAG ATC GCC GAA ATG AAG GGA ACA TGT GAG TTC ATA 1008 Gin Ala Ala Underworld Glu How much Ala Glu Underworld Lys Gly Thr Cys Glu Phe How much 325 330 335 GTG ACT AGC CTC GAG CCT GAC CGT CTC ACT GGT ACG AAG AGA ACA TCA 1056 Val Thr Cheese Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Cheese 340 345 350 CCG GGT GTA AAG ATA GCA CCT TTC AGA ATC CTA GAA GAG CAT CAA AGT 1104 Pro Gly Val Lys How much Ala Pro Phe Arg How much Leu Glu Glu His Gin Cheese 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 Cheese Lys Thr Val Glu Leu Gly Lys Arg Phe Phe 370 375 380

PL 191 355 B1

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

Pro Arg Cys Ser Ala Val Leu Asp Gin Ile Met Asn Cys *

385 390 395 (2) INFORMATION FOR SEQ. ID NO: 14:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 398 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 14

Underworld 1 Asp Cheese Val Val 5 Thr Val Leu Ala Tyr 10 Val Tyr Cheese Cheese Arg 15 Val Arg Pro Pro Pro twenty Lys Gly Val Cheese Glu 25 Cys Ala Asp Glu Asn thirty Cys Cys His Val Ala 35 Cys Arg Pro Ala Val 40 Asp Phe Underworld Leu Glu 45 Val Leu Tyr Leu Ala 50 Phe How much Phe Lys How much 55 Pro Glu Leu How much Thr 60 Leu Tyr Gin Arg His 65 Leu Leu Asp Val Val 70 Asp Lys Val Val How much 75 Glu Asp Thr Leu Val 80 How much Leu Lys Leu Ala 85 Asn How much Cys Gly Lys 90 Ala Cys Underworld Lys Leu 95 Leu Asp Arg Cys Lys 100 Glu How much How much Val Lys 105 Cheese Asn Val Asp Underworld 110 Val Cheese Leu Glu Lys 115 Cheese Leu Pro Glu Glu 120 Leu Val Lys Glu How much 125 How much Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Cheese

130 135 140

100

PL 191 355 B1

PL 191 355 B1

101

Pro Arg Cys Ser Ala Val Leu Asp Gin Ile Met Asn Cys * 385 390 395 (2) INFORMATION FOR SEQ. ID NO: 15:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 786 base pairs (B) TYPE: Nucleic acid (C) STRAND: Single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix> FEATURE:

(A) NAME / KEY: CDS (B) ITEM: 1..786 (D) OTHER INFORMATION: / product = "NIM1 altered form / note =" NIM1 ankyrin domains "(xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 15:

ATG GAC TCC AAC AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG Lys GAG Glu 15 ATT How much 48 Underworld 1 Asp Cheese Asn Asn 5 Thr Ala Ala Val Lys 10 Leu Glu Leu GCC AAG GAT TAC GAA GTC GGT TTC GAT TCG GTT GTG ACT GTT TTG GCT 96 Ala Lys Asp Tyr twenty Glu Val Gly Phe Asp 25 Cheese Val Val Thr Val thirty Leu Ala TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT GAA 144 Tyr Val Tyr 35 Cheese Cheese Arg Val Arg 40 Pro Pro Pro Lys Gly 45 Val Cheese Glu TGC GCA GAC GAG AAT TGC TGC CAC GTG GCT TGC CGG CCG GCG GTG GAT 192 Cys Ala 50 Asp Glu Asn Cys Cys 55 His Val Ala Cys Arg 60 Pro Ala Val Asp TTC ATG TTG GAG GTT CTC TAT TTG GCT TTC ATC TTC AAG ATC CCT GAA 240 Phe 65 Underworld Leu Glu Val Leu 70 Tyr Leu Ala Phe How much 75 Phe Lys How much Pro Glu 80

102

PL 191 355 B1

TTA Leu ATT ACT CTC TAT Tyr 85 CAG AGG Gin Arg CAC His TTA TTG GAC GTT GTA GAC AAA GTT 288 How much Thr Leu Leu Leu 90 Asp Val Val Asp Lys 95 Val GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT GGT 336 Val How much Glu Asp Thr Leu Val How much Leu Lys Leu Ala Asn How much 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 Underworld Lys Leu Leu Asp Arg Cys Lys Glu How much How much Val Lys 115 120 125 TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA GAG CTT 432 Cheese Asn Val Asp Underworld Val Cheese Leu Glu Lys Cheese 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 How much How much 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 Cheese Asn Val His Lys Ala Leu Asp Cheese Asp 165 170 175 GAT ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG GAT CAC ACC AAT CTA 576 Asp How much 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 Underworld Arg Lys Glu 225 230 235 240 CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA GAA 768

PL 191 355 B1

103

Pro Gin Leu How much Leu Leu cheese Leu Glu Lys Gly Ala Ala Cheese Glu Cheese 245 250 255 GCA ACT TTG GAA GGT TGA Ala Thr Leu Glu Gly ★ 260

(2) INFORMATION FOR SEQ. ID NO: 16:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 262 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 16

Met Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu Ile

1 5 10 15 Ala Lys Asp Tyr twenty Glu Val Gly Phe Asp 25 Cheese Val val Thr Val thirty Leu Ala Tyr Val Tyr 35 Cheese Cheese Arg Val Arg 40 Pro Pro Pro Lys Gly 45 Val Cheese Glu Cys Ala 50 Asp Glu Asn Cys Cys 55 His Val Ala Cys Arg 60 Pro Ala Val Asp Phe 65 Underworld Leu Glu Val Leu 70 Tyr Leu Ala Phe How much 75 Phe Lys How much Pro Glu 80 Leu How much Thr Leu Tyr Θ5 Gin Arg His Leu Leu 90 Asp Val Val Asp Lys 95 Val Val How much Glu Asp 100 Thr Leu Val How much Leu 105 Lys Leu Ala Asn How much 110 Cys Gly Lys Ala Cys Underworld Lys Leu Leu Asp Arg Cys Lys Glu How much How much Val Lys

115 120 125

104

PL 191 355 B1

Cheese Asn 130 Val Asp Met Val Leu Glu Cheese Lys Leu Cheese Pro Glu Glu Leu 135 140 Val Lys Glu How much How much Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro 145 150 155 160 Lys Val Lys Lys His Val Cheese Asn Vał His Lys Ala Leu Asp Cheese Asp 165 170 175 Asp How much 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 Underworld Arg Lys Glu 225 230 235 240 Pro Gin Leu How much Leu Cheese Leu Leu Glu Lys Gly Ala Cheese Ala Cheese Glu 245 250 255 Ala Thr Leu Glu Gly *

260 (2) INFORMATION FOR SEQ. ID NO: 17:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 41 amino acids (B) TYPE: amino acid (C) STRAND: unbound (D) TOPOLOGY: unbound (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 17

PL 191 355 B1

105

How much 1 Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile Glu Leu Val 5 10 15 Lys Leu Underworld Val Underworld Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala twenty 25 thirty Val His Tyr Ala Val Gin His Cys Asn 35 40

(2) INFORMATION FOR SEQ. ID NO: 18:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 38 amino acids (B) TYPE: amino acid (C) STRAND: unbound (D) TOPOLOGY: unbound (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 18

Pro 1 Thr Gly Lys Thr 5 Ala Leu His Leu Ala 10 Ala Glu Underworld Val Cheese 15 Pro Asp Underworld Val Cheese Val Leu Leu Asp His His Ala Asp Xaa Asn Phe Arg twenty 25 thirty

Thr Xaa Asp Gly Val Thr 35 (2) INFORMATION FOR SEQ. ID NO: 19 (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 41 amino acids (B) TYPE: amino acid (C) STRAND: unbound (D) TOPOLOGY: unbound (ii) TYPE OF MOLECULE: protein

106

PL 191 355 B1 <xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 19:

How much 1 Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile Glu Leu Val 5 10 15 Lys Leu Underworld Val Underworld Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala twenty 25 thirty Val His Tyr Ala Val Gin His Cys Asn 35 40

(2) INFORMATION FOR SEQ. ID NO: 20:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 27 amino acids (B) TYPE: amino acid (C) STRAND: unbound (D) TOPOLOGY: unbound (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 20:

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

Ser Pro Asp Met Val Ser Val Leu Leu Asp Gin 20 25 (2) INFORMATION FOR SEQ. ID NO: 21 (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 41 amino acids (B) TYPE: amino acid (C) STRAND: unbound (D) TOPOLOGY: unbound

PL 191 355 B1

107 (ii) MOLECULE TYPE: protein (Xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 21

How much Arg Arg Underworld Arg Arg Ala Leu Asp Ala Ala Asp How much Glu Leu Val 1 5 10 15 Lys Leu Underworld Val Underworld Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala twenty 25 thirty Val His Tyr Ala Val Gin His Cys Asn 35 40

(2) INFORMATION FOR SEQ. ID NO: 22:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 27 amino acids (B) TYPE: amino acid (C) STRAND: unbound (D) TOPOLOGY: unbound (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 22:

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

Ser Pro Asp Met Val Ser Val Leu Leu Asp Gin 20 25 (2) INFORMATION FOR SEQ. ID NO: 23 (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 41 amino acids (B) TYPE: amino acid (C) STRAND: unbound

108

PL 191 355 B1 (D) TOPOLOGY: unbound (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 23

How much Arg Arg Underworld Arg Arg Ala Leu Asp Ala Ala Asp How much Glu Leu Val 1 5 10 15 Lys Leu Underworld Val Underworld Gly Glu Gly Leu Asp Leu Asp ASp Ala Leu Ala twenty 25 thirty Vał His Tyr Ala Val Gin His Cys Asn 35 40

(2) INFORMATION FOR SEQ. ID NO: 24:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) STRAND: unbound (D) TOPOLOGY: unbound (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 24:

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

Asp Met Val (2) INFORMATION FOR SEQ. ID NO: 25 (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 35 base pairs

PL 191 355 B1

109 (B) TYPE: nucleic acid (C) STRAND: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / description = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 25:

CAACAGCTTC GAAGCCGTCT TTGACGCGCC GGATG 35 (2) INFORMATION FOR SEQ. ID NO: 26:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRAND: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / description = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 26:

CATCCGGCGC GTCAAAGACG GCTTCGAAGC TGTTG 35 (2) INFORMATION FOR SEQ. ID NO: 27:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRAND: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / description = "oligonucleotide"

110

PL 191 355 B1 (xi> SEQUENCE DESCRIPTION: SEQ ID NO: 27

GGAATTCAAT GGATTCGGTT GTGACTGTTT TG (2) INFORMATION FOR SEQ. ID NO: 28:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRAND: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / description ~ "oligonucleotide (xi) DESCRIPTION SEQUENCE: SEQ. ID NO: 28:

GGAATTCTAC AAATCTGTAT ACCATTGG (2) INFORMATION FOR SEQ. ID NO: 29:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRAND: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / description = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 29:

CGGAATTCGA TCTCTTTAAT TTGTGAATTT C (2) INFORMATION FOR SEQ. ID NO: 30 (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 29 base pairs (B) TYPE: Nucleic acid (C) STRAND: single (D) TOPOLOGY: linear

PL 191 355 B1

111 (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: / description = "oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 30:

GGAATTCTCA ACAGTTCATA ATCTGGTCG 29 (2) INFORMATION FOR SEQ. ID NO: 31:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRAND: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / description = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 31:

GGAATTCAAT GGACTCCAAC AACACCGCCG C 31 (2) INFORMATION FOR SEQ. ID NO: 32:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRAND: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / description = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 32:

GGAATTCTCA ACCTTCCAAA GTTGCTTCTG ATG 33

Claims (35)

Patent claims A method of protecting a plant against pathogen attack by imparting synergistic disease resistance to the plant, comprising providing an immunomodulated plant having a first level of disease resistance, and applying to said immunomodulated plant at least one microbicide which imparts a second level of disease resistance to it, whereby the application of a microbicidal agent to an already immunomodulated plant gives it a synergistically enhanced third level of disease resistance which is greater than the sum of the first and second levels of disease resistance, characterized in that the immunomodulated plant is: (i) a plant of the mutant (cim) with constitutive resistance, (ii) a mutant mutant plant of damage, or (iii) a plant obtained by recombinant expression of a SAR gene. 2. The method according to p. The method of claim 1, wherein the cim mutant of a plant is selected from the plant population and then (a) evaluating the expression of SAR genes in uninfected plants that are phenotypically normal by not having a damage mimicking phenotype, and sequentially (b) selecting uninfected plants which constitutively express SAR genes in the absence of viral, bacterial or fungal infection. 3. The method according to p. The method of claim 1, wherein a plant that is a mutant damage mimic is selected from the plant population, and then (a) the expression of SAR genes in uninfected plants that have a damage mimicking phenotype is assessed, and then (b) uninfected plants that constitutively express SAR genes are selected in the absence of viral, bacterial or fungal infection. 4. The method according to p. The method of claim 1, wherein the SAR gene is a functional form of the NIM1 gene encoding the NIM1 protein associated with a signal transduction cascade leading to acquired systemic resistance in plants. 5. The method according to p. The process of claim 4, wherein the NIM1 protein is produced with the amino acid sequence shown in SEQ ID NO: 2. 6. The method according to p. The process of claim 4, wherein the NIM1 protein is produced which is encoded by a DNA molecule comprising the coding sequence shown as SEQ ID NO: 1. 7. The method according to p. The method of claim 1, wherein the SAR gene is used which encodes an altered form of the NIM1 protein that constitutively expresses the SAR gene in the transgenic plant. 8. The method according to p. The method of claim 7, wherein an altered form of the NIM1 protein is produced which has alanines instead of serines at amino acid positions corresponding to positions 55 and 59 in SEQID NO: 2. 9. The method according to p. The method of claim 8, wherein an altered form of the NIM1 protein is produced which comprises the amino acid sequence shown in SEQ ID NO: 8. 10. The method according to p. The process of claim 8, wherein an altered form of the NIM1 protein is produced which is encoded by a DNA molecule which comprises the nucleotide sequence shown as SEQID NO: 7. 11. The method according to p. The method of claim 7, wherein an altered form of the NIM1 protein is produced which has cleaved amino acids at the N-terminus corresponding approximately to amino acid positions 1-125 in SEQ ID NO: 2. 12. The method according to p. The method of claim 11, wherein an altered form of the NIM1 protein is produced which comprises the amino acid sequence shown as SEQ ID NO: 10. 13. The method according to p. The method of claim 11, wherein an altered form of the NIM1 protein is produced which is encoded by a DNA molecule which comprises the nucleotide sequence shown as SEQ ID NO: 9. 14. The method according to p. The method of claim 7, wherein an altered form of the NIM1 protein is produced which has cleaved amino acids at the C-terminus corresponding approximately to amino acid positions 522-593 in SEQ ID NO: 2. 15. The method according to p. The method of claim 14, wherein an altered form of the NIM1 protein is produced which comprises the amino acid sequence shown in SEQ ID NO: 12. 16. The method according to p. The method of claim 14, wherein an altered form of the NIM1 protein is produced which is encoded by a DNA molecule which comprises the nucleotide sequence shown as SEQ ID NO: 11. PL 191 355 B1 113 17. The method according to p. The method of claim 7, wherein an altered form of the NIM1 protein is produced which has amino acids truncated at the N-terminus corresponding approximately to amino acid positions 1-125 of SEQ ID NO: 2 and having amino acids truncated at the C-terminus corresponding to approximately amino acid positions 522 -593 in SEQ ID NO: 2. 18. The method according to p. The method of claim 17, wherein an altered form of the NIM1 protein is produced which comprises the amino acid sequence shown as SEQ ID NO: 14. 19. The method according to p. The method of claim 17, wherein an altered form of the NIM1 protein is produced which is encoded by a DNA molecule which comprises the nucleotide sequence shown as SEQ ID NO: 13. 20. The method according to p. The method of claim 7, wherein an altered form of the NIM1 protein is produced which consists essentially of ankyrin motifs corresponding to approximately amino acid positions 103-362 in SEQ ID NO: 2. 21. The method according to p. The method of claim 20, wherein an altered form of the NIM1 protein is produced which comprises the amino acid sequence shown in SEQ ID NO: 16. 22. The method according to p. The method of claim 20, wherein an altered form of the NIM1 protein is produced which is encoded by a DNA molecule comprising the nucleotide sequence shown as SEQ ID NO: 15. 23. The method according to p. The method of claim 6, 10, or 13, or 16, or 19, or 22, characterized in that the DNA molecule hybridizes to the nucleotide sequence shown as SEQ ID NO 1, 7, 9, 11, 13 or 15 under the following conditions: 1% BSA; 520 mM NaPO4, pH 7.2; 7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM NaCl at 55 ° C for 18-24 hours. and washing in 6xSSC for 15 min (X3) 3xSSC for 15 min (X1) at 55 ° C. 24. The method according to p. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22, characterized in that the microbicide is a fungicide selected from the group consisting of: 4- [3- (4-chlorophenyl) -3- (3,4-dimethoxyphenyl) acryloyl] morpholine ("dimethomorph"); 5-methyl-1,2,4-triazolo [3,4-b] [1,3] benzothiazole ("tricyclazole"); 3-allyloxy-1,2-benzothiazole-1,1-dioxide ("probonazole"); α- [2- (4-chlorophenyl) ethyl] - α (1,1-dimethylethyl) -1H-1,2,4-triazole-1-ethanol ("tebuconazole"); 1- {[3- (2-chlorophenyl) -2- (4-fluorophenyl) oxiran-2-yl] methyl} 1H-1,2,4-triazole ("epoxiconazole"); a- (4-chlorophenyl) -a- (1-cyclopropylethyl-1H-1,2,4-triazole-1-ethanol ("cyproconazole"); 5- (4-chlorobenzyl) -2-2-dimethyl-1- (1H-1,2,4-triazol-1-ylmethyl) cyclopentanol ("metconazole"); 2- (2,4-dichlorophenyl) -3- (1H-1,2,4-triazol-1-yl) propyl-1,1,2,2-tetrafluoroethylether ("tetraconazole"); methyl- (E) -2- {2- [6- (2-cyanophenoxy) pyrimidin-4-yloxy] phenyl} -3-methoxyacrylate ("ICI A 5504", "azoxystrobin"); methyl (E) -2-methoxyimino-2- [α- (o-tolyloxy) -o-tolyl] acetate ("BAS 490 F", "methyl cresoxime"); 2- (2-phenoxyphenyl) - (E) -2-methoxyimino-N-methylacetamide; [2- (2,5-dimethylphenoxymethyl) phenyl] - (E) -2-methoxyimino-N-methyl acetamide; (1R, 3S / 1S, 3R) -2,2-dichloro-N - [(R) -1- (4-chlorophenyl) ethyl] -1-ethyl-3-methylcyclopropane carboxamide ("KTU 3616"); polymeric manganese ethylene bis (dithiocarbamate) complex with zinc salts ("mancozeb"); 1- [2- (2,4-dichlorophenyl) -4-propyl-1,3-dioxolan-2-ylmethyl] -1H-1,2,4-triazole ("propiconazole"); 1- [2- [2-chloro-4- (4-chlorophenoxy) phenyl] -4-methyl-1,3-dioxolan-2-yl-methyl] -1H-1,2,4-triazole ("diphenoconazole" ); 1- [2- (2,4-dichlorophenyl] pentyl-1H-1,2,4-triazole ("penconazole"); cis-4- [3- (4-tert-butylphenyl) -2-methylpropyl] -2 , 6-dimethylmorpholine ("fenpropimorph"); 1- [3- (4-tert-butylphenyl) -2-methylpropyl] piperidine ("fenpropidin"); 4-cyclopropyl-6-methyl-N-phenyl-2-pyrimidinamine ("cyprodinyl"); (RS) -N- (2,6-dimethylphenyl-N- (methoxyacetyl) -alanine ("metalaxyl") methyl ester; (R) -N- (2,6-dimethylphenyl-N- (methoxyacetyl) -alanine) methyl ester ("R-metalaxyl"); 1,2,5,6-tetrahydro-4H-pyrrolo [3,2,1,1j] quinoline-4-one ("pyroquilone") and ethyl hydride phosphonate ("fosetyl"). 25. The method according to p. The process of claim 24, wherein the fungicide is metalaxyl. 26. The method according to p. The method of claim 23, wherein one of the microbicides is a fungicide selected from the group consisting of: 4- [3- (4-chlorophenyl) -3- (3,4-dimethoxyphenyl) acryloylmorpholine ("dimethomorph"); 114 PL 191 355 B1 5-methyl-1,2,4-triazolo [3,4-b] [1,3] benzothiazole ("tricyclazole"); 3-allyloxy-1,2-benzothiazole-1,1-dioxide ("probonazole"); α- [2- (4-chlorophenyl) ethyl] - α (1,1-dimethylethyl) -1H-1,2,4-triazole-1-ethanol ("tebucanozole"); 1- {[3- (2-chlorophenyl) -2- (4-fluorophenyl) oxiran-2-yl] methyl} -1H-1,2,4-triazole ("epoxiconazole"); a- (4-chlorophenyl) -a- (1-cyclopropylethyl) -1H-1,2,4-triazole-1-ethanol ("cyproconazole"); 5- (4-chlorobenzyl) -2-2-dimethyl-1- (1H-1,2,4-triazol-1-ylmethyl) cyclopentanol, ("metconazole"); 2- (2,4-dichlorophenyl) -3- (1H-1,2,4-triazol-1-yl) -propyl-1,1,2,2-tetrafluoroethyl ether, ("tetraconazole"); methyl- (E) -2- {2- [6- (2-cyanophenoxy) pyrimidin-4-yloxy] phenyl} -3-methoxyacrylate, ("ICI A 5504", "azoxystrobin"); methyl (E) -2-methoxyimino-2- [α- (o-tolyloxy) -o-tolyl] acetate ("BAS 490 F", "methyl cresoxime"); 2- (2-phenoxyphenyl) - (E) -2-methoxyimino-N-methylacetamide; [2- (2,5-dimethylphenoxymethyl) phenyl] - (E) -2-methoxymino-N-methylacetamide; (1R, 3S / 1S, 3R) 2,2-dichloro-N - [(R) -1- (4-chlorophenyl) ethyl] -1-ethyl-3-methylcyclopropanecarboxamide ("KTU 361 6"); polymeric manganese ethylene bis (dithiocarbamate) complex with zinc salts ("mancozeb"); 1- [2- (2,4-dichlorophenyl) -4-propyl-1,3-dioxolan-2-ylmethyl] -1H-1,2,4-triazole ("propiconazole"); 1- {2- [2-chloro-4- (4-chlorophenoxy) phenyl] -4-methyl-1,3-dioxolan-2-yl-methyl} -1H-1,2,4-triazole ("diphenoconazole" ); 1- [2- (2,4-dichlorophenyl} pentyl-1H-1,2,4-triazole ("penconazole"); cis-4- [3- (4-tert-butylphenyl) -2-methylpropyl] -2 , 6-dimethylmorpholine ("fenpropimorph"); 1- [3- (4-tert-butylphenyl) -2-methylpropylpiperidine ("fenpropidine"); 4-cyclopropyl-6-methyl-N-phenyl-2-pyrimidinamide ("cyprodinyl"); (RS) -N- (2,6-dimethylphenyl-N- (methoxyacetyl) -alanine ("metalaxyl") methyl ester; (R) -N- (2,6-dimethylphenyl-N- (methoxyacetyl) -alanine) methyl ester ("R-metalaxyl"); 1,2,5,6-tetrahydro-4H-pyrrolo [3,2,1,1j] quinoline-4-one ("pyroquilone") and ethyl hydride phosphonate ("fosetyl"). 27. The method according to p. The process of claim 26, wherein the fungicide is metalaxyl. 28. The method according to p. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19, 20, 21 or 22, characterized in that either a benzothiadiazole compound, an isonicotinic acid compound or a salicylic acid compound is used as the microbicide. 29. The method according to p. The process of claim 23, wherein the microbicide is either a benzothiadiazole compound, an isonicotinic acid compound, or a salicylic acid compound. 30. A method according to claim 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17, or 18, or 19, or 20, or 21 or 22, characterized in that two microbicides are applied simultaneously to the immunomodulated plant. 31. The method according to p. The method of claim 30, wherein one of the microbicides is a fungicide selected from the group consisting of: 4- [3- (4-chlorophenyl) -3- (3,4-dimethoxyphenyl) acryloylmorpholine ("dimethomorph"); 5-methyl-1,2,4-triazolo [3,4-b] [1,3] benzothiazole ("tricyclazole"); 3-allyloxy-1,2-benzothiazole-1,1-dioxide ("probonazole"); α- [2- (4-chlorophenyl) ethyl] -α (1,1-dimethylethyl) -1H-1,2,4-triazole-1-ethanol ("tebucanozole"); 1- {[3- (2-chlorophenyl) -2- (4-fluorophenyl) oxiran-2-yl] methyl} -1H-1,2,4-triazole ("epoxiconazole"); α- (4-chlorophenyl) -α- (1-cyclopropylethyl) -1H-1,2,4-triazole-1-ethanol ("cyproconazole"); 5- (4-chlorobenzyl) -2-2-dimethyl-1- (1H-1,2,4-triazol-1-ylmethyl) cyclopentanol, ("metconazole"); 2- (2,4-dichlorophenyl) -3- (1H-1,2,4-triazol-1-yl) -propyl-1,1,2,2-tetrafluoroethyl ether, ("tetraconazole"); methyl- (E) -2- {2- [6- (2-cyanophenoxy) pyrimidin-4-yloxy] phenyl} -3-methoxyacrylate, ("ICI A 5504", "azoxystrobin"); methyl (E) -2-methoxyimino-2- [α- (o-tolyloxy) -o-tolyl] acetate ("BAS 490 F", "methyl cresoxime"); 2- (2-phenoxyphenyl) - (E) -2-methoxyimino-N-methylacetamide; [2- (2,5-dimethylphenoxymethyl) phenyl] - (E) -2-methoxymino-N-methylacetamide; (1R, 3S / 1S, 3R) 2,2-dichloro-N - [(R) -1- (4-chlorophenyl) ethyl] -1-ethyl-3-methylcyclopropanecarboxamide ("KTU 3616"); polymeric manganese ethylene bis (dithiocarbamate) complex with zinc salts ("mancozeb"); 1- [2- (2,4-dichlorophenyl) -4-propyl-1,3-dioxolan-2-ylmethyl] -1H-1,2,4-triazole ("propiconazole"); PL 191 355 B1 115 1- {2- [2-chloro-4- (4-chlorophenoxy) phenyl] -4-methyl-1,3-dioxolan-2-yl-methyl} -1H-1,2,4-triazole ("diphenoconazole" ); 1- [2- (2,4-dichlorophenyl} pentyl-1H-1,2,4-triazole ("penconazole"); cis-4- [3- (4-tert-butylphenyl) -2-methylpropyl] -2 , 6-dimethylmorpholine ("Fenpropimorph"); 1- [3- (4-tert-butylphenyl) -2-methylpropylpiperidine ("Fenpropidine"); 4-cyclopropyl-6-methyl-N-phenyl-2-pyrimidinamide ("cyprodinyl"); (RS) -N- (2,6-dimethylphenyl-N- (methoxyacetyl) -alanine ("metalaxyl") methyl ester; (R) -N- (2,6-dimethylphenyl-N- (methoxyacetyl) -alanine) methyl ester ("R-metalaxyl"); 1,2,5,6-tetrahydro-4H-pyrrolo [3,2,1,1j] quinoline-4-one ("pyroquilone") and ethyl hydride phosphonate ("fosetyl"). 32. The method according to p. 31, characterized in that said fungicide is a metalaxyl, the second microbicide is a benzothiadiazole compound, 33. The method according to p. 23. The method according to claim 23, characterized in that two microcybids are applied simultaneously to the immodulated plant. 34. The method according to p. The method of claim 33, wherein one of the microbicides is a fungicide selected from the group consisting of: 4- [3- (4-chlorophenyl) -3- (3,4-dimethoxyphenyl) acryloylmorpholine ("dimethomorph"); 5-methyl-1,2,4-triazolo [3,4-b] [1,3] benzothiazole ("tricyclazole"); 3-allyloxy-1,2-benzothiazole-1,1-dioxide ("probonazole"); α- [2- (4-chlorophenyl) ethyl] - α (1,1-dimethylethyl) -1H-1,2,4-triazole-1-ethanol ("tebucanozole"); 1- {[3- (2-chlorophenyl) -2- (4-fluorophenyl) oxiran-2-yl] methyl} -1H-1,2,4-triazole ("epoxiconazole"); a- (4-chlorophenyl) -a- (1-cyclopropylethyl) -1H-1,2,4-triazole-1-ethanol ("cyproconazole"); 5- (4-chlorobenzyl) -2-2-dimethyl-1- (1H-1,2,4-triazol-1-ylmethyl) cyclopentanol, ("metconazole"); 2- (2,4-dichlorophenyl) -3- (1H-1,2,4-triazol-1-yl) -propyl-1,1,2,2-tetrafluoroethyl ether, ("tetraconazole"); methyl- (E) -2- {2- [6- (2-cyanophenoxy) pyrimidin-4-yloxy] phenyl} -3-methoxyacrylate, ("ICI A 5504", "azoxystrobin"); methyl (E) -2-methoxyimino-2- [α- (o-tolyloxy) -o-tolyl] acetate ("BAS 490 F", "methyl cresoxime"); 2- (2-phenoxyphenyl) - (E) -2-methoxyimino-N-methylacetamide; [2- (2,5-dimethylphenoxymethyl) phenyl] - (E) -2-methoxymino-N-methylacetamide; (1R, 3S / 1S, 3R) 2,2-dichloro-N - [(R) -1- (4-chlorophenyl) ethyl] -1-ethyl-3-methylcyclopropanecarboxamide ("KTU 3616"); polymeric manganese ethylene bis (dithiocarbamate) complex with zinc salts ("mancozeb"); 1- [2- (2,4-dichlorophenyl) -4-propyl-1,3-dioxolan-2-ylmethyl] -1H-1,2,4-triazole ("propiconazole"); 1- {2- [2-chloro-4- (4-chlorophenoxy) phenyl] -4-methyl-1,3-dioxolan-2-yl-methyl} -1H-1,2,4-triazole ("diphenoconazole" ); 1- [2- (2,4-dichlorophenyl} pentyl-1H-1,2,4-triazole ("penconazole"); cis-4- [3- (4-tert-butylphenyl) -2-methylpropyl] -2 , 6-dimethylmorpholine ("fenpropimorph"); 1- [3- (4-tert-butylphenyl) -2-methylpropylpiperidine ("Fenpropidine"); 4-cyclopropyl-6-methyl-N-phenyl-2-pyrimidinamide ("cyprodinyl"); (RS) -N- (2,6-dimethylphenyl-N- (methoxyacetyl) -alanine ("metalaxyl") methyl ester; (R) -N- (2,6-dimethylphenyl-N- (methoxyacetyl) -alanine) methyl ester ("R-metalaxyl"); 1,2,5,6-tetrahydro-4H-pyrrolo [3,2,1, ij] quinoline-4-one ("pyroquilone") and ethyl hydride phosphonate ("fosetyl") and the other microbicide is either a benzothiadiazole compound or an isonicotinic acid compound or a salicylic acid compound. 35. The method according to p. The process of claim 34, wherein said fungicide is metalaxyl and the second microbicide is a benzothiadiazole compound.