CA2275854A1 - Method for protecting plants - Google Patents

Abstract

The present invention concerns a method of protecting plants from pathogen attack through synergistic disease resistance attained by applying a conventional microbicide to immunomodulated plants. Immunomodulated plants are those in which SAR is activated and are therefore referred to as "SAR-on"
plants. Immunomodulated plants may be provided in at least three different ways: by applying to plants a chemical inducer of SAR such as BTH, INA, or SA;
through a selective breeding program based on constitutive expression of SAR
genes and/or a disease-resistant phenotype; or by transforming plants with one or more SAR genes such as a functional form of the N/M1 gene. By concurrently applying a microbicide to an immunomodulated plant, disease resistance is unexpectedly synergistically enhanced; i.e., the level of disease resistance is greater than the expected additive levels of disease resistance.

Description

METHOD FOR PROTECTING PLANTS
The present invention relates to a method for protecting a plant against pathogen attack through synergistic disease-resistance attained by applying a microbicide to an immunomodulated plant.
Plants are constantly challenged by a wide variety of pathogenic organisms including viruses, bacteria, fungi, and nematodes. Crop plants a: a particularly vulnerable because they are usually grown as genetically-uniform monocultures; when disease strikes, losses can be severe. However, most plants have their own innate mechanisms of defense against pathogenic organisms. Natural variation for resistance to plant pathogens has been identified by plant breeders and pathologists and bred into many crop plants.
These natural disease resistance genes often provide high levels of resistance to or immunity against pathogens.
Systemic acquired resistance (SAR) is one component of the complex system plants use to defend themselves from pathogens (Hunt and Ryals) Crit; Rev. in Plant Sci. 15) 583-606 (1996), incorporated by reference herein in its entirety; Ryais et al., Planf Ce118, 1809-1819 (i 996), incorporated by reference herein in its entirety). See also, U.S. Patent No.
5,614,395) incorporated by reference herein in its entirety. SAR is a particularly important aspect of plant-pathogen responses because it is a pathogen-inducibie) systemic resistance against a broad spectrum of infectious agents, including viruses) bacteria, and fungi. When the SAR signal transduction pathway is blocked) plants become more susceptible to pathogens that normally cause disease, and they also become susceptible to some infectious agents that would not normally cause disease (Gaffney et al.) Science 261, 754-756 (1993), incorporated by reference herein in its entirety; Delaney et al., Science 266, 1247-1250 (1994), incorporated by reference herein in its entirety; Delaney et al.) Proc.
Natl. Acad. Sci. USA 92, 6602-6606 (1995)) incorporated by reference herein in its entirety;
Delaney, Plant Phys. 113) 5-12 (1997), incorporated by reference herein in its entirety; Bi et al., Plant J. 8, 235-245 (1995), incorporated by reference herein in its entirety; Mauch-Mani and Slusarenko, Plant CellB, 203-212 {1996), incorporated by reference herein in its entirety). These observations indicate that the SAR signal transduction pathway is critical for maintaining plant health.
Conceptually) the SAR response can be divided into two phases. In the initiation phase, a pathogen infection is recognized, and a signal is released that travels through the phloem to distant tissues. This systemic signal is perceived by target cells, which react by expression of both SAR genes and disease resistance. The maintenance phase of SAR
refers to the period of time, from weeks up to the entire life of the plant, during which the plant is in a quasi steady state) and disease resistance is maintained (Ryals et al., 1996).
Salicylic acid (SA) accumulation appears to be required for SAR signal transduction.
Plants that cannot accumulate SA due to treatment with specific inhibitors, epigenetic repression of phenylalanine ammonia-iyase, or transgenic expression of salicylate hydroxylase, which specifically degrades SA, also cannot induce either SAR
gene expression or disease resistance {Gaffney et al.) 1993; Delaney et ai., 1994;
Mauch-Mani and Slusarenko 1996; Maher et al., Proc. Natl. Acad. Sci. USA 91, 7802-7806 (1994), incorporated by reference herein in its entirety; Pallas et al., Plant J. 10) 281-293 (1996), incorporated by reference herein in its entirety). Although it has been suggested that SA
might serve as the systemic signal, this is currently controversial and, to date, all that is known for certain is that if SA cannot accumulate, then SAR signal transduction is blocked (Pallas et al., 1996; Shulaev et al., Plant Cell7, 1691-1701 (1995)) incorporated by reference herein in its entirety; Vernooij et al., Plant Cell6, 959-965 {i 994), incorporated by reference herein in its entirety).
Recently, Arabidopsis has emerged as a model system to study SAR (Uknes et al.) Plant Cell4, 645-656 (1992), incorporated by reference herein in its entirety;
Uknes et al., MoL Plant-Microbe Interact. 6, 692-698 (1993), incorporated by reference herein in its entirety; Cameron et al., Plant J. 5, 715-725 (1994), incorporated by reference herein in its entirety; Mauch-Mani and Slusarenko, Mol. Plant-Microbe Interact. 7) 378-383 (1994), incorporated by reference herein in its entirety; Dempsey and Klessig, Bulletin de L'Instifut Pasteur93, 167-186 (1995), incorporated by reference herein in its entirety).
It has been demonstrated that SAR can be activated in Arabidopsis by both pathogens and chemicals, such as SA) 2,6-dichloroisonicotinic acid (INA) and benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester {BTH) (Uknes et al., 1992; Vernooij et al., Mol. Plant Microbe Interact 8, 228-234 (1995), incorporated by reference herein in its entirety; Lawton et al., Plant J. 10, 71-82 (1996), incorporated by reference herein in its entirety). FoNowing treatment with either INA or BTH or pathogen infection, at least three pathogenesis-related (PR) protein genes, namely, PR-1, PR-2, and PR-5 are coordinately induced concomitant with the onset of resistance (Uknes et al., 1992, 1993). In tobacco, the best characterized species, treatment with a pathogen or an immunization compound induces the expression of at least nine sets of genes (Vllard et al., Plant Cell 3, 1085-1094 (1991 )) incorporated by reference herein in its entirety). Transgenic disease-resistant plants have been created by transforming plants with various SAR genes (U.S. Patent No. 5,614,395).
A number of Arabidopsis mutants have been isolated that have modified SAR
signal transduction _(Delaney, 1997) The first of these mutants are the so-called Isd (lesions _simulating disease) mutants and acd2 Lccelerated _cell _death) (Dietrich et al., Ce1177, 565-577 (1994), incorporated by reference herein in its entirety; Greenberg et al., Cell77, 551-563 (1994), incorporated by reference herein in its entirety). These mutants all have some degree of spontaneous necrotic lesion formation on their leaves, elevated levels of SA) mRNA accumulation for the SAR genes) and significantly enhanced disease resistance. At least seven different Isd mutants have been isolated and characterized (Dietrich et aL, 1994; Weymann et al., Plant Cell7, 2013-2022 (1995), incorporated by reference herein in its entirety). Another interesting class of mutants are cim Lonstitutive immunity) mutants (Lawton et al., 'The molecular biology of systemic aquired resistance°
in Mechanisms of Defence Responses in Plants, B. Fritig and M. Legrand, eds (Dordrecht, The Netherlands:
Kluwer Academic Publishers), pp. 422-432 (1993)) incorporated by reference herein in its entirety). See also, International PCT Application WO 94/16077, which is incorporated by reference herein in their entireties. Like !sd mutants and acd2, cim mutants have elevated SA and SAR gene expression and resistance, but in contrast to Isd or acd2, do not display detectable lesions on their leaves. cpri (constitutive expresser of PR genes) may be a type of cim mutant; however, because the presence of microscopic lesions on the leaves of cprl has not been ruled out, cprl might be a type of Isd mutant (Bowling et al., Plant Cell6, 1845-1857 (1994)) incorporated by reference herein in its entirety).
Mutants have also been isolated that are blocked in SAR signaling. ndrl Lon-race-specific disease resistance) is a mutant that allows growth of both Pseudomonas syringae containing various avirulence genes and also normally avirulent isolates of Peronospora parasitica (Century et al., Proc. Natl. AcadSci. USA 92, 6597-6601 (1995)) incorporated by reference herein in its entirety). Apparently this mutant is blocked early in SAR signaling.
npri t nonexpresser of PR genes) is a mutant that cannot induce expression of the SAR
signaling pathway following INA treatment (Cao et al., Plant Ce116, 1583-1592 (1994), incorporated by reference herein in its entirety). eds Lnhanced disease susceptibility) mutants havb been isolated based on their ability to support bacterial infection following inoculation of a low bacterial concentration (Glazebrook et al.) Genetics 143, -4- .
(1996), incorporated by reference herein in its entirety; Parker et al., Plant CellB) 2033-2046 (1996), incorporated by reference herein in its entirety). Certain eds mutants are phenotypically very similar to npr~, and, recently, eds5 and eds53 have been shown to be allelic to npri (Glazebrook et al., 1996). nim i (n_oninducible immunity) is a mutant that supports P. parasitica (i.e., causal agent of downy mildew disease) growth following INA
treatment (Delaney et al., 1995; W094/16077). Although nim 1 can accumulate SA
following pathogen infection, it cannot induce SAR gene expression or disease resistance, suggesting that the mutation blocks the pathway downstream of SA. nim i is also impaired in its ability to respond to INA or BTH, suggesting that the block exists downstream of the action of these chemicats (Delaney et al., 1995; Lawton et al., 1996).
Recently, two allelic Arabidopsis genes have been isolated and characterized) mutants of which are responsible for the nim 1 and npri phenotypes) respectively (Ryals et al., Plant Cell 9, 425-439 (1997), incorporated by reference herein in its entirety; Cao et al., Cell88, 57-63 (1997), incorporated by reference herein in its entirety). The wild-type NIM1 gene product is involved in the signal transduction cascade leading to both SAR and gene-for-gene disease resistance in Arabidopsis (Ryals et al., 1997). Ryals et al., 1997 also report the isolation of five additional alleles of niml that show a range of phenotypes from weakly impaired in chemically induced PR-1 gene expression and fungal resistance to very strongly blocked. Transformation of the wild-type NPRi gene into nprl mutants not only complemented the mutations) restoring the responsiveness of SAR induction with respect to PR-gene expression and disease resistance, but also rendered the transgenic plants more resistant to infection by P. syringae in the absence of SAR induction (Cao et aL, 1997).
NF-xB/IxB signal transduction pathways have been implicated in disease resistance responses in a range of organisms from Drosophila to mammals. in mammals, NF-xB/IxB
signal transduction can be induced by a number of different stimuli including exposure of cells to lipopolysaccharide, tumor necrosis factor) interleukin 1 (IL-1 ), or virus infection (Baeuerle and Baltimore, Cell87, 13-20 (1996); Baldwin, Annu. Rev. Immunol.
14, 649-681 (1996)). The activated pathway leads to the synthesis of a number of factors involved in inflammation and immune responses, such as IL-2, IL-6) IL-8 and granulocyte/macrophage-colony stimulating factor (deMartin et al., Gene 152, 253-255 (1995)). In transgenic mouse studies, the knock-out of NF-xB/IxB signal transduction leads to a defective immune response including enhanced susceptibility to bacterial and viral pathogens (Beg and Baltimore, Science 274, 782-784 (199fi); Van Antwerp et al., Science 274, 787-789 {1996);
Wang et al., Science 274, 784-787 (1996); Baeuerle and Baltimore (1996)). In Arabidopsls) SAR is functionally analogous to inflammation in that normal resistance processes are potentiated following SAR activation leading to enhanced disease resistance (Bi et al., 1995; Cao et al., 1994; Delaney et al., 1995; Delaney et al., 1994; Gaffney et al., 1993;
Mauch-Mani and Slusarenko 1996; Delaney, 1997). Furthermore, inactivation of the pathway leads to enhanced susceptibility to bacterial) viral and fungal pathogens.
Interestingly, SA has been reported to block NF-xB activation in mammalian cells (Kopp and Ghosh, Science 265, 956-959 (1994)), while SA activates signal transduction in Arabidopsis. Bacterial infection of Drosophila activates a signal transduction cascade leading to the synthesis of a number of antifungal proteins such as cercropin B, defensin, diptericin and drosomycin (Ip et al., Cell75, 753-763 (1993); Lemaitre et al., Cell86) 973-983 {1996)). This induction is dependent on the gene product of dorsal and dif, hero NF-xB
homologs, and is repressed by cactus, an IxB homolog, in the fly. Mutants that have decreased synthesis of the antifungal and antibacterial proteins have dramatically lowered resistance to infection.
Despite much research and the use of sophisticated and intensive crop protection measures, including genetic transformation of plants, losses due to disease remain in the billions of dollars annually. Therefore, there is a continuing need to develop new crop protection measures based on the ever-increasing understanding of the genetic basis for disease resistance in plants.
In view of the above) a preferred aspect of the present invention pertains to a novel method of protecting plants from pathogen attack through synergistic disease resistance attained by applying a microbicide to immunomodulated plants. Immunomodulated plants are those in which SAR is activated, typically exhibiting greater-than-wild-type SAR gene expression, and are therefore referred to as "SAR-on° plants.
Immunomodulated plants for use in the method of the invention may be obtained in at least three different ways: by applying to plants a chemical inducer of SAR such as BTH, INA, or SA; through a selective breeding program in which plants are selected based on constitutive expression of SAR
genes and/or a disease-resistant phenotype; or by genetically engineering plants by transforming them with one or more SAR genes such as a functional form of the NlMi gene.

-6- _ The microbicide applied to the immunomodulated plants may be either a conventional microbicide such as the fungicide metalaxyl or, if applied to immunomoduiated plants obtained through selective breeding or genetic engineering) the microbicide may be a chemical inducer of SAR such as BTH, INA, or SA.
Immunomodulation provides a certain level of disease resistance in a plant.
Similarly, application of a microbicide to a plant provides a certain level of disease resistance. The expected result of combining immunomedulation with microbicide application would be a level of control reflecting the additive levels of control provided by the individual methods of providing disease resistance. However, by concurrently applying a microbicide to an immunomodulated plant, the disease resistance is unexpectedly synergistically enhanced;
i.e.) the level of disease resistance is greater than the expected additive levels of disease resistance.
Accordingly, the present invention concerns the cultivation of immunomodulated plants and the application of a suitable amount of a conventional microbicide thereto.
Especially preferred embodiments of the invention concern plants genetically engineered to contain and express a functional form of the NIM1 gene or a homologue or variant thereof.
The method of the invention results in greater pathogen control than is achieved through either immunomodulation or microbicide application alone.
Immunomodulation provides a certain level of disease resistance in a plant. Similarly, application of a microbicide to a plant provides a certain lave! of disease resistance. The expected result of combining immunomodulation with microbicide application would be a level of control reflecting the additive levels of control provided by the individual methods of providing disease resistance. However, by concurrently applying a microbicide to an immunomodulated plant, the control of pathogenic disease is unexpectedly synergistically enhanced; i.e., the level of disease control is greater than the expected additive levels of disease resistance.
In addition to greater disease resistance, another advantage of the present invention is that less microbicide is required to achieve the level of disease resistance provided by the method of the invention than is required for use with ordinary, wild-type plants. The result of this is both lower economic costs of microbicide) as well as less chance of adverse environmental consequences resulting from toxicity of some microbicides.
Furthermore) the inventive method of protecting plants by combining the effects of immunomodulation and application of a microbicide results in a longer duration of antipathogenic action and altogether higher crop yields. Another advantage of this method is that because the two 7 PCT/EP97I0?253 _7.
combined modes of action of pathogen control are completely different from one another, the threat of resistance developing is effectively prevented.
Thus the present invention relates to a method for protecting a plant from pathogen attack' through synergistic disease resistance, comprising the steps of:
(a) providing an immunomoduiated plant having a first level of disease resistance;
and (b) applying to said immunomodulated plant a least one microbicide that confers a second level of disease resistance;
(c) whereby application of said microbicide to said immunomodulated plant confers a synergistically enhanced third level of disease resistance that is greater than the sum of the first and second levels of disease resistance.
Preferred is a method according to the invention, wherein said immunomodulated plant is a constitutive immunity (cim) mutant plant.
In particularly preferred is a method according to the invention, wherein said cim mutant plant is selected from a population of plants according to the following steps:
(a) evaluating the expression of SAR genes in uninfected plants that are phenotypically normal in that said uninfected plants lack a lesion mimic phenotype; and (b) selecting uninfected plants that constitutively express SAR genes in theabsence of viral) bacterial) or fungal infection.
Also preferred is a method according to the invention, wherein said immunomodulated plant is a lesion mimic mutant plant.
In particularly preferred is a method according to the invention, wherein said lesion mimic mutant plant is selected from a population of plants according to the following steps:
(a) evaluating the expression of SAR genes in uninfected plants that have a lesion mimic phenotype; and (b) selecting uninfected plants that constitutively express SAR genes in the absence of viral) bacterial, or fungal infection.
Also preferred is a method according to the invention) wherein said immunomodulated plant is obtained by recombinant expression in a plant of an SAR gene.
In particularly preferred is a method according to the invention, wherein said SAR gene is a functional form of a NIM1 gene.

-8- .
More preferred is method according to the invention, wherein said NIM1 gene encodes a NIM1 protein involved in the signal transduction cascade leading to systemic acquired resistance in plants.
Especially preferred is a method according to the invention, wherein said NIM1 protein comprises the amino acid sequence set forth in SEQ ID N0:2.
Especially preferred is a method according to the invention, wherein said NIM1 gene hybridizes under the following conditions to the coding sequence set forth in SEQ ID NO:1:
hybridization in 1 %BSA; 520mM NaP04, pH7.2; 7% lauryi sulfate, sodium salt; 1 mM EDTA;
250 mM sodium chloride at 55°C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC
for 15 min. (X1 ) ai 55°C.
Especially preferred is a method according to the invention, wherein said NIMi gene comprises the coding sequence set forth in SEA ID N0:1 and all DNA molecules hybridizing therewith using moderate stringent conditions.
In particularly preferred is a method according to the invention) wherein said SAR gene encodes an altered form of a NIM1 protein that acts as a dominant-negative regulator of the SAR signal transduction pathway.
More preferred is method according to the invention, wherein said altered form of the NIM1 protein has alanines instead of serines in amino acid positions corresponding to positions 55 and 59 of SEQ ID N0:2.
Especially preferred is a method according to the invention) wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEA ID N0:8.
Especially preferred is a method according to the invention) wherein said DNA
molecule comprises the nucleotide sequence shown in SEQ ID N0:7 and all DNA
molecules hybridizing therewith using moderate stringent conditions.
Especially preferred is a method according to the invention, wherein said DNA
molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID N0:7: hybridization in 1 %BSA; 520mM NaPO,, pH7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55°C for 18-24h) and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1 ) at 55°C.
More preferred is a method according to the invention) wherein said altered form of the NIM1 protein has an N-terminal truncation of amino acids corresponding approximately to amino acid positions 1-125 of SEGO ID N0:2.
Especially preferred is a method according to the invention wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO:10.

Especially preferred is a method according to the invention wherein said DNA
molecule comprises the nucleotide sequence shown in SEQ ID N0:9 and all DNA
molecules hybridizing therewith using moderate stringent conditions.
Especially preferred is a method according to the invention, wherein said DNA
molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID N0:9: hybridization in 1 %BSA; 520mM NaP04, pH7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55°C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1 ) at 55°C.
Especially preferred is a method according to the invention, wherein said altered form of the NIM1 protein has a C-terminal truncation of amino acids corresponding approximately to amino acid positions 522-593 of SEQ ID N0:2.
Especially preferred is a method according to the invention) wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID N0:12.
Especially preferred is a method according to the invention, wherein said DNA
molecule comprises the nucleotide sequence shown in SEQ ID N0:11 and all DNA
molecules hybridizing therewith using moderate stringent conditions.
Especially preferred is a method according to the invention, wherein said DNA
molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID N0:11: hybridization in 1 %BSA; 520mM NaPOa) pH7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55°C for 18-24h) and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1 ) at 55°C.
More preferred is a method according to the invention, wherein said altered form of the NIM1 protein has an N-terminal truncation of amino acids corresponding approximately to amino acid positions 1-125 of SEQ ID N0:2 and a C-terminal truncation of amino acids corresponding approximately to amino acid positions 522-593 of SEQ ID N0:2.
Especially preferred is a method according to the invention, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID N0:14.
Especially preferred is a method according to the invention, wherein said DNA
molecule comprises the nucleotide sequence shown in SED ID N0:13 and all DNA
molecules hybridizing therewith using moderate stringent conditions.
Especially preferred is a method according to the invention, wherein said DNA
molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID N0:13: hybridization in 1 %BSA; 520mM NaP04, pH7.2; 7% lauryl sulfate, sodium - 10 - _.
salt; 1 mM EDTA; 250 mM sodium chloride at 55°C for f 8-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1 ) at 55°C.
More preferred is a method according to the invention, wherein said altered form of the NIM1 protein consists essentially of ankyrin motifs corresponding approximately to amino acid positions 103-362 of SEQ ID N0:2.
Especially preferred is a method according to the invention, wherein said altered form of the NIM1 protein ccmprises the amino acid sequence shown in SEQ ID N0:16.
Especially preferred is a method according to the invention, wherein said DNA
molecule comprises the nucleotide sequence shown in SEQ ID NOa 5 and all DNA
molecules hybridizing therewith using moderate stringent conditions.
Especially preferred is a method according to the invention, wherein said DNA
molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID N0:15: hybridization in 1 %BSA; 520mM NaP04, pH7.2; 7% lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM sodium chloride at 55°C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1 ) at 55°C.
Examples of target crops for the areas of indication disclosed herein comprise, without limitation, the following species of plants: cereals (maize, wheat, barley) rye, oats, rice, sorghum and related crops); beet (sugar beet and fodder beet); pomes, stone fruit and soft fruit (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and blackberries);
leguminous plants (beans) lentils, peas) soybeans); oil plants (rape, mustard, poppy) olives) sunflowers) coconut, castor oil plants, cocoa beans, groundnuts); cucumber plants (marrows) cucumber, melons); fibre plants (cotton, flax, hemp, jute); citrus fruit (oranges, lemons, grapefnrit, mandarins); vegetables (spinach, lettuce, asparagus, cabbages, carrots) onions) tomatoes, potatoes, paprika); lauraceae (avocados) cinnamon, camphor}; or plants such as tobacco, nuts, coffee) sugar cane) tea, vines, hops, bananas and natural rubber plants) as well as ornamentals (flowers, shnrbs) broad-leaved trees and evergreens, such as conifers). This list does not represent any limitation.
The method of the present invention can be used to confer resistance to a wide array of plant pathogens, which include, but are not limited to the following:
viruses or viroids such as tobacco or cucumber mosaic virus, ringspot virus or necrosis virus, pelargonium leaf curl virus) red clover mottle virus, tomato bushy stunt virus, and like viruses; Ascomycete fungi such as of the genera Venturia, Podosphaera, Erysiphe, Monolinia, Mycosphaerella, and Uncinula; Basidiomycete fungi such as from the genera Hemileia, Rhizoctonia, and Puccinia; Fungi imperfecti such as the genera Botrytis, Helminthosporium, Rhynchosporium, Fusarium (i.e., F. monoliforme), Septoria, Cenrospora, Altemaria, Pyricularia) and Pseudocercosporella (i.e.) P. herpotrichoides);
Oomycete fungi such as from the genera Phytophthora (i.e., P. parasnica)) Peronospore (i.e, P. tabacina), Bremia, Pythium, and Plasmopara; as well as other fungi such as Scleropthora macrospora) Sclerophthora rayissiae, Sclerospora graminicola) Peronosclerospora sorghi, Peronosclerospora phihppinensis, Peronosclerospora saccharf and Peronosclerospora maydis, Physopella zeae, Cercospora zeae-maydis, Colletotrichum gramintcota, Gibberella zeae, Exserohilum turcicum, Kabattellu zeae, and Bipolaris maydis; bacteria such as Pseudomonas syringae, Pseudomonas tabaci, and Erwinia stewartii; insects such as aphids, e.g. Myzus persicae; and lepidoptera such as Heliothus spp.; and nematodes such as Meloidogyne incognita.
Obtaining Immunomodulated Plants All three of the following general routes for obtaining immunomodulated plants are related in that they all fit into the SAR signal transduction pathway model set forth in Ryals et al., (1996). Upon activating the SAR signal transduction pathway to achieve disease resistance, the same set of SAR genes is fumed "on" and disease resistance results, regardless of which of the three below-described routes is taken. The differences among these three routes pertain only to which point in the pathway SAR is turned on; the end result is same among these three routes. Therefore, analyses and results observed with regard to immunomodulated plants attained through one route may be extrapolated and applied to immunomoduiated plants attained through a different route.
A. Application of a Chemical Inducer of Systemic Acquired Resistance A first route for obtaining immunomodulated plants involves applying to a plant a chemical capable of inducing SAR. Particularly potent chemical inducers of SAR
are benzothiadiazoles such as benzo[1 (2,3]thiadiazole-7-carbothioic acid-S-methyl ester (BTH).
Derivatives of benzothiadiazoles that may further be used as regulators are described in U.S. Patent Nos. 5,523,311 and 5,614,395, both of which are incorporated herein by reference. BTH-induced SAR) which supplies protection in the field against a broad spectrum of diseases in a variety of crops 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 Goriach et al., Plani CellB, 629-643 (1996)) each of which is incorporated herein by reference.
Other chemical inducers of SAR that may 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 the lower alkyl esters thereof, as well as salicylic acid compounds (SA). Examples of suitable INA and SA compounds are described in U.S.
Patent No. 5,614,395.
B. Breeding Constitutive Immunity (CIM) Mutant Plants A second route for obtaining immunomodulated plants is through a selective breeding program based on constitutive expression of SAR genes and/or a disease-resistant phenotype. Considerable data shows a tight correlation between the expression of SAR genes and systemic acquired resistance itself (Ward et at. (1991 ); Uknes et al.
(1992); Uknes et al.
{1993); Lawton) et at. (1993); and Alexander et al. (1993) PNAS USA 90) 7327-7331 ) herein incorporated by reference. In Arabidopsis) examples of well characterized SAR
genes are PR-1, PR-2 and PR-5, with PR-1 expressed at the highest level with the lowest background.
To identify and select plants that constitutively express SAR genes, Northern analysis is performed to detect expression of SAR genes. Known SAR DNA sequences can be utilized in cross-hybridization experiments as described in Uknes et al. (1992). Methods for the hybridization and cloning of nucleic acid sequences are well known in the art.
(See, for example, Molecular Cloning, A Laboratory Manual, 2nd Edition, Vol. 1-3, Sambrook et aL
(eds.) Cold Spring Harbor Laboratory Press (1989) and the references cited therein). At least two classes of SAR signal transduction mutants that constitutiveiy express SAR
genes have been isolated. One class has been designated as "Isd' mutants (isd = lesion simulating disease), which are also referred to as "cim Class I" mutants. See WO 94/16077. Isd (am Class I) mutants form spontaneous lesions on the leaves, accumulated elevated concentrations of SA, 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). A second class has been designated as "cim" (am = constitutive immunity) mutants, which are also referred to as "cim Class It"
mutants. See, WO
94/16077. cim mutants have all the characteristics of Isd mutants except spontaneous lesions.
That is, cim mutants are visibly phenotypically normal.
Once plants that constitutively express SAR genes are selected) they can be utilized in breeding programs to incorporate constitutive expression of the SAR genes and resistance to -13- .
pathogens into plant lines. Descendants for further crossing are selected based on expression of the SAR genes and disease resistance as well as for other characteristics important for production and quality accorcling to methods well known to those skilled in the art of plant breeding. For example, because lsd mutants display lesion fom~ation and necrosis, cim mutants with their normal phenotypes are preferable for use in such breeding programs and in the method of the present invention, although lsd mutants could be used if desired.
C. Transforming Plants with SAR Genes A third route for obtaining immunomodulated plants is by transforming plants with an SAR gene) preferably a functional form of the NlM1 gene.
1. Recombinant Expression of the Wild-Type NIM1 Gene Recombinant overexpression of the wild-type form of NIM1 (SEQ ID NO:1 ) gives rise to transgenic plants with a disease resistant phenotype. See, co-pending U.S.
Patent Application Serial No. 08/880,179, incorporated herein by reference. Increased levels of the active NIM1 protein produce the same disease-resistance effect as chemical induction with inducing chemicals such as BTH, INA, and SA. Preferably, the expression of the NIM1 gene is at a level that is at least two-fold above the expression level of the NIM1 gene in wild-type plants and is more preferably at least tenfold above the wild-type expression level.
The section below entitled "Recombinant DNA Technology" sets forth protocols that may be used to recombinantly express the wild-type NIM1 gene in transgenic plants at higher-than-wild-type levels. Alternately) plants can be transformed with the wild-type NPR1 gene to produce disease resistant plants as described in Cao, ef 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 the alteration of the NIM1 gene exploits both the recognition that the SAR pathway in plants shows functional parallels to the NF-xB/IxB regulation scheme in mammals and flies, as well as the discovery that the NIM1 gene product is a structural homologue of the mammalian signal transduction factor Ix8 subclass a. See, co-pending PCT
application "METHODS OF USING THE NIM1 GENE TO CONFER DISEASE RESISTANCE IN
PLANTS' incorporated herein by reference.

The sequence of the NIM1 gene (SEQ ID N0:1 ) was used in BLAST searches, and matches were identified based on homology of one rather highly conserved domain in the NIM1 gene sequence to ankyrin domains found in a number of proteins such as spectrins, ankyrins, NF-xB and IxB (Michaeiy and Bennett) Trends Cell Biol. 2) 127-129 (1992)). Pair-wise visual inspections between the NIM1 protein (SEGl ID N0:2) and 70 known ankyrin-containing proteins were carried out, and striking similarities were found to members of the fxBa class of transcription regulators (Baeuerle and Baltimore 1996; Baldwin 1996). As shown in Figure 1, the NIM1 protein {SEA ID N0:2) shares significant homology with IxBa proteins from mouse, rat, and pig (SEQ ID NOs: 3, 4, and 5, respectively).
NIM1 contains several important structural domains of IxBa throughout the entire length of the protein, including ankyrin domains (indicated by the dashed underscoring in Figure 1 ), 2 amino-terminal serines (amino acids 55 and 59 of NIM1 ) , a pair of lysines (amino acids 99 and 100 in NIM1 ) and an acidic C-terminus. Overall, NIM1 and ixBa share identity at 30% of the residues and conservative replacements at 50% of the residues. Thus, there is homology between ixBa and NIM1 throughout the proteins, with an overall similarity of 80%.
One way in which IxBa protein functions in signal transduction is by binding to the cytosolic transcription factor NF-xB and preventing it from entering the nucleus and altering transcription of target genes (Baeuerle and Baltimore, 1996; Baldwin) 1996).
The target genes of NF-xB regulate (activate or inhibit) several cellular processes, including antiviral) antimicrobiaf and cell death responses (Baeuerle and Baltimore) 1996). When the signal transduction pathway is activated, IxBa is phosphorylated at two serine residues (amino acids 32 and 36 of Mouse IlcBa). This programs ubiquitination at a double lysine (amino acids 21 and 22 of Mouse IxBa). Following ubiquitination, the NF-xB/IxB
complex is routed through the proteosome where IxBa is degraded and NF-xB is released to the nucleus.
The phosphorylated serine residues important in IxBa function are conserved in NIM1 within a large contiguous block of conserved sequence from amino acids 35 to 84 (Figure 1 ). In contrast to IxBa, where the double lysine is located about 15 amino acids toward the N-terminus of the protein, in NIM1 a double lysine is located about 40 amino acids toward the C-terminal end. Furthermore, a high degree of homology exists between NIM1 and IxBa in the serine/threonine rich carboxy terminal region which has been shown to be important in basal turnover rate (Sun ef al., Mol. Cell. Biol. 16, 1058-1065 (1996)).
According to the present invention based on the analysis of structural homology and the -15- _.
presence of elements known to be important for IxBa function, NIM1 is expected to function like the IxBa, having analogous effects on plant gene regulation.
Plants containing the wild-type NIMi gene when treated with inducer chemicals are predicted to have more NIM1 gene product (1xB homology or less phosphorytation of the NIM1 gene product (IxB homology. In accordance with this model, the result is that the plant NF-xB homolog is kept out of the nucleus, and SAR gene expression and resistance responses are allowed to occur. In the niml mutant plants) a non-functional NIMi gene product is present. Therefore, in accordance with this model, the NF-xB
homolog is free to go to the nucleus and repress resistance and SAR gene expression.
Consistent with this idea, animal cells treated with salicylic acid show increased stability/abundance of IxB and a reduction of active NF-x8 in the nucleus (Kopp and Ghosh, 1994). Mutations of IxB are known that act as super-repressors or dominant-negatives (Britta-Mareen Traenckner et al., EMBO 14: 2876-2883 (1995); Brown et al., Science 267:
1485-1488 ( 1996); Brockman et al., Molecular and Cellular Biology 15: 2809-2818 ( 1995);
Wang et al., Science 274: 784-787 (1998)). These mutant forms of IxB bind to NF-xB but are not phosphorylated or ubiquitinated and therefore are not degraded. NF-xB
remains bound to the IxB and cannot move into the nucleus.
In view of the above, altered forms of NIM7 that act as dominant-negative regulators of the SAR signal transduction pathway can be created. Plants transformed with these dominant-negative forms of NlM1 have the opposite phenotype as nim 1 mutant plants in that the plants transformed with altered forms of NIM1 exhibit constitutive SAR gene expression and therefore a CIM phenotype; i.e) the transgenic plants are immunomodulated. Because of the position the NIMI gene holds in the SAR signal transduction pathway, it is expected that a number of alterations to the gene, beyond those specifically disclosed herein, will result in constitutive expression of SAR
genes and, therefore, a CIM phenotype. The section below entitled "Recombinant DNA
Technology°
sets forth protocols that may be used to recombinantly express the altered forms of the NIM1 gene in transgenic plants at higher-than-wild-type levels. Below are described several altered forms of the NIMi gene that act as dominant-negative regulators of the SAR signal transduction pathway.

a. Changes of Serine Residues 55 and 59 to Alanine Residues:
Phosphorylation of serine residues in human IxBa is required for stimulus activated degradation of IxBa thereby activating NF-xB. Mutagenesis of the serine residues (S32 and S36) in human IxBa to alanine residues inhibits stimulus-induced phosphorylation, thus blocking IxBa proteosome-mediated degradation (Traenckner et al., 1995; Brown et al.) 1996; Brockman et al.) i 995; Wang et al.) 1996). This altered form of IxBa can function as a dominant-negative form by retaining NF-xB in the cytoplasm thereby blocking downstream signaling events. Based on the amino acid sequence comparison between NIM1 and IxB
shown in Figure 1, serines 55 (S55) and 59 (S59) in NIM1 (SEGO ID N0:2) are homologous to S32 and S36 in human IxBa. 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 NIMi gene is altered so that the encoded product has alanines instead of serines in the amino acid positions corresponding to positions 55 and 59 of the Arabidopsis NIM1 amino acid sequence (SEQ ID N0:2).
b. N-terminal Deletion:
Deletion of amino acids 1-36 (Brockman et al., 1995; Sun et al., 1996) or 1-72 (Sun et al., 1996) of human IkBa, which includes ubiquination lysine residues IC21 and K22 as well as phosphorylation sites S32 and S36, results in a dominant-negative IkBa phenotype in transfected human cell cultures. An N-terminal deletion of the first 125 amino acids of the NIM1 gene product will remove eight lysine residues that could serve as ubiquination sites as well as the putative phosphorylation sites at S55 and S59 discussed above.
Thus, in a preferred embodiment, the NIM1 gene is altered so that the encoded product is missing approximately the first 125 amino acids compared to the native Arabidopsis NIM1 amino acid sequence (SEQ ID N0:2).
c. C-Terminal Deletion:
Deletion of amino acids 261-317 of human IkBa may result in enhanced intrinsic stability by blocking constitutive phosphorylation of serine and threonine residues in the C-terminus. This altered form of IxBa is expected to function as a dominant-negative form. A
region rich in serine and threonine is present at amino acids 522-593 in the C-terminus of NIM1. Thus, in a preferred embodiment) the NIM1 gene is altered so that the encoded product is missing approximately its Gtenninal portion, including amino acides 522-593, compared to the native Arabidopsis NIM1 amino acid sequence (SEQ ID N0:2).
d. N-terminaUC-terminal Deletion Chimera and Ankyrin Domains Altered forms of the NIMi gene product may also be produced as a result of C-terminal and N-terminal segment deletions or chimeras. In yet another embodiment of the present invention, constructs comprising the ankyrin domains from the NIM1 gene are provided.
3. Recombinant Expression of Other SAR Genes immunomodulated plants for use in the method of the present invention can also be created by recombinant expression of various SAR genes such as those described in Ward et al. (1991 ). See, for example, U.S. Patent No. 5,614,395, which describes disease resistant plants created by overexpression of one or more PR-protein genes.
Although it refers to recombinant expression of forms of the NIMi gene particularly) the section below entitled NRecombinant DNA Technology" sets forth protocols that may also be used to recombinantly express other SAR genes such as PR-protein genes in transgenic plants at higher-than-wild-type levels.
Recombinant DNA Technology The wild-type or altered form of the NIM1 gene conferring disease resistance to plants by enhancing SAR gene expression can be incorporated into plant cells using conventional recombinant DNA technology. Generally, this involves inserting DNA molecule encoding the selected form of NIM1 described above into an expression system to which the DNA
molecule is heterologous (i.e., not normally present) using standard cloning procedures known in the art. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences. A large number of vector systems known in the art can be used, such as plasmids, bacteriophage viruses and other modified viruses. Suitable vectors include, but are not limited to) viral vectors such as lambda vector systems 7~gt11, ~,gtl0 and Charon 4; plasmid vectors such as pBl121, pBR322) pACYC177) pACYC184, pAR series, pKK223-3, pUCB, pUC9) pUClB, pUCl9, pLG339) pRK290) pKC37, pKC101, pCDNAI I; and other similar systems. The components of the expression _18_ system may also be modffied to increase expression. For example) truncated sequences) nucleotide substitutions or other modifications may be employed. The expression systems described herein can be used to transform virtually any crop plant cell under suitable conditions. Transformed cells can be regenerated into whole plants such that the chosen form of the NIMf gene activates SAR in the transgenic plants.
A. Construction of Plant Expression Cassettes Gene sequences intended for expression in transgenic plants are first assembled in expression cassettes behind a suitable promoter expressible in plants. The expression cassettes may also comprise any futher sequences required or selected for the expression of the transgene. Such sequences include, but are not restricted to, transcription terminators, extraneous sequences to enhance expression such as introns) vital sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments. These expression cassettes can then be easily transferred to the plant transformation vectors described infra. The following is a description of various components of typical expression cassettes.
1. Promoters The selection of the promoter used in expression cassettes will determine the spatial and temporal expression pattern of the transgene in the transgenic plan!.
Selected promoters will express transgenes in specific cell types (such as leaf epidermal cells) mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example) and the selection will reflect the desired location of accumulation of the gene product. Alternatively, the selected promoter may drive expression of the gene under various inducing conditions. Promoters vary in their strength, i.e., ability to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters can be used. The following are non-limiting examples of promoters that may be used in the expression cassettes.
a. Constitutive Expression, the CaMV 35S Promoter:
Construction of the plasmid pCGNi761 is described in the published patent application EP 0 392 225 (Example 23), which is hereby incorporated by reference.
pCGN1761 contains the "double' CaMV 35S. promoter and the tml transcriptional terminator with a unique EcoRl site between the promoter and the terminator and has a pUC-type backbone. A derivative of pCGN1761 is constructed which has a modified polylinker which includes Notl and Xhol sites in addition to the existing EcoRl site. This derivative is designated pCGN1761 ENX. pCGN1761 ENX is useful for the cloning of cDNA
sequences or gene sequences (including microbial ORF sequences) within its polylinker for the purpose of their expression under the control of the 35S promoter in transgenic plants. The entire 35S promoter-gene sequence-tml tem~inator cassette of such a construction can be excised by Hindlll, Sphl, Sall, and Xbal sites 5' to the promoter and Xbal, BamHl and Bgll sites 3' to the terminator for transfer to transformation vectors such as those described below. Furthermore, the double 35S promoter fragment can be removed by 5' excision with Hindlll, Sphl, Sall, Xbal, or Pstl, and 3' excision with any of the polylinker restriction sites (EcoRl, Notl or Xhon for replacement with another promoter. If desired, modifications around the cloning sites can be made by the introduction of sequences that may enhance translation. This is particularly useful when overexpression is desired. For example, pCGN1761 ENX may be modified by optimization of the translational initiation site as described in Example 37 of U.S. Patent No. 5,639,949, incorporated herein by reference.
b. Expression under a Chemicaliy/Pathogen Regulatable Promoter:
The double 35S promoter in pCGN1761 ENX may be replaced with any other promoter of choice that will result in suitably high expression levels. By way of example) one of the chemically regulatable promoters described in U.S. Patent No. 5,614,395 may replace the double 35S promoter. The promoter of choice is preferably excised from its source by restriction enzymes, but can alternatively be PCR-amplified using primers that carry appropriate terminal restriction sites. Should PCR-amplification be undertaken, then the promoter should be re-sequenced to check for amplification errors after the cloning of the amplified promoter in the target vector. The chemically/pathogen regulatable tobacco PR-1a promoter is cleaved from plasmid pCIB1004 (for construction, see example 21 of EP 0 332 104, which is hereby incorporated by reference) and transferred to plasmid pCGN1761 ENX (Uknes et al., 1992). pC181004 is cleaved with Ncol and the resultant 3' overhang of the linearized fragment is rendered blunt by treatment with T4 DNA
polymerase. The fragment is then cleaved with Hindlll and the resultant PR-i a promoter-containing fragment is gel purified and cloned into pCGN1761 ENX from which the double 35S promoter has been removed. This is done by cleavage with Xhol and blunting with T4 polymerase, followed by cleavage with Hindlll and isolation of the larger vector-terminator containing fragment into which the pCIB1004 promoter fragment is cloned. This generates a pCGN1761 ENX derivative with the PR-1a promoter and the tml terminator and an intervening polylinker with unique EcoR! and Notl sites. The selected coding sequence can be inserted into this vector, and the fusion products (l.e. promoter-gene-terminator) can subsequently be transferred to any selected transformation vector, including those described intra. Various chemical regulators may be employed to induce expression of the selected coding sequence in the plants transformed according to the present invention, including the benzothiadiazole, isonicotinic acid, and salicylic acid compounds disclosed in U.S. Patent Nos. 5,523,311 and 5,614,395.
c. Constitutive Expression, the Actin Promoter:
Several isoforms of actin are known to be expressed in most cell types and consequently the actin promoter is a good choice for a constitutive promoter.
In particular, the promoter from the rice Acfl gene has been cloned and characterized (McElroy et aL
Plant Cell 2: 163-171 (1990)). A 1.3kb fragment of the promoter was found to contain all the regulatory elements required for expression in rice protoplasts.
Furthermore, numerous expression vectors based on the Act! promoter have been constructed specifically for use in monocotyledons (McElroy et al. Mol. Gen. Genet. 231: 150-160 (1991 )). These incorporate the Act!-intron 1 ( Adhl 5' flanking sequence and AdhJ-intron 1 (from the maize alcohol dehydrogenase gene) and sequence from the CaMV 35S promoter. Vectors showing highest expression were fusions of 35S and Act! intron or the Actl5' flanking sequence and the Acfl intron. Optimization of sequences around the initiating ATG (of the GUS reporter gene) also enhanced expression. The promoter expression cassettes described by McElroy et al. (Mol. Gen. Genet. 231: 150-160 (1991 )) can be easily modified for gene expression and are particularly suitable for use in monocotyledonous hosts. For example, promoter-containing fragments is removed from the McElroy constructions and used to replace the double 35S promoter in pCGN1761 ENX, which is then available for the insertion of specific gene sequences. The fusion genes thus constructed can then be transferred to appropriate transformation vectors. In a separate report, the rice Act! promoter with its first intron has also been found to direct high expression in cultured barley cells (Chibbar et al. Plant Cell Rep. 12: 506-509 (1993)).

d. Constitutive Expression, the Ubiquitin Promoter:
Ubiquitin is another gene product known to accumulate in many cell types and its promoter has been cloned from several species for use in transgenic plants (e.g. sunflower - Binet et al. Plant Science 79: 87-94 (1991 ) and maize - Christensen et al.
Plant Molec.
Biol. 12: 619-632 (1989)). The maize ubiquitin promoter has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent pubiicatian EP 0 342 92S (to Lubrizol) which is herein incorporated by reference. Taylor et aI. (Plant Cell Rep. ,1~: 491-495 (1993)) describe a vector (pAHC25) that comprises the maize ubiquitin promoter and first intron and its high activity in cell suspensions of numerous monocotyledons when introduced via microprojectile bombardment. The ubiquitin promoter is suitable for gene expression in transgenic plants, especially monocotyledons. Suitable vectors are derivatives of pAHC25 or any of the transformation vectors described in this application) modified by the introduction of the appropriate ubiquitin promoter and/or intron sequences.
e. Root Specific Expression:
Another pattern of gene expression is root expression. A suitable root promoter is described by de Framond (FEES X90: 103-106 (1991 )) and also in the published patent application EP 0 452 269, which is herein incorporated by reference. This promoter is transferred to a suitable vector such as pCGN1761 ENX for the insertion of a selected gene and subsequent transfer of the entire promoter-gene-terminator cassette to a transformation vector of interest.
f. Wound-Inducible Promoters:
Wound-inducible promoters may also be suitable for gene expression. Numerous such promoters have been described (e.g. Xu et al. Plant Molec. Biol. ~2: 573-588 (1993), Logemann et aG Plant Cell 1: 151-158 (1989), Rohrmeier & Lehle, Plant Molec.
Biol. .~2:
783-792 (1993), Firek et al. Plant Molec. Biol. 2~: 129-142 (1993), Warner et al. Plant J. _3:
191-201 ( 1993)) and all are suitable for use with the instant invention.
Logemann ef al.
describe the 5' upstream sequences of the dicotyledonous potato wunl gene. Xu et al.
show that a wound-inducible promoter from the dicotyledon potato (pin2~ is active in the monocotyledon rice. Further, Rohrmeier & Lehle describe the cloning of the maize INipl cDNA which is wound induced and which can be used to isolate the cognate promoter using - 22 - _ standard techniques. Similar, Firek et al. and Warner et al. have described a wound-induced gene from the monocotyledon Asparagus officina~is) which is expressed at focal wound and pathogen invasion sites. Using cloning techniques well known in the art, these promoters can be transferred to suitable vectors, fused to the genes pertaining to this invention) and used to express these genes at the sites of plant wounding.
g. Pith-Preferred Expression:
Patent Application WO 93/07278, which is herein incorporated by reference, describes the isolation of the maize irpA gene, which is preferentially expressed in pith cells. The gene sequence and promoter extending up to -1726 by from the start of transcription are presented. Using standard molecular biological techniques, this promoter) or parts thereof, can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a foreign gene in a pith-preferred manner. In fact, fragments containing the pith-preferred promoter or parts thereof can be transferred to any vector and modified for utility in transgenic plants.
h. Leaf-Specific Expression:
A maize gene encoding phosphoenol carboxylase (PEPC) has been described by Hudspeth & Grula (Plant Moiec Biol 12: 579-589 (1989)). Using standard molecular biological techniques the promoter for this gene can be used to drive the expression of any gene in a leaf-specific manner in transgenic plants.
2. Transcriptional Terminators A variety of transcriptional terminators are available for use in expression cassettes.
These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Appropriate transcriptional temninators are those that are known to function in plants and include the CaMV 35S terminator, the tml terminator) the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons.
3. Sequences for the Enhancement or Regulation of Expression Numerous sequences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes of this invention to increase their expression in transgenic plants.

- 23 - -~
Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adhl gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells. Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis ei al., Genes Develop. 1_: 1183-1200 (1987)). In the same experimental system, the intron from the maize bronze 1 gene had a similar effect in enhancing expression. Intra~
sequences have been routinely incorporated into plant transfom~ation vectors, typically within the non-translated leader.
A number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells.
Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the "W-sequence"), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15:
8693-8711 (1987);
Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990)).
4. Targeting of the Gene Product Within the Cell Various mechanisms for targeting gene products are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterized in some detail. For example) the targeting of gene products to the chloroplast is controlled by a signal sequence found at the amino terminal end of various proteins which is cleaved during chloroplast import to yield the mature protein (e.g. Comai et aL J.
Biol. Chem. 263:
15104-15109 (1988)). These signal sequences can be fused to heterologous gene products to effect the import of heterologous products into the chloroplast (van den Broeck, et al. Nature 313: 358-363 (i 985)). DNA encoding for appropriate signal sequences can be isolated from the 5' end of the cDNAs encoding the RUBISCO protein, the CAB
protein, the EPSP synthase enzyme, the GS2 protein and many other proteins which are known to be chloroplast localized. See also, the section entitled "Expression With Chloroplast Targeting°
in Example 37 of U.S. Patent No. 5,639,949.
Other gene products are localized to other organelles such as the mitochondrion and the peroxisome ,(e.g. Unger ef al. Plant Molec. Biol. 13: 411-418 (1989)). The cDNAs encoding these products can also be manipulated to effect the targeting of heterologous gene products to these organelles. Examples of such sequences are the nuclear-encoded -24- _ ATPases and specific aspartate amino transferase isoforms for mitochondria.
Targeting cellular protein bodies has been described by Rogers et al. (Proc. Natl. Acad.
Sci. USA 82:
6512-6516 (1985)).
in addition, sequences have been characterized which cause the targeting of gene products to other cell compartments. Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler 8 Ho, Plant Cell 2: 769-783 (1990)). Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).
By the fusion of the appropriate targeting sequences described above to transgene sequences of interest it is possible to direct the transgene product to any organelle or cell compartment. For chloroplast targeting, for example, the chloroplast signal sequence from the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused in frame to the amino terminal ATG of the transgene. The signal sequence selected should include the known cleavage site, and the fusion constructed should take into account any amino acids after the cleavage site which are required for cleavage. in some cases this requirement may be fulfilled by the addition of a small number of amino acids between the cleavage site and the transgene ATG or) alternatively, replacement of some amino acids within the transgene sequence. Fusions constructed for chloroplast import can be tested for efficacy of chloroplast uptake by in vitro translation of in vitro transcribed constructions followed by in vitro chloroplast uptake using techniques described by Bartlett et al. In:
Edelmann ef al. (Eds.) Methods in Chloropiast Molecular Biology, Elsevier pp (1982) and Wasmann et al. Mol. Gen. Genet. 205: 446-453 (1986). These construction techniques are well known in the art and are equally applicable to mitochondria and peroxisomes.
The above-described mechanisms for cellular targeting can be utilized not only in conjunction with their cognate promoters) but also in conjunction with heterologous promoters so as to effect a specific cell-targeting goal under the transcriptional regulation of a promoter that has an expression pattern different to that of the promoter from which the targeting signal derives.
B. Construction of Plant Transformation Vectors Numerous transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts) and the genes pertinent to this -invention can be used in conjunction with any such vectors. The selection of vector will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the npfll gene) which confers resistance to kanamycin and related antibiotics (Messing ~
Vierra.
Gene 19: 259-268 (i 982); Bevan et al.) Nature ~Q4-:184-187 (1983)), the bargene, which confers rc~sisiance io the herbicide phosphinothricin (White et al., Nucl.
Acids Res 18: 1062 (1990), Spencer et a1. Theor. Appl. Genet 79: 625-631 (1990)), the hph gene) which confers resistance to the antibiotic hygromycin (Blochinger 8~ Diggelmann, Mol Cell Biol 4: 2929-2931 )) and the dhfrgene, which confers resistance to methatrexate (Bourouis et al., EMBO
J. 2 7 : 1099-1 i 04 (1983))) and the EPSPS gene, which confers resistance to glyphosate (U.S. Patent Nos. 4,940,935 and 5,188,642).
1. Vectors Suitable for Agrobacterium Transformation Many vectors are available for transformation using Agrobacterium tumefaciens.
These typically carry at least one T-DNA border sequence and include vectors such as pBINl9 (Bevan, Nucl. Acids Res. (1984)) and pXYZ. Below, the construction of two typical vectors suitable for Agrobacterium transformation is described.
a. pCIB200 and pCIB2001:
The binary vectors pCIB200 and pCIB2001 are used for the construction of recombinant vectors for use with Agrobacterium and are constructed in the following manner. pTJS75kan is created by Narl digestion of pTJS75 (Schmidhauser ~
Helinski) J.
Bacteriol. 164: 446-455 (1985)) allowing excision of the tetracycline-resistance gene, followed by insertion of an Accl fragment from pUC4K carrying an NPTII
(Messing ~ Vierra, Gene 19: 259-268 (1982): Bevan et al., Nature 304: 184-187 (1983): McBride et al.) Plant Molecular Biology 14: 266-276 (1990)). Xhol linkers are ligated to the EcoRVfragment of PCIB7 which contains the left and right T-DNA borders, a plant selectable nosJnptll chimeric gene and the pUC polylinker (Rothstein et al., Gene 53: 153-161' (1987)), and the Xhol digested fragment are cloned into Salt-digested pTJS75kan to create pCIB200 (see also EP
0 332 104) example 19). pCIB200 contains the following unique polylinker restriction sites:
EcoRl, Ssfl, Kpnl) Bglll, Xbal, and Sall. pCIB2001 is a derivative of pCIB200 created by the insertion into the polylinker of additional restriction sites. Unique restriction sites in the polylinker of pCIB2001 are EcoRl, Sstl, Kpnl, Bglll, ~Cbal, Sall, Mlul, BcII, Avrll, Apal, Hpal, and Stul. pCIB2001, in addition to containing these unique restriction sites also has plant and bacterial kanamycin selection, left and right T-DNA borders for Agrobacterium-mediated transformation) the RK2-derived trfA function for mobilization between E. coli and other hosts, and the OriTand OriVfunctions also from RK2. The pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals.
b. pCIBlO and Hygromycin Selection Derivatives thereof:
The binary vector pCIBlO contains a gene encoding kanamycin resistance for selection in plants and T-DNA right and left border sequences and incorporates sequences from the wide host-range plasmid pRK252 allowing it to replicate in both E, coli and Agrobacterium. Its construction is described by Rothstein et al. (Gene 53: 153-161 (1987)).
Various derivatives of pCIBlO are constructed which incorporate the gene for hygromycin B
phosphotransferase described by Gritz et al. (Gene 25: 179-188 (1983)). These derivatives enable selection of transgenic plant cells on hygromycin only (pCIB743), or hygromycin and kanamycin (pCIB715) pCIB717).
2. Vectors Suitable for non-Agrobacterium Transformation Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protopiast uptake (e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Below, the construction of typical vectors suitable for non-Agrobacterium transformation is described.
a. pCIB3064:
pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques in combination with selection by the herbicide basta (or phosphinothricin). The plasmid pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator and is described in the PCT
published application WO 93/07278. The 35S promoter of this vector contains two ATG
sequences 5' of the start site. These sites are mutated using standard PCR techniques in such a way as to remove the ATGs and generate the restriction sites Sspl and Pvull. The new restriction sites are 96 and 37 by away from the unique Sall site and 101 and 42 by away from the actual start site. The resultant derivative of pCIB246 is designated pCIB3025.
The GUS
gene is then excised from pCIB3025 by digestion with Sall and Sacl, the termini rendered blunt and religated to generate plasmid pCIB3060. The plasmid pJIT82 is obtained from the John Innes Centre, Norwich and the a 400 by Smal fragment containing the bargene from Streptomyces viridochromogenes is excised and inserted into the Hpal site of pCIB3060 (Thompson et al. EMBO J _6: 2519-2523 (1987)). This generated pCIB3064) which comprises the bargene under the control of the CaMV 35S promoter and terminator for herbicide selection, a gene for ampicillin resistance (for selection in E.
cold and a polylinker with the unique sites Sphl, Psil, Hindlll, and BamHl. This vector is suitable for the cloning of plant expression cassettes containing their own regulatory signals.
b. pSOGl9 and pSOG35:
pSOG35 is a transformation vector that utilizes the E. coli gene dihydrofolate reductase (DFR) as a selectable marker conferring resistance to methotrexate.
PCR is used to amplify the 35S promoter (-800 bp), intron 6 from the maize Adh1 gene (-550 bp) and 18 by of the GUS untranslated leader sequence from pSOGlO. A 250-by fragment encoding the E. coli dihydrofolate reductase type II gene is also amplified by PCR and these two PCR fragments are assembled with a Sacl-Pstl fragment from pB1221 (Clontech) which comprises the pUCl9 vector backbone and the nopaline synthase terminator.
Assembly of these fragments generates pSOGl9 which contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene and the nopaline synthase terminator. Replacement of the GUS leader in pSOGl9 with the leader sequence from Maize Chlorotic Mottle Virus (MCMV) generates the vector pSOG35, pSOGl9 and pSOG35 carry the pUC gene for ampicillin resistance and have Hindlll, Sphl, Pstl and Eco<31 sites available for the cloning of foreign substances.
C. Transformation Once the coding sequence of interest has been cloned into an expression system, it is transformed into a plant cell. Methods for transformation and regeneration of plants are well known in the art. For example, Ti piasmid vectors have been utilized for the delivery of -28- .
foreign DNA, as well as direct DNA uptake, liposomes) electroporation, micro-injection) and microprojectiles. In addition, bacteria from the genus Agrobacterium can be utilized to transform plant cells. Below are descriptions of representative techniques for transforming both dicotyledonous and monocotyledonous plants.
1. Transformation of Dicotyledons Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium. Non-Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake) particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described by Paszkowski et al., EMBO J 3_: 2717-2722 (1984), Potrykus et aL, Mol. Gen.
Genet. 199: 169-177 (1985), Reich et al., Biotechnology 4_: 1001-1004 (1986), and Klein et al., Nature 327: 70-73 (1987). In each case the transformed cells are regenerated to whole plants using standard techniques known in the art.
Agrobacterium-mediated transformation is a preferred technique for transformation of dicotyiedons because of its high efficiency of transformation and its broad utility with many different species. Agrobacferium transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest (e.g. pCIB200 or pCIB2001 ) to an appropriate Agrobacterium strain which may depend of the complement of virgenes carried by the host Agrobacterium strain either on a co-resident Ti pfasmid or chromosomally (e.g.
strain CIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5_: 159-i69 (1993)).
The transfer of the recombinant binary vector to Agrobacterium is accomplished by a triparental mating procedure using E. coli carrying the recombinant binary vector, a helper E. coli strain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Aitematively) the recombinant binary vectflr can be transferred to Agrobacterium by DNA transformation (Hofgen 8~
Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).
Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with expiants from the plant and follows protocols well known in the art. Transformed tissue is regenerated on selectable medium carrying the antibiotic or herbicide resistance marker present between the binary plasmid T-DNA borders.

Another approach to transforming plant cells with a gene involves propelling inert or biologically active particles at plant tissues and cells. This technique is disclosed in U.S.
Patent Nos. 4,945,050) 5,036,006) and 5,100,792 all to Sanford et al.
Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the desired gene.
Alternatively) the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried yeast cells, dried bacterium or a bacteriophage, each containing DNA sought to be introduced) can also be propelled into plant cell tissue.
2. Transformation of Monocotyledons Transformation of most monocotyledon species has now also become routine.
Preferred techniques include direct gene transfer into protoplasts using PEG
or electroporation techniques, and particle bombardment into callus tissue.
Transformations can be undertaken with a single DNA species or multiple DNA species (i.e. co-transformation) and both these techniques are suitable for use with this invention. Co-transformation may have the advantage of avoiding complete vector construction and of generating transgenic plants with unlinked loci for the gene of interest and the selectable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable. However) a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al. Biotechnology 4: 1093-1096 (1986)).
Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describe techniques for the preparation of callus and protoplasts from an elite inbred line of maize, transformation of protoplasts using PEG or electroporation) and the regeneration of maize plants from transformed protoplasts. Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and Fromm et al. (Biotechnology 8: 833-839 (1990)) have published techniques for transformation of A188-derived maize line using particle bombardment.
Furthermore, WO 93/07278 and Koziel et al. (Biotechnology 11: 194-200 (1993)) describe techniques for the transformation of elite inbred lines of maize by particle bombardment.
This technique utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after pollination and a PDS-1000He Biolistics device for bombardment.

WO 98129537 PCTlEP97/07253 Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment. Protopiast-mediated transformation has been described for Japonica-types and Indica-types (Zhang et al. Plant Cell Rep 7:

(1988); Shimamoto et al. Nature 338: 274-277 (1989); Datta et al.
Biotechnology 8: 736-740 (1990)). Both types are also routinely transformable using particle bombardment (Christou et al. Biotechnology _9: 957-962 (1991 )).
Patent Application EP 0 332 581 describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Furthermore, wheat transformation has been described by Vasil et al. (Biotechnology 10: 667-674 (1992)) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et aI.
(Biotechnology 11:
1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102: 1077-1084 (1993)) using particle bombardment of immature embryos and immature embryo-derived callus. A
preferred technique for wheat transformation, however, involves the transformation of wheat by particle bombardment of immature embryos and includes either a high sucrose or a high maltose step prior to gene delivery. Prior to bombardment, any number of embryos (0.75-1 mm in length) are plated onto MS medium with 3% sucrose (Murashiga 8~ Skoog, Physiologia Pfantarum 15: 473-497 (1962)) and 3 mgll 2,4-D for induction of somatic embryos) which is allowed to proceed in the dark. On the chosen day of bombardment, embryos are removed from the induction medium and placed onto the osmoticum (i.e.
induction medium with sucrose or maltose added at the desired concentration, typically 15%}. The embryos are allowed to plasmolyze for 2-3 h and are then bombarded.
Twenty embryos per target plate is typical, although not critical. An appropriate gene-carrying plasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer size gold particles using standard procedures. Each plate of embryos is shot with the DuPont Biolistics~
helium device using a burst pressure of .-1000 psi using a standard 80 mesh screen. After bombardment) the embryos are placed back into the dark to recover for about 24 h (still on osmoticum). After 24 hrs, the embryos are removed from the osmoticum and placed back onto induction medium where they stay for about a month before regeneration.
Approximately one month later the embryo explants with developing embryogenic callus are transferred to regeneration medium (MS + 1 mglliter NAA, 5 mglliter GA), further containing the appropriate selection agent (10 mg/l baste in the case of pCIB3064 and 2 mg/l methotrexate in the case of pSOG35). After approximately one month, developed shoots are transferred to larger sterile containers known as 'GA7s' which contain half-strength MS, 2% sucrose, and the same concentration of selection agent.
More recently) tranformation of monocotyledons using Agrobacterium has been described. See, WO 94/00977 and U.S. Patent No. 5,591,616, both of which are incorporated herein by reference.
Breeding The immunomodulated plants obtained via tranformation with an SAR gene such as a form of the NIMi gene can be any of a wide variety of plant species, including those of monocots and dicots; however, the immunomodulated plants used in the method of the invention are preferably selected from the list of agronomically important target crops set forth supra. The expression of the chosen 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) Second Edition, Ciarendon 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).
The genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in descendants plants. Generally said maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting. Specialized processes such as hydroponics or greenhouse technologies can also be applied. As the growing crop is vulnerable to attack and damages caused by insects or infections as well as to competition by weed plants, measures are undertaken to control weeds) plant diseases, insects) nematodes) and other adverse conditions to improve yield. These include mechanical measures such a tillage of the soil or removal of weeds and infected plants, as well as the application of agrochemicals such as herbicides, fungicides) gametocides, nematicides, growth regulants, ripening agents and insecticides.

Use of the advantageous genetic properties of the transgenic plants and seeds according to the invention can further be made in plant breeding, which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or stress, improved nutritional value, increased yield, or improved stnrcture causing less loss from lodging or shattering. The various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental tines, or selecting appropriate descendants plants. Depending on the desired properties, different breeding measures are taken. The relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding) variety blend, interspecific hybridization) aneuploid techniques, etc.
Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical) or biochemical means. Cross pollination of a male sterile plant with pollen of a different line assures that the genome ~of the male sterile but female fertile plant wili uniformly obtain properties of both parental lines.
Thus, the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines, that for example, increase the effectiveness of conventional methods such as herbicide or pestidice treatment or allow one to dispense with said methods due to their modified genetic properties. Alternatively new crops with improved stress tolerance can be obtained, which, due to their optimized genetic °equipment", yield harvested product of better quality than products that were not able to tolerate comparable adverse developmental conditions.
In seeds production, germination quality and uniformity of seeds are essential product characteristics, whereas germination quality and uniformity of seeds harvested and sotd by the farmer is not important. As it is difficult to keep a crop free from other crop and weed seeds) to control seedborne diseases, and to produce seed with gaol germination, fairly extensive and well-defined seed production practices have been developed by seed producers, who are experienced in the, art of growing, conditioning and marketing of pure seed. Thus, it is common practice for the farmer to buy certified seed meeting specific quality standards instead of using seed harvested from his own crop.
Propagation material to be used as seeds is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures thereof.
Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTDm), metaiaxyl (Apron'), and pirimiphos-methyl (Actellic~. !f desired) these compounds are formulated together with further carriers) surfactants or application-promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests. The protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods of application are also possible such as treatment directed at the buds or the fruit.
It is a further aspect of the present invention to provide new agricultural methods, such as the methods exemplified above, which are characterized by the use of transgenic plants, transgenic plant material, or transgenic seed according to the present invention.
The seeds may be provided in a bag, container or vessel comprised of a suitable packaging material, the bag or container capable of being closed to contain seeds. The bag) container or vessel may be designed for either short term or long term storage) or both, of the seed. Examples of a suitable packaging material include paper, such as kraft paper, rigid or pliable plastic or other polymeric material, glass or metal.
Desirably the bag, container, or vessel is comprised of a plurality of layers of packaging materials, of the same or differing type. In one embodiment the bag, container or vessel is provided so as to exclude or limit water and moisture from contacting the seed. In one example, the bag, container or vessel is sealed, for example heat sealed, to prevent water or moisture from entering. In another embodiment water absorbent materials are placed between or adjacent to packaging material layers. In yet another embodiment the bag, container or vessel, or packaging material of which it is comprised is treated to limit, suppress or prevent disease, contamination or other adverse affects of storage or transport of the seed.
An example of such treatment is sterilization, for example by chemical means or by exposure to radiation. Comprised by the present invention is a commercial bag comprising seed of a transgenic plant comprising a form of a NIMi gene or a NIM1 protein that is expressed in said transformed plant at higher levels than in a wild type plant, together with a suitable carrier, together with table instructions for the use thereof for conferring broad spectrum disease resistance to plants.
Application Of A Microbicide To Immunomodulated Plants As described herein, the inventive method of protecting plants involves two steps:
first, activating the SAR pathway to provide an immunomodulated plant) and second, applying a microbicide to such immunomodulated plants to attain synergistically enhanced disease resistance.

A. Conventional Microbicides According to the method of the present invention, any oommeraal or conventional microbicide may be applied to immunomodulated plants obtained through any of the three above-described routes. Examples of suitable microbicides include) but are not limited to, the following fungiades: 4-[3-(4-chlorophenyl)-3-(3,4-dimethoxyphenyl)acryloyl]morphoEine ("dimethomorph'), (reference: C. Tomlin {Editor): The Pesticide Manual, 10th edition, Famhan) UK, 1994, pages 351-352); 5-methyl-1,2,4-triazolo[3,4-b][1,3]benzothiazole ("tricyclazole'), (reference: C. Tomiin (Editor): The Pesticide Manual,10th edition, Farnham, UK,1994, pages 1017-1018); 3-allyloxy-1,2-benzothiazole-1,1-dioxide {"probonazole") ) (reference: C. Tomlin (Editor): The Pesticide Manual,10th edition, Farnham, UK,1994) pages 831-832);
a-[2-(4-chlorophenyl )ethyl]--a-( 1,1-dimethyiethyl)-1 H-1,2,4-triazoie-1-ethanol, ("tebuconazole"), (reference: EP-A-40 345);1-[[3-(2-chlorophenyl)-2-{4-fluorophenyl)oxiran-2-yl]methyl]-1 H-1,2,4-triazoie, (epoxyconazole"), (reference: EP-A-196 038); p-(4-chlorophenyl)-N-(1-cyclopropylethyl)-i H-1,2,4-triazole-1-ethanol, ("cyproconazole'), (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-(1 H-1,2,4-triazol-1-yl)-propyl-1,1,2,2-tetrafluoroethyl-ether) ("tetraconazole"), (reference: EP-A-234 242); methyl-(E)-2-{2-[6-(2-cyanophenoxy)pyrimidin--4-yloxy]phenyl}-3-methoxyacrylate) ("ICI A 5504', "azoxystrobin'), (reference: EP-A-382 375); methyl-(E)-2-methoximino--2-[a-(o-tolyloxy)--o-tolyl]acetate, ("BAS
490 F", "kresoxime methyl"), (reference: EP-A-400 417); 2-(2-phenoxyphenyl)-(E)-2-methoximino--N-methylacetamide, (reference: EP-A-398 692); [2-(2,5-dimethyiphenoxymethyl}-phenyi]-(E)-2-methoximino-N-methylacetamide, (reference: EP-A-398 692); (1 R,3S/1 S,3R)-2,2-dichloro--N-[(R)-1-(4-chlorophenyl)ethylj-1-ethyl-3-methylcyclopropanecarboxamide, ("KTU
3616"), (reference: EP-A-341 475); manganese ethylenebis(dithiocarbamate)polymer zinc complex, ("mancozeb'), (reference: US 2 974156); 1-[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-ylmethylJ-1 H-1,2,4-triazole) ("propiconazole'), (reference: GB-1522657); i-{2-[2-chloro-4-(4-chlorophenoxy)phenyl]-4-methyl-1,3-dioxolan-2-ylmethy I)-1 H-1,2,4-triazole, ("difenoconazoie"), (reference: GB-209860); 1-[2-(2,4-dichlorophenyl)pentyl-1 H-1,2,4-triazole, ("penconazole"), (reference: GB-1589852); cis-4-(3-(4-tert-butylphenyl)-2-methylpropyl]-2,6-dimethyimorpholine, ('fenpropimorph'), (reference: DE 2752135); 1-[3-(4-tert-butylphenyl~2-methyfpropyl]-piperidine) ("fenpropidin")) (reference: DE2752135); 4-cyclopropyl-6-methyl-~-phenyl-2-pyrimidinamine ("cyprodinil") (reference: EP-A-310550); (RS)-N-(2,6-dimethylphenyl-N-(methoxyacetyl)-alanine methyl ester ("metalaxyl')) (reference: G&1500581 );
(R}-N-(2,&
dimethyiphenyl--N-(methoxyacetyl)-atanine methyl ester ("R-metalaxyl'), (reference: G&
1500581 );1,2,5,&tetrahydro--4H-pyrroio[3,2,1-ij)quinoiin-4-one ("pyroquilon')) (reference: GB-i 394373); ethyl hydrogen phosphonate ("fosetyf~, (reference: C. Tomlin (Editor): The Pestiade Manual, 10th edition, Famhan, UK, 1994, pages 530-532); and copper hydroxide (reference: C.
Tomlin (Editor): The Pestiade Manual, 10th edition, Famhan) UK) 1994, pages 229-230).
The chosen microbiade is Nraferably applied to the immunomodulated plants to be protected in the form of a composition with further carriers, surfactants or other application-promoting adjwants customarily employed in formulation technology. Suitable carriers and adjwants can be solid or liquid and are the substances ordinarily employed in formulation technology) e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tack'rfiers, thickeners, binders or fertilizers.
A preferred method of applying a microbicidal composition is application to the parts of the plants that are above the soil, especially to the leaves (foiiar application).
The frequency and rate of application depend upon the biological and climatic living conditions of the pathogen. The microbicide can, however, also penetrate the plant through the roots via the soil or via the water (systemic action) if the locus of the plant is impregnated with a liquid fomlulation (e.g. in rice culture) or if the microbicide is introduced in solid form into the soil, e.g.
in the form of granules (soil application). In order to treat seed, the microbiade can also be applied to the seeds (coating), either by impregnating the tubers or grains with a liquid formulation of the microbicide, or by coating them with an already combined wet or dry formulation. In addition) in special cases, other methods of application to plants are possible) for example treatment directed at the buds or the fruit trusses.
The microbicide may be used in unmod~ed form or, preferably, together with the adjwants conventionally employed in formulation technology) and is therefore formulated in known manner e.g. into emulsifiable concentrates) coatable pastes) directly sprayable or dilutable solutions, dilute emulsions, wettable powders) soluble powders, dusts) granules, or by encapsulation in e.g. polymer substances. As with the nature of the compositions, the methods of application, such as spraying, atomising, dusting) scattering, coating or pouring, are chosen in accordance with the intended objectives and the prevailing circumstances.
Advantageous rates of application of the microbicide are normally from 50 g to 2 kg a.iJha, preferably from 100 g to 1000 g a.iJha, especially from 150 g to 700 g a.i./ha. In the case of the treatment of seed, the rates of application are from 0.5 g to 1000 g, preferably from 5 g to 100 g, a.i. per 100 kg of seed.

The formulations are prepared in known manner, e.g. by homogeneously mixing and/or grinding the microbicide with extenders, e.g. solvents) solid caniers and) where appropriate) surface-active compounds (surtactants).
Suitable solvents are: aromatic hydrocarbons, preferably the fracfions containing 8 to 12 carbon atoms, e.g. xylene mixtures or substituted naphthalenes, phthalates, such as dibutyl phthalate or dioctyl phthalate) aliphatic hydrocarbons) such as cyclohexane or paraffins, alcohols and glycols and their others ~d esters, such as ethanol, ethylene glycol, ethylene glycol monomethyl or monoethyl ether, ketones, such as cyclohexanone, strongly polar solvents) such as N-methyl-2-pyrrolidone, dimethyl sutfoxide or dimethylformamide, as well as vegetable oils or epoxidised vegetable oils, such as epoxidised coconut oil or soybean oil; or water.
The solid carriers used) e.g. for dusts and dispersible powders, are normally natural mineral fillers, such as calcite, talcum, kaolin, montmorillonite or attapulgite. In order to improve the physical properties it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. Suitable granulated adsorptive carriers are porous types, for example pumice, broken brick, sepiolite or bentonite, and suitable nonsorbent carriers are, for example, calcite or sand. In addition) a great number of pregranulated materials of inorganic or organic nature can be used, e.g. especially dolomite or pulverised plant residues.
Depending upon the nature of the microbicide) suitable surface-active compounds are non-ionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties. The term "surfactants" will also be understood as comprising mixtures of surfactants.
Particularly advantageous application-promoting adjuvants are also natural or synthetic phospholipids of the cephalin and lecithin series, e.g.
phosphatidylethanotamine) phos-phatidylserine, phosphatidylglycerol and lysolecithin.
The agrochemical compositions generally comprise 0.1 to 99 %, preferably 0.1 to 95 %, active microbicidal ingredient) 99.9 to 1 %, preferably 99.9 to 5 %, of a solid or liquid adjwant and 0 to 25 %, preferably 0.1 to 25 %) of a surfactant.
Whereas commercial products wilt preferably be formulated as concentrates, the end user will normally employ dilute formulations.
B. Plant Activating Microbicides If applied to immunomodulated plants obtained through the second or third above-described route (selective breeding or genetic engineering), the microbicide may alternately be a chemical inducer of SAR (plant activating microbicide) such as a benzothiadiazole compound, an isonicotinic acid compound) or a salicylic acid compound) which are -37- .
described in U.S. Patent Nos. 5,523,311 and 5,614,395. Hence, two methods of immunomodulation are concurrently employed. By applying plant activating microbicides to immunomodutated plants obtained through either a selective breeding route or a genetic engineering route, °extra-immunomodulation" results) and synergistically enhanced disease resistance is achieved.
As described below, transgenic immunomodulated plants overexpressing NIMi responded much faster and to much lowar doses of BTH, as shcwn by PR-1 gene expression and resistance to P. parasitica) than wild-type plants. See) Example 35 and the Northern blots in Figure 3. Synergistically enhanced disease resistance in overexpressors can be achieved with only lOwM BTH application) a concentration normally insufficient for ariy efficacy at alt. Normally phytotoxic or otherwise undesirable concentrations of SAR-inducing chemicals can be avoided by taking advantage of this synergy. In addition, one can take advantage of the alteration of the time-course of SAR
activation that occurs when SAR-inducing chemicals are applied to already-immunomodulated plants such as NIMi-overexpressors. Furthermore, economic gains can be realized as a result of the decreased quantity of SAR-inducing chemicals required to provide a given level of protection to plants.
C. Conventional Microbicides In Conjunction With Plant Activating Microbicides For even greater disease resistance) both a conventional microbicide and a plant activating rivcrobicide may be applied to immunomodulated plants obtained through either a selective breeding route or a genetic engineering route. This results in an even higher level of synergistic disease resistance compared to the level of disease resistance obtained through immunomodulation alone, through immunomoduiation plus only one type of microbicide, or through the simultaneous application of both types of microbicides (conventional and plant activating). See, for example) Table 35 in Example 19.
Disease Resistance Evaluation Disease resistance evaluation 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 Cell 8:629-643; Alexander et al., Proc. Natl. Acad. Sci. USA 90: 7327-7331 (1993). For example, several representative disease resistance assays are described below.

-38-.
A. Phytophthora parasitica (Black shank) Resistance Assay Assays for resistance to Phytophthora paras'rtica) the causative organism of black shank) are performed on six-week-old plants grown as described in Alexander et al., Proc.
Natl. Acad. Sci. USA 90: 7327-7331 (1993). Plants are watered, allowed to drain well, and then inoculated by applying 10 ml of a sporangium suspension (300 sporangia/ml) to the soil. Inoculated plants are kept in a greenhouse maintained at 23-25°C
day temperature, and 20-22°C night temperature. The wilt index used for the assay is as follows: 0=no symptoms; 1=no symptoms; 1=some sign of wilting, with reduced turgidity;
2=clear wilting symptoms, but no rotting or stunting; 3=clear wilting symptoms with stunting, but no apparent stem rot; 4=severe wilting, with visible stem rot and some damage to root system;
5=as for 4, but plants near death or dead, and with severe reduction of root system. All assays are scored blind on plants arrayed in a random design.
B. Pseudomonas syringae Resistance Assay Pseudomonas syringae pv. tabaci strain #551 is injected into the two lower leaves of several 6-7-week-old plants at a concentration of 106 or 3 x 10s per ml in H20. Six individual plants are evaluated at each time point. Pseudomonas tabaci infected plants are rated on a point disease severity scale) 5=100% dead tissue, 0=no symptoms. A T-test (LSD) is conducted on the evaluations for each day and the groupings are indicated after the Mean disease rating value. Values followed by the same letter on that day of evaluation are not statistically significantly different.
C. Cercospora nicotianae Resistance Assay A spore suspension of Cercospora nicotianae (ATCC #18366) (100,000-150,000 spores per ml) is sprayed to imminent run-off onto the surface of the leaves.
The plants are maintained in 100% humidity for five days. Thereafter the plants are misted with water 5-10 times per day. Six individual plants are evaluated at each time point.
Cercospora nicotianae is rated on a % leaf area showing disease symptoms basis. A T-test (LSD) is conducted on the evaluations for each day and the groupings are indicated after the Mean disease rating value. Values followed by the same letter on that day of evaluation are not statistically significantly different.

D. Peronospora parasitica Resistance Assay Assays for resistance to Peronospora parasltica are performed on plants as described in Uknes et al) (1993). Plants are inoculated with a combatible isolate of P.
parasitica by spraying with a conidial suspension (approximately 5 x 10° spores per milliliter). Inoculated plants are incubated under humid conditions at 17° C in a growth chamber with a 14-hr day/10-hr night cycle. Plants are examined at 3-14 days, preferably 7-12 days) after inoculation for the presence of conidiophores. In addition, several plants from each treatment are randomly selected and stained with lactophenol-trypan blue (Keogh et al.) Trans. Br. MycoL Soc. 74: 329-333 (1980)) for microscopic examination.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 is a sequence alignment of the NIM1 protein sequence with IxBa from mouse, rat, and pig. Vertical bars (1) above the sequences indicate amino acid identity between NIM1 and the IxBa sequences {matrix score equals 1.5); double dots (:) above the sequences indicate a similarity score >0.5; single dots (.) above the sequences indicate a similarity score <0.5 but >0.0; and a score <0.0 indicates no similarity and has no indicia above the sequences (see Examples). Locations of the mammalian IxBa ankyrin domains were identified according to de Martin et al., Gene 152, 253-255 (1995). The dots within a sequence indicate gaps between NIM1 and IxBa proteins. The five ankyrin repeats in IxBa are indicated by the dashed lines under the sequence. Amino acids are numbered relative to the NIM1 protein with gaps introduced where appropriate. Plus signs (+) are placed above the sequences every 10 amino acids.
FIGURE 2 is an amino acid sequence comparison of regions of the NIM1 protein (numbers correspond to amino acid positions in SEA ID N0:2) and rice EST protein products (SEQ ID NOs: 17-24).
FIGURE 3 presents the results of Northern analysis showing the time course of PR-1 gene expression in wild-type and NIMt-overexpressing lines following treatment with water or BTH. RNA was prepared from treated plants and analyzed as described in the Examples. "Ws" is the wild-type Arabidopsis thaliana Ws ecotype. "3A", "5B", "6E", and "7C" are individual NIMt-overexpressing plant lines produced according to Example 21. "0 BTH" is water treatment; "10 BTH" is 10 N.M BTH treatment; "100 BTH° is 100 pM BTH treatment. °0" is day zero control samples;
°1 ", "3") and °5° are samples at days 1, 3, and 5.

SEA ID N0:1 is a 5855-by genomic sequence comprising the coding region of the wild-type Arabidopsis tl~afiana h'!M1 gene.
SE4 ID NO:2 is the amino acid sequence of the wild-type Arabidopsis thalisna NIM1 protein encoded by the coding region of SEQ ID N0:1.
SEQ ID N0:3 is the mouse IxBa amino acid sequence from Figure 1.
SEGZ ID N0:4 is the rat IxBa amino acid sequence from Figure 1.
SEA ID N0:5 is the pig IxBa amino acid sequence from Figure 1.
SEQ ID N0:6 is the cDNA sequence of the Arabidopsis thaliana NIM1 gene.
SEQ ID NO's:7 and 8 are the DNA coding sequence and encoded amino acid sequence, respectively, of a dominant-negative form of the NIM1 protein having alanine residues instead of serine residues at amino acid positions 55 and 59.
SEQ ID NO's:9 and 10 are the DNA coding sequence and encoded amino acid sequence, respectively, of a dominant-negative form of the NIM1 protein having an N-terminal deletion.
SEA ID NO's:11 and 12 are the DNA coding sequence and encoded amino acid sequence, respectively, of a dominant-negative form of the NIM1 protein having a C-terminal deletion.
SEA ID NO's:13 and 14 are the DNA coding sequence and encoded amino acid sequence, respectively, of an altered form of the NIMI gene having both N-terminal and C-terminal amino acid deletions.
SEQ 1D NO's:15 and 16 are the DNA coding sequence and encoded amino acid sequence) respectively, of the anlryrin domain of NIMI.
SEQ 1D N0:17 is the Rice-1 AA sequence 33-155 from Figure 2.
SEA ID N0:18 is the Rice-1 AA sequence 215-328 from Figure 2.
SEQ 1D N0:19 is the Rice-2 AA sequence 33-155 from Figure 2.
SEQ ID N0:20 is the Rice-2 AA sequence 208-288 from Figure 2.
SEA ID N0:21 is the Rice-3 AA sequence 33-155 from Figure 2.
SEA ID N0:22 is the Rice-3 AA sequence 208-288 from Figure 2.

_ 41 _ _.
SEG1 ID N0:23 is the Rice-4 AA sequence 33-i 55 from Figure 2.
SEQ ID N0:24 is the Rice-4 AA sequence 215-271 from Figure 2.
SEQ ID NOs:25 through 32 are oligonucleotide primers.
DEFINITIONS
The following definitions will assist in the understanding of the present invention:
Plant Cell: the structural and physiological unit of plants) consisting of a protoplast and the cell wall. The term °piant cell" refers to any cell which is either part of or derived from a plant. Some examples of cells include differentiated cells that are part of a living plant; differentiated cells in culture; undifferentiated cells in culture; the cells of undifferentiated tissue such as callus or tumors; differentiated cells of seeds) embryos, propagules and pollen.
Plant Tissue: a group of plant cells organized into a stnrctural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants) plant organs, plant seeds) tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of) any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.
Protoplast: a plant cell without a cell wall.
Descendant Plant: a sexually or asexually derived future generation plant which includes, but is not limited to, progeny plants.
Transgenic Plant: a plant having stably incorporated recombinant DNA in its genome.
Recombinant DNA: Any DNA molecule formed by joining DNA segments from different sources and produced using recombinant DNA technology.
Recombinant DNA Technology: Technology which produces recombinant DNA in vitro and transfers the recombinant DNA into cells where it can be expressed or propagated (See, Concise Dictionary of Biomedicine and Molecular Biology) Ed. Juo, CRC
Press, Boca Raton (1996))) for example, transfer of DNA into a protoplast(s) or cells) in various forms, including, for example, (1 ) naked DNA in circular, linear or superooiled forms, (2) DNA contained in nucieosomes or chromosomes or nuclei or parts thereof, (3) DNA complexed or associated with other molecules) (4) DNA enclosed in liposomes, spheroplasts, cells or protoplasts or (5) DNA transferred from organisms other than the host organism (ex. Agrobacterium tumefiaciens). These and other various methods of introducing the recombinant DNA into cells are known in the art and can be used to produce the transgenic cells or transgenic plants of the present invention.
Recombinant DNA technology also includes the homologous recombination methods described in Treco et al., WO 94/12650 and Treco et al., WO 95/3 560 which can be applied to increasing peroxidase activity in a monocot.
Specifically) regulatory regions (ex. promoters) can be introduced into the plant genome to increase the expression of the endogenous peroxidase.
Also included as recombinant DNA technology is the insertion of a peroxidase coding sequence lacking selected expression signals into a monocot and assaying the transgenic monocot plant for increased expression of peroxidase due to endogenous control sequences in the monocot. This would result in an increase in copy number of peroxidase coding sequences within the plant.
The initial insertion of the recombinant DNA into the genome of the R°
plant is not defined as being accomplished by traditional plant breeding methods but rather by technical methods as described herein. Following the initial insertion, transgenic descendants can be propagated using essentially traditional breeding methods.
Chimeric Gene: A DNA molecule containing at least two heterologous parts, e.g., parts derived from pre-existing DNA sequences which are not associated in their pre-existing states) these sequences having been preferably generated using recombinant DNA technology.
Expression Cassette: a DNA molecule comprising a promoter and a terminator between which a coding sequence can be inserted.
Coding Sequence: a DNA molecule which) when transcribed and translated) results in the formation of a polypeptide or protein.
Gene: a discrete chromosomal region comprising a regulatory DNA sequence responsible for the control of expression, i.e. transcription and translation, and of a coding sequence which is transcribed and translated to give a distinct polypeptide or protein.
acd accelerated cell death mutant plant AFLP: Amplified Fragment Length Polymorphism avrRpt2: aviruience gene Rpt2, isolated from Pseudomonas syringae - .
BAC: Bacterial Artificial Chromosome BTH: benzo[1,2,3Jthiadiazole-7-carbothioic acid-S-methyl ester CIM: _Constitutive IMmunity phenotype (SAR is constitutively activated) cim: constitutive immunity mutant plant cM: centimorgans cprl: constitutive expresser of Phi genes mutant plant Col-O: Arabidopsis ecotype Columbia ECs: Enzyme combinations Emwa: Peronospora parasifica isolate compatible in the Ws-O ecotype of Arabidopsis EMS: ethyl methane sulfonate INA: 2,6-dichloroisonicotinic acid Ler: Arabidopsis ecotype Landsberg erects Isd. lesions simulating _disease mutant plant nahG: salicylate hydroxylase Pseudomonas putida that converts salicylic acid to catechol NahG: Arabidopsis line transformed with nahG gene ndr. non-race-specific _disease resistance mutant plant nim: non-inducible immunity mutant plant NlMI: the wild type gene) involved in the SAR signal transduction cascade NIMt: Protein encoded by the wild type NIMi gene niml: mutant allele of NlM7, conferring disease susceptibility to the plant;
also refers to mutant Arabidopsis ihaliana plants having the niml mutant allele of NIM1 Noco: Peronospora parasifica isolate compatible in the Col-O ecotype of Arabidopsis ORF: open reading frame PCs: Primer combinations PR: Pathogenesis Related SA: salicylic acid SAR: Systemic Acquired Resistance 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 Susceptible phenotype Wela: Peronospora parasitica isolate compatible in the Weiningen ecotype of Arabidopsis Ws-O: Arabidopsis ecotype Issilewskija WT: wild type YAC: Yeast Artificial Chromosome EXAMPLES
The invention is illustrated in further detail by the following detailed procedures) preparations) and examples. The examples are for illustration only, and are not to be construed as limiting the scope of the present invention.
Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, et al., Molecular Clonina, eds.) Cold Spring Harbor Laboratory Press) Cold Spring Harbor, NY (1989) and by T.J.
Silhavy) M.L.
German, and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and by Ausubel, F.M. et al., Current Protocols in Molecular Bioloav, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987).
I. Synergistic Disease Resistance Effects Achieved By Coordinate Application To Plants Of A
Chemical Inducer Of Systemic Acquired Resistance With A Conventional Microbicide In this set of examples, SAR was induced in plants by application of a chemical inducer of SAR such as a benzothiadiazole. In addition) conventional microbicides were applied to the plants. Plants were then subjected to disease pressure from various pathogens.
The combination of both methods of combating pathogens (inducing chemical +
microbicide) produced, greater-than-additive, i.e., synergistic, disease resistance. This was determined as the synergy factor (SF), i.e., the ratio of observed (O) effect to expected (E) effect.
The expected effect (E) for a given combination of active ingredients can be described by the so-called Colby formula and can be calculated as foltows (Colby, S.R.) "Calculating synergistic and antagonistic responses of herbicide combination". Weeds, Vol.15) pages 20-22 (1967)):
ppm = milligrams of active ingredient (= a.i.) per liter of spray mixture, X = % action caused by active ingredient I at a rate of application of p ppm of active ingredient, Y = % action caused by active ingredient It at a rate of application of q ppm active ingredient, E = expected effect of active ingredients I + II at a rate of application of p + q ppm of active ingredient (additive action).
Colby's formula reads E = X + Y - ~Y .

Example 1: Action Against Erysiphe graminis On Barley Residual-protective action: Barley plants about 8 cm in height were sprayed to drip point with an aqueous spray mixture (mex. 0.02 % active ingredient) and were dusted 3 to 4 days later with conidia of the fungus. The infected plants were stood in a greenhouse at 22°. Fungus infestation was generally evaluated 10 days after infection.
Systemic action: Barley plants about 8 cm in height were watered with an aqueous spray mixture (max. 0.002 % active ingredient, based on the volume of the soil).
Care was taken that the spray mixture did not come into contact with parts of the plants above the soil. The plants were dusted with conidia of the fungus 3 to 4 days later. The infected plants were stood in a greenhouse at 22°. Fungus infestation was generally evaluated i0 days after infection.
Table 1 Action against Erysiphe graminis on barley component I: benzothiadiazole-7-carboxylic acid component II: metconazole Test mg a.i. I:ll % action SF
no. per litre (ppm) comp. O (observed) O/E
I comp. E (expected) II

1 0.6 0 4 0.6 10 8 0.6 0.6 1:1 37 10 3.7 9 0.6 2 1:3 59 40 1.5 0.6 6 1:10 81 51 1.6 11 0.6 20 1:30 78 65 1.2 12 2 6 1:3 78 71 1.1 13 2 20 1:10 98 79 1.2 -46- w Table 2 Action against Erysiphe graminis on barley component t: benzothiadiazole-7-carboxylic acid component Il: tetraconazoie Test mg a.i. I:II % action SF
no. per Litre (ppm) comp. O (observed) O/E
I comp. E (expected) II

1 0.8 14 3 0.6 45 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 Table 3 Action against Erysiphe graminis on barley component t: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester component II: metconazole Test mg a.i. I:II % action SF
no. per litre (ppm) comp. O (observed) O/E
I comp. E (expected) 1 o.s o 6 0.6 6 1:10 33 17 1.9 7 0.6 20 1:30 50 33 1.5 8 0.6 60 1:100 83 50 1.7 _47-Example 2: Action Against Colleiotrichum lagenarium On Cucumis sabws L.
After a cultivation period of 10 to 14 days) cucumber plants were sprayed with a spray mixture prepared from a wettable powder formulation of the test compound.
After 3 to 4 days, the plants were infected with a spore suspension (1.0 x 105 sporeslml) of the fungus and incubated for 30 hours at high humidity and a temperature of 23°C.
Incubation was then continued at normal humidity and 22°C to 23°C. Lvaluation of protect'sve action was made 7 to days after infection and was based on fungus infestation.
After a cultivation period of 10 to 14 days, cucumber ptants were treated by soil application with a spray mixture prepared from a wettabie powder formulation of the test compound. After 3 to 4 days, the plants were infected with a spore suspension (1.5 x 105 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 and 22°C. Evaluation of protective action was made 7 to 10 days after infection and was based on fungus infestation.
Table 4 Action Against Colletotrichum lagerrarium On Cucumis sativus L. / Foliar Application component I: benzothiadiazole-7-carboxylic acid component II: azoxystrobin Test mg a.i. I:II % action SF
no. per litre (ppm) comp. O (observed) O/E
I comp. E (expected) II

1 0.06 0 2 0.2 5 4 0.06 5 5 0.2 9 6 0.6 12 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 Table 5 Action Against Colletotrichum lagenarium On Cucumis sativus L. l Soil Application component I: benzothiadiazole-7-carboxylic acid component II: azoxystrobin Test mg a.i. I:II % action SF
no. per litre (ppm) comp. O (observed) O/E
I comp. E (expected) II

1 0.006 0 2 0.02 40 3 0.06 49 4 0.2 91 0.2 0 6 0.6 9 s s ss 9 0.006 0.2 1:30 11 0 0.6 1:100 30 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 synergy factor SF cannot be calculated Table 6 Action Against Colletotrichum lagenarium On Cucumis sativus L. l Foliar Application component I: benzothiadiazole-7-carboxylic acid component Il: kresoxime methyl Test mg a.i. I:II % action SF
no. per litre (ppm) comp. O (observed) O/E
I comp. E (expected) II

1 0.2 3 2 0.6 51 5 0.2 2 1:10 15 3 5 6 0.2 20 1:100 61 43 1.4 Table 7 Action Against Colletotrichum lagenarium On Cucumis sativus L. / Foliar Application component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester component I1: azoxystrobin Test mg a.i. I:II % action SF
no. per litre (ppm) comp. I O (observed) O/E
comp. E (expected) II

1 0.06 16 2 0.2 22 6 0.06 2 1:30 43 31 1.4 7 0.2 2 1:10 57 36 1.6 Table 8 Action Against Colletotrichum lagenarium On Cucumis sativus L. / Soil Application component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester component II: azoxystrobin Test mg a.i. I:II % action SF
no. per litre (ppm) comp. I O (observed) O/E
comp. E (expected) II

1 0.006 0 2 0.02 6 3 0.06 23 4 0.2 3fi 5 0.02 1 6 0.06 5 7 0.6 27 s 2 s1 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 92 70 i .3 i 7 0.2 2 1:10 93 75 1.2 Example 3: Action Against Ceraospore nicobanae On Tobacco Plants Tobacco plants (6 weeks old) were sprayed with a formulated solution of the test compound (concentration: max. 0.02 % active ingredient). Four days after treatment, the plants were inoculated with a sporangia suspension of Cercospora nico~anae (150,000 spores/ml) and kept at high humidity for 4 to 5 days and then incubated further under a normal day/night sequence.
Evaluation of the symptoms in the tests was based on the leaf surface infested with fungus.
Table 9 Action Against Cercospora nicotianae On Tobacco Plants component t: benzo(1,2,3nhiadiazole-7-carbothioic acid-S-methyl ester component It: tebuconazole Test mg a.i. l:ll % action SF
no. per litre (ppm) comp. I O (observed} O/E
comp. E (expected) ll 1 0.2 0 7 0.2 2 1:10 87 0 8 0.2 6 1:30 97 0 9 2 2 1:1 87 17 5.1 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 20 2 10:1 97 78 1.2 14 20 6 3:1 97 78 1.2 WO 98/29537 PCT/EP97l07253 -51 - w Table 10 Action Against Cercospora nicotianae On Tobacco Plants component I: benzo[1,2,3Jthiadiazole-7-carbothioic acid-rmethyl ester component II: cyproconazole Test mg a.i. I:II % action SF
no. per litre (ppm) comp. O (observed) O/E
I comp. E (expected) II

1 0.2 0 6 6 ~ 0 7 0.2 2 1:10 78 0 8 0.2 6 1:30 84 0 ' 9 2 2 1:1 90 17 5.3 2 6 1:3 94 i 7 5.5 11 6 2 3:1 87 55 1.6 12 6 6 1:1 93 55 1.7 13 20 2 10:1 100 78 1.3 14 20 6 3:1 100 78 1.3 Table 11 Action Against Cercospora nicotianae On Tobacco Plants component I: benzothiadiazole-7-carboxylic acid component II: fenpropimorph Test kg of 1:11 % action SF
no. a.i. O (observed) O/E
per ha E (expected}
comp.
I comp.
II

0 -- -- 0 (control) 1 0.2 0 2 0.6 3 5 ~ 2 I 13 8 0.2 2 1:10 52 13 4 9 0.2 6 1:30 61 23 2.7 0.6 2 1:3 71 16 4.4 11 6 6 1:1 100 83 1.2 Table 12 Action Against Cercospora nicotianae On Tobacco Plants Component I: benzothiadiazole-7-carboxylic acid Component II: difenoconazole Test kg of I:II % action SF
no. a.i.
per ha comp. O (observed) O/E
I comp. E (expected) II

0 - -- 0 (control) 4 0.6 3 7 2 0.6 3:1 90 70 i .3 8 6 0.6 10:1 100 80 1.3 Example 4: Action Against Pyricularta oryzae On Rice Plants Rice plants about 2 weeks old were placed together with the soil around the roots in a container filled with spray mixture (max. 0.006 % active ingredient). 96 hours later) the rice plants were infected with a conidia suspension of the fungus. Fungus infestation was evaluated after incubating the infected plants for 5 days at 95-100 % relative humidity and about 24°C.

Table 13 Action Against Pyricularia oryzae On Rice Plants component I: benzo[1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester component II: KTU 3616 Test mg a.i. I:II % action SF
no. per litre (ppmj comp. I O (observed) O/E
comp. E (expected) II

2 0.02 0 3 0.06 28 0.2 47 O.fi 79 8 6 0.02 300:1 42 15 2.8 9 6 0.06 100:1 76 39 1.9 6 0.2 30:1 98 55 1.8 1 i 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 On a 12m2 plot, rice plants were sprayed with a spray mixture prepared with a wettable powder of the active ingredient. Infection was naturally. For evaluation, the leaf area infested with the fungus was measured 44 days post-application. The following results were obtained:
Table 14 Action Against Pyricularia oryzae On Rice Plants in the open Component I: benzo[1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester Component II: pyroquilon Test kg of a.i. I:II % action SF
no. per ha ' comp. comp. II O (observed)E (expected)O/E
I

- -- -- 0 (control) _~_ 1 0.25 22 2 0.5 50 3 0.75 46 4 1.5 82 0.25 0.75 1:3 80 58 1.4 6 0.5 0.75 1:1.5 85 73 1.2 Rice plants about 2 weeks old were placed together with the soil around the roots in a container filled with spray mixture. Fungus infestation was evaluated 36 days later. Infestation of the untreated plants corresponded to 0 % action.
Table 15 Action Against Pyricularia oryzae On Rice Plants Component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component II: tricyclazole Test mg a.i. l:ll % action SF
No. per litre (ppm) comp. O (observed) O/E
I comp. E (expected) II

1 0.5 65 2 0.25 39 3 0.1 18 4 0.05 5 8 0.5 71 7 0.25 48 8 0.1 32 9 0.25 0.25 1:1 75 68 1.1 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 Example 5: Action Against Colletotrichum sp. (Anthracnose) and Cercospora sp. (Leaf Spot) On Chili Effects on crop yield: In a plot of land about 10 m2 (test location: Cikampek, Java, Indonesia), chili plants were sprayed a total of 7 times at intervals of about 7 days with 500-700 litres of spray mixture per hectare. Three days after the first spraying, the plants were infected artificially with the fungus.
Table 16 Action Against Colletotrichum: Evaluation was made by assessing infestation on the chili fruits after the fifth spraying.
component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester component II: mancozeb Test mg a.i. I:II % action SF
no. per litre (ppm) comp. I O (observed) O/E
comp. E (expected) II

3 5 100 1:20 77 59 1.3 Table 17 Action Against Cercospora: Evaluation was made by assessing infestation on the leaves after the sixth spraying.
Component I: benzo[1,2,3Jthiadiazole-7-carbothioic acid-S-methyl ester Component II: mancozeb Test mg a.i. I:II % action SF
no. per litre (ppm) comp. I O (observed) OIE
comp. E (expected) II

3 5 100 1:20 87 78 1.1 Table 18 Action On Crop Yield: The chills were harvested after the sixth spraying.
Component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component II: mancozeb Test mg a.i. I:II Crop yield SF
no. per litre in kg per (ppm) hectare comp. I O (observed) O/E
comp. E (expected) II

3 5 100 1:20 1400 ca 460 ca Example 6: Action Against Puccinia recondite In Wheat 7-day-old wheat plants were sprayed to drip point with a spray mixture prepared from a formulated active ingredient, or combination of active ingredients. After 4 days, the treated plants were infected with a conidia suspension of the fungus, and the treated plants were subsequen~y incubated for 2days at a relative atmospheric humidity of 90-100% and 20 C.10 days post-infection, the fungus infestation was assessed.
Table 19 Action Against Puccinia recondite In Wheat Component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component II: propiconazole Test mg of a.i. I:II % action SF
no. per litre O (observed) O/E
comp. I E (expected) comp.
II

- - -- 0 (control) 3 100 5 20:1 79 56 1.4 - 57 _ _ Table 20 Action Against Puccinia recondita In Wheat Component t: benzothiadiazole-7-carboxylic acid Component II: fenpropidine Test kg of a.i. I:II % action SF
no. per ha I comp. I O {observed) OIE
comp. E (expected) II
-- _ _- 0 {~ntrol) 4 fi0 60 6 20 1:3 73 52 1.4 6 6 20 1:10 75 68 1.1 Example 7: Action Against Erysiphe graminis In wheat In field trials (10m2), winter wheat in the growth phase was sprayed with a spray mixture prepared with a wettable powder of the active ingredient. Infection was naturally. 10 days post-infection) the fungus infestation was assessed. The following results were obtained:
Table 21 Action Against Erysiphe graminis In Wheat Component I: benzo[i ,2,3)thiadiazole-7-carbothioic acid-S-methyl ester Component I1: propiconazole Test g of a.i. 1:11 % action SF
no. per ha comp. I O {observed) O/E
comp. E (expected) ll - - - 0 (control) 4 5 50 1:10 49 32 1.5 5 5 100 1:20 59 51 1.2 WO 98/2953? PCT/EP97/0?253 Table 22 Action Against Erysiphe graminis In Wheat Component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component I1: cyprodinil Test g of a.i. till % action SF
no. per ha comp. I O (observed) O/E
comp. E (expected}
il - -- -- 0 (control) 4 5 50 1:10 49 32 1.5 5 100 1:20 59 51 12 Example 8: Action Against Mycosphaerella fijiensis In Bananas 40 banana plants in a 300m2 plot were sprayed at 17-19 day intervals with a spray mixture prepared with the wettable powder of the active ingredient; in total 6 times.
Infection was naturally. For evaluation, the leaf infested with the fungus was measured. The following results were obtained:
Table 23 Action Against Mycosphaerella fijiensis fn Bananas Component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component II: propiconazole Test g of a.i. I:II % action SF
no. per ha O (observed) OlE
comp. I E (expected) comp.
II

- -- -- 0 (control) 3 50 50 1:1 46 40 1.15 Example 9: Action Against Altemaria solani In Tomatoes Tomato plants on a 7m2 plot were sprayed at 7-day intervals with a spray mixture prepared with a wettable powder of the active ingredient; in total 9 times. Infection was naturally. For evaluation, the leaf infested with the fungus was measured. The following results were obtained:
Table 24 Action Against Altemaria solani in Tomatoes in the open Component I: benzo[1,2,3Jthiadiazole-7-carbothioic acid-S-methyl ester Component II: cyprodinil Test g of a.i. l:ll % action SF
no. per ha comp. O (observed) O/E
I comp. E (expected) ll _ ~ - 0 (control) 1 2.5 32 2 12.5 30 4 2.5 12.5 1:5 79 53 1.5 2.5 25 1:10 80 67 1.2 Example 10: Action Against Phytophthora infestans In Tomatoes Tomato plants cv. 'Rotor Gnom' were sprayed to drip point with a spray mixture prepared with the fomlulated active ingredient, or combination of active ingredients. After 4 days, the treated plants were sprayed with a sporangia suspension of the fungus and subsequently incubated in a cabinet for 2 days at 18-20 C and a retative atmospheric humidify of 90-100%. 5 days post-infection, the fungus infestation was assessed. The following results were obtained:

Tabis 25 Action Against Phytophthora infestans in Tomatoes Component 1: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component tl: metalaxyl Test mg of a.i. I:II % action SF
no. per litre comp. I O (observed) O/E
comp. E {expected) II

- - - 0 (control) 0.1 13 9 5 0.1 50:1 50 25 2.0 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 1.1 100 50 2:1 95 88 1.1 16 500 10 50:1 97 82 1.2 Table 26 Action Against Phytophthora infestans In Tomatoes Component i: benzothiadiazole-7-carboxylic acid Component II: metalaxyl Test mg of a.i. 1:11 % action SF
no. per litre comp. I comp. II O (observed)E (expected)O/E

- - -- 0 (control) 1 0.1 0 2 0.5 9 0.1 1 1:10 36 13 2.8 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 in Cucumbers 16-19-day-old cucumber plants ("Wisconsin") were sprayed to drip point with a spray mixture prepared with the formulated active ingredient) or combination of active ingredient) or combination of active ingredients. After 4 days, the treated plants were infected with sporangia of Pseudoperonospora cubenswas (strain 365) Ciba; max. 5000 per ml)) and the treated plants were subsequently incubated for 1-2 days at 18-20 C and a relative atmospheric humidity of 70-90%. 10 days post-infection, the fungus infestation was assessed and compared with the infestation on untreated plants. The following results were obtained:
Table 27 Action Against Pseudoperonospora cubensis In Cucumbers Component l: benzothiadiazole-7-carboxylic acid Component II: metalaxyl Test mg of I:II % action SF
no. a.i.
per litre comp. O (observed) O/E
I comp. E (expected) II

- -- -- 0 (control) 1 0.05 0 2 0.5 6 4 0.5 31 7 0.05 0.5 ~ 1:10 66 31 2.1 8 0.05 ~ 1:100 83 66 1.3 9 0.5 0.5 1:1 83 35 2.4 0.5 5 1:10 83 68 1.2 Example 12: Action Against Peronospora tabacina On Tobacco Plants Tobacco plants (6 weeks old) were sprayed with a formulated solution of the test compound. Four days after treatment) the plants were inoculated with a sporangia suspension of the fungus) kept at high humidity for 4 to 5 days and then incubated further under a normal day/-night sequence. Evaluation of the symptoms in the tests was based on the leaf surface infested with fungus. The infestation of the untreated plants corresponded to 0 %
action.
Table 28 Action Against Peronospora tabacina On Tobacco Plants Component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component II: dimethomorph Test mg a.i. I:II % action SF
No. per litre (ppm) comp. I O (observed) O/E
comp. E (expected) II

1 0.03 14 2 0.1 34 3 0.3 88 4 0.3 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 i 3: Action Against Peronospora parasitica In Arabidopsis thaliana The fungicides metalaxyl) fosetyl, and copper hydroxide, and the SAR activator benzo(1,2,3)-thiadiazole-7-carbothioc acid S-methyl ester (BTH), formulated as 25%, 80%, 70%) and 25% active ingredient (ai) respectively) with a wettable powder carrier, were applied as fine mist to leaves of three week-old plants. The wettable powdQr alone was applied as a control. Three days later, plants were inoculated with a Peronospora parasidca conidial suspension as described in Delaney et al. (1995). Ws plants were inoculated with the compatible P. parasitica isolate Emwa (1-2 x 105spores/ml); Col plants were inoculated with the compatible P. parasitica isolate Noco2 (0.5-1 x 105 sporesJml). Following inoculation) plants were covered to maintain high humidity and were placed in a Percival growth chamber at 17°C
with a 14-hr day/10-hr night cycle (Uknes et al.,1993). Tissue was harvested 8 days after inoculation.
Fungal infection progression was followed for 12 days by viewing under a dissecting microscope to score development of conidiophores (Delaney, et aL (1994);
Dietrich, et al.
(1994)). Lactophenoltrypan blue staining of individual leaves was carried out to observe fungal growth within leaf tissue. Fungal growth was quantified using a rRNA fungal probe that was obtained by PCR according to White et al. (1990; PCR Protocols: A guide to Methods and Application, 315-322) using primers NS1 and NS2 and P. parasit<ca EmWa DNA as templates.
RNA was purified from frozen tissue by phenoUchloroform extraction following lithium chloride precipitation (Lagrimini et al) 1987: PNAS, 84: 7542-7546). Samples (7.5 ug) were separated by electrophoresis through formaldehyde agarose gels and blotted to nylon membranes (Hybond-N+, Amersham) as described by Ausbel et al. (1987). Hybridizations and washing were according to Church and Gilbert (1984, PNAS, 81: 1991-1995). Relative amounts of the transcript were determined using a Phosphor Imager ( Molecular Dynamics, Sunnyvale, CA
following manufacturers instructions. Sample loading was normalized by probing stripped filter blots with the constitutively expressed b-tubulin Arabidopsis cDNA. The infestation of the untreated plants corresponded to 0 % fungal growth inhibition. The following results were obtained:

Table 29 Action Against Perr~nospora parasitica NoCo2 In Arabidopsis thaliana {Col-0) Component 1: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component If: metalaxyl Test Components Fungal Growth Synergy no. Inhibition Factor BTH metalaxyl % O/E

O (observed}
E (expected) control-- -- 0 1 0.01 mM -- 0 2 -- 0.1 mg/I 0 3 0.01 mM 0.1 mg/I 40.7 0 Table 30 Action Against Peronospora parasitica Emwa In Arabidopsis fhaliana (Ws) Component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component If: metalaxyl Test Components Fungal Growth Synergy no. Inhibition %

Factor BTH metaiaxyl O (observed) O/E
E (expected) control-- -- 0 1 0.01 mM 20 2 0.003 mM 0 3 2.5 mg/I 75 4 0.5 mg/l 50 0.1 mg/l 50 6 0.01 mM 2.5 mg/1 100 90 1.1 7 0.01 mM 0.5 mg/l 95 70 1.4 8 0.01 mM 0.1 mg/I 88 70 ~ 1.3 9 0.003 mM 2.5 mg/I 100 75 1.3 Table 31 Action Against Peronospora parasitica Emwa In Arabidopsis thaliana (Ws) Component t: benzo[1 (2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component II: fosetyl Test Components Fungal Growth Synergy no. Inhibition %

Factor BTH fosetyl O (observed) O/E
E (expected) control - - 0 1 0.01 mM 30 1.0 g/I 40 3 0.2 g/I 10 4 0.04 g/I 0 0.01 m 1.0 g/I 100 70 1.4 M

6 0.01 m 0.2 gll 100 40 2.5 M

7 0.01 mM 0.04 g/I 95 30 3.2 Table 32 Action Against Peronospora parasidca Emwa In Arabidopsis thaliana (Ws) Component I: benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester Component li: copper hydroxide Test Components Fungal Growth Synergy no. Inhibition Factor BTH Cu(OH)Z % O/E

O (observed) E (expected) control -- -- 0 1 0.01 m - 30 M

2 0.01 g/I 0 3 0.01 mM 0.01 g/l 85 30 2.8 As can be seen in Table 29, synergistic disease-resistant effects were demonstrated in the wild-type Arabidopsis Col-0 plants. No fungal growth inhibition was observed by separately applying either 0.01 mM BTH or 0.0001 g/L metalaxyl to the plants, because these concentrations are normally insufficient for efficacy. However, by applying both of these compounds to the plants at these normally insufficient concentrations, 40.7% fungal growth inhibition was observed) which is clearly a synergistic effect. Tables 30-32 show synergistic disease-resistant effects in wild-type Arabidopsis Ws plants. Only 20-30%
fungal growth inhibition was observed by applying 0.01 mM BTH to the Ws plants.
However) by simultaneously applying BTH and either metalaxyl, fosetyl) or copper hydroxide to the plants, synergistic disease resistance was observed. These combined antifungal effects, which result in a decrease in the effective concentration of the fungicide and BTH required for pathogen control) allow the reduction of the chemical dose needed to stop fungal growth and therefore mitigate the incidence of foliar damage due to chemical tolerance.
I I. Synergistic Disease Resistance Effects Achieved By Application Of Conventional Microbicides and/or Chemical Inducers of Systemic Acquired Resistance To Constitutive Immunity (CIM) Mutant Plants In this set of examples, a high-throughput Northern blot screen was developed to identify mutant plants having high concentrations of PR-1 mRNA during normal growth, with the idea that these mutants also exhibit systemic acquired resistance. A number of mutants have been isolated using this screen and they have been shown to accumulate not only PR-1 but also PR-2 and PR-5 mRNAs (Lawton et al. (1993); Dietrich et al. (1994); and Weymann et al. (1995).
These mutants also have elevated levels of SA and are resistant to pathogen infection, confirming that this approach can be used to isolate SAR signal transduction mutants.
Two classes of SAR signal transduction mutants have been isolated using this screen.
One class has been designated as Isd mutants (Isd = lesion simulating disease). This class of mutants is also referred to as "cim Class I" as disclosed in WO 94/16077 the disclosure of which is hereby incorporated by reference in its entirety. . This Isd class (aka am Class I) fom~ed spontaneous lesions on the leaves, accumulated elevated concentrations of SA, high levels of PR-1, PR-2 and PR-5 mRNA and was resistant to fungal and bacterial pathogens (Dietrich et al., 1994; Weymann et al.) 1995).
The second class, called cim (cim = constitu~ve immunity), is described below and has all the characteristics of the Isd mutants except spontaneous lesions. This second class (am) corresponds to the "cim Class II° mutants discussed WO 94/16077. The cim3 mutant plant line described below fails into this cim class (cim Class I I) and is a dominant mutation with wild-type appearance that expresses stable) elevated levels of SA, SAR gene mRNA and has broad spectrum disease resistance.

-s7-Example 14: Isolation and Characterization of cim Mutants With Constitutive SAR Gene Expression 1100 individual M2 mutagenized (EMS) Arabidopsis plants were grown in Aracon trays (Lehle Seeds, Round Rock, TX) in sets of approximately 100. Plank were grown as described in Uknes et al.) 1993, supra) with special attention given to avoid over-watering and pathogen infection. Briefly, Metro Mix 360 was saturated with water and autoclaved three times for 70 minutes in 10-liter batches. The potting mix was stirred thoroughly in between each autoclaving.
Seeds were surface sterilized in 20% Clorox for 5 minutes and washed with seven changes of sterile water before sowing. Planted seeds were vemalized for 3-4 days followed by growth in chambers with a 9 hour day and 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) mini-RNA preparation (Verwoerd et al. (1989) Nua Acid Res.17, 2362).
PR-1 gene expression was analyzed by Northern blot analysis (Lagrimini et al.
(1987) Proc. Natl.
Acad. Sci. USA 84, 7542-7546; Ward et al.,1991 ). Each set of plants also contained a non-treated A. thaliana Col-0 and a 2-day INA-treated (0.25 mglml) control. All plants were maintained as described in Weymann et al.) (1995).
80 putative mutants accumulating elevated levels of PR-1 mRNA were identified.
Following descendants testing, five were chosen for further characterization.
Putative cim mutants displayed elevated SAR gene expression in the absence of pathogen or inducing treatment. Descendants testing of the putative cim mutants confirmed that constitutive PR-1 expression was heritable. Of the cim mutants, two, cim2 and cim3, with the highest, most stable expression of PR-1 were characterized further.
Back crosses to Columbia utilized the recessive glabrous trait as a marker for identfication of Fi descendants. Col-gl1 flower buds were emasculated prior to pollen shed) and pollen from the mutants was applied immediately and the following day. F1 plants were grown in soil and the out crossed plants were identified by the presence of trichomes.
Following crosses of cim2 and cim3 to ecotype Col-0 or La-er, a large proportion of Fi plants were identified with high SAR gene expression, suggesting these traits were dominant. In the case of cim2, some, but not all) F1 plants had constitutive SAR gene expression. Such a result would be expected ff the cim2 mutant were dominant and carried as a heterozygote in the parent. Further genetic testing of cim2 showed continued variable segregation in the F2 generation, consistent with incomplete penetrance.
am3 demonstrated a 1:1 segregation in the F1 generation whereupon two individual Fi plants expressing a high level of PR-1 mRNA were selfed to form an F2 population. F2 segregation) obtained by scoring PR-1 mRNA accumulation,, showed 93 F2 plants with high PR-1 mRNA and 25 F2 plants without sign~cant PR-1 mRNA accumulation giving a 3.7:1 ratio (c2 =
1.77; 0.5 > P > 0.1 )) which is consistent with the hypothesis that cin:3 ss a dominant, single gene mutation. Subsequent outcrosses confirmed that cim3 was inherited as a dominant mutation.
For cim3, the original M2 plant identified in the screen and the M3 population appeared normal. However, as the cim3 plants were selfed some of the best expressing lines had low fertility. Following the back cross to Col-gl1, plants with normal appearance and fertility and strong PR-1 expression were obtained.
When initially identified, cim3 also appeared slightly dwarted with thin, distorted leaves.
However, F2 plants resulting from a cross with ecotype Col-gl1 retained high SAR gene expression and could not be distinguished from wild-type plants. This suggested that the dwarfed, distorted-leaf phenotype was caused by an independent mutation that was not associated with constitutive SAR gene expression. The cim3 mutant phenotype was also observed when plants were grown in sterile conditions confirming that PR-1 mRNA accumulation was not caused by a 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. Levels of SAR gene expression varied between the descendants, but were always more than 10-fold higher than the untreated control and similar to the levels obtained following a resistance-inducing INA (0.25 mg/ml) treatment of wild-type plants.
Example i 6: Salicylic Acid Analysis Endogenous concentrations of SA have been shown to increase following pathogen-induced necrosis in Arabidopsis (Uknes et aL) 1993, supra). Salicylic acid and its glucose conjugate were analyzed as described in Uknes et al.) 1993. Leaf tissue was harvested from 10 cim3 and 10 control, 4 week-old plants. Leaves from individual plants were harvested and analyzed for PR-1 gene expression. SA levels were measured from plants expressing PR-1. The concentration of free SA in cim3 was 3.4-fold higher than in non-infected wild-type An~bidopsis (233135 vs. 6918 nglg fresh weight, respectively). The glucose conjugate of SA
(SAG) was 13.1 fold higher in cim3 than in non-infected wild-type Arabidopsis (45191473 vs. 344158 ng/g fresh weight, respectively). These increased levels of SA and SAG are comparable to the levels that have been reported for either pathogen-infected tissue or the cpr mutant.
Example 17: ~ Disease Resistance cim3 was evaluated for resistance to Peronospora parasitica (NoCo2)) the causal agent of downy mildew disease of Arabidopsis. Thirty cim3 (confirmed by PR-1 RNA
expression) and thirty control plants (ecotype Columbia)) each about 4 weeks oki, were inoculated with P.
parasitica, as described in Uknes, et al.1992, supra. Seven days later, plants were analyzed for sporulation and stained with trypan blue to visualize fungal stnrctures, as described in Keogh et al. (1980) Trans. Br. Mycol. Soc. 74) 329-333, and in Koch and Slusarenko (1990) Plant Cell2) 437-445. Wild-type (Col-0) plants support the growth of hyphae, conidia, and oospores, while wild type plants treated with INA (0.25 mg/ml) and cim3 plants showed no fungal growth. The cim3-mediated resistance is typically seen as a small group of dead cells at the site of pathogen infection. This type of resistance is similar to that seen in Isd mutants (Dietrich et al., 1994, supra; Weymann et al., 1995, supra), or in wild-type plants in which SAR has been induced (Uknes et al.) 1992) supra). Occasionally) intermediate resistance phenotypes were observed, including trailing necrosis in the wake of the hyphal tip in cim3 plants. This trailing necrosis is similar to that found in wild-type ptants treated with low doses of SA or INA
(Uknes et al., 1992, supra; Uknes et al.,1993, supra). However, sporutation was never observed on cim3 plants while all control plants showed sponrlation. No spontaneous Lesions were observed on uninoculated cim3 leaves when stained with trypan blue.
In addition to resistance to the fungal pathogen P. pargsitica, cim3 was also resistant to infection with the bacterial pathogen Pseudomonas syringae DC3000. Six-week-old wild-type (t INA treatment), and cim3 plants were inoculated with a suspension of P.
syringae DC3000 and the progress of the disease was followed by monitoring the growth of the bacteria extracted from infected leaves over time. The difference in bacterial titers between Col-O) Col-O + INA and cim3 at either day 0 or day 2 was not statistically significant. However) by day four, there was a 31-fold decrease in bacterial growth between wild-type and am3 plants (P<
0.003; Sokal and Rohlf,1981 ). The plants were also visually inspected for disease symptoms.
Leaves from wild-type plants were severely chlorotic with disease symptoms spreading well beyond the initial zone of injection. In contrast, either wild-type plants pretreated with INA or am3 plants were nearly devoid of disease symptoms.
For this example) cultures of Pseudomonas syringae pv. tomato strain DC3000 were grown on King's B media (agar plates or liquid) plus rifampicin (50 Ng/ml) at 28°C (Walen et al.
(199i) Plant Cell3, 49-59). An overnight culture was diluted and resuspended in 10 mM MgCl2 to a density of 2-5 x 105 cells per ml and injected into Arabidopsis leaves.
Injecfions were carried out by creating a small hole with a 28 gauge needle midway up the leaf and then injecting approximately 250 NI of the diluted bacterial solution with a 1 cc syringe. At various time points) random samples consisting of 3 random leaf punches from a #1 cork borer were taken from 10 plants from each treatment. The 3 leaf punches were placed in an eppendorf tube with 300 NI of 10 mM MgCl2 and ground with a pestle. The resul~ng bacterial suspension was appropriately diluted and plated on King's B media plus rifampicin (50 Ng/ml) and grown for 4 days at 28°C. Bacterial colonies were counted and the data were subjected to Student's t statistical analysis (Sokal and Rohlf (1981 ), Biometry) 2"~ ed. New York:
W.H. Freeman and Company).
Also for this example, 2,6-Dichloroisonicotinic acid (INA) was suspended in sterile, distilled water as a 25% active ingredient formulated in a wetable powder (0.25 mglml, 325 NM;
Kessmann et al. (1994) Annu. Rev. Phytopaihol. 32, 439-59). All plants were sprayed with water or INA solutions to the point of imminent runoff.
Example 18: The Role of SA in SAR Gene Expression and Disease Resistance To investigate the relationship between SA, SAR gene expression and resistance in cim3) crosses were carried out with Arabidopsis plants expressing the salicylate hydroxylase (nahG) gene (Deianey et al.,1994). These "NahG plants" were made by transformation of the 35S driven nahG gene into Arabidopsis using Agrobacteriunrmediated transformation. See, Huang, H. Ma, H. (1992) Plant Mol. 9iol. Rep. 10, 372-383) herein incorporated by reference;
Gaffney, et al. (1993) Science 261, 754-756, herein incorporated by reference;
and Dslaney, et al. (1994) Science 266,1247-1250, herein incorporated by reference. Col-nahG
Arabidopsis carries a dominant kanamycin resistance gene in addition to the dominant nahG
gene, so Col-nahG was used as the pollen donor. F1 seed was hydrated in water for 30 minutes and then surface sterilized in 10% Clorox, .05% Tween 20 for five minutes and washed thoroughly in sterile water. Seeds were plated onto germination media (GM, Murashige and Skoog medium containing 10g/L sucrose buffered with 0.5 gIL 2-(N-morpholino) ethanesulfonic aad, pH 5.7 with KOH) containing 25 mglmi kanamyan to select for F, plants. See Valvekens et al. (1988) Proc.
Natl. Aced. Sci) USA 85) 5536-5540. Kanamyan resistant F~ plants were transferred to soil after 18 days. The presence of the nahG gene and PR-1 expression was confirmed in all experiments by Northern blot analysis.
Because both the ~m3 mutant and nahG phenotypes are dominant, epistasis between the two genes could be analyzed in F1 plants. Seventy F1 plants from a am3 X
nahG cross were analyzed for PR-1 and nahG gene expression. In Northern blot analysis of mRNA
expression, the presence of the nahG gene correlated with suppressed SAR gene expression.
The presence of am3 in each Fi was confirmed by assessing PR-1 mRNA in the resulting F2 segregants.
To determine if the cim3 mutation was epistatic to nahG with respect to disease resistance, 5 F1 plants from the cim3 X nahG cross, which had been confirmed for the presence of nahG and absence of PR-1 mRNA) were selfed and 20-30 F2 seed were planted.
Expression of nahG and PR-1 mRNA was analyzed in individuals from this F2 population, which were then challanged with P. parasitica (NoCo2) to assess their disease susceptibility. Disease resistance conferred by cim3 was eliminated by the presence of the nahG gene, demonstrating that nahG is epistatic to cim3 for the SAR gene expression and disease resistance phenotypes.
Example 19: Synergistic Disease-Resistance Attained by Applying Microbicide and/or BTH to cim Mutants Three days before pathogen inoculation) the chemical inducer of systemic acquired resistance BTH ( benzo[1,2,3Jthiadiazole-7-carbothioic acid-S~methyl ester) formulated as 25% active ingredient (ai) with a wettable powder carrier (Metraux et al.,1991 ) and/or the microbicide metalaxyl (CGA 48988) formulated as 25% ai, or the wettable powder alone was applied as a fine mist to leaves of 4 week-o!d plants. Plants were inoculated with a conidial suspension (1.8 x 105 sporeslml) of the compatible pathogen Peronospora parasitica NoCo2.
Following inoculation) plants were covered to maintain high humidity and were placed in a Percival growth chamber at 17°C with a 14-hr day/10-hr night cycle (Uknes et al.) 1993). Tissue was harvested 8 days after inoculation.

Fungal growth was detemlined using a rRNA fungal probe that was obtained by PCR
according to White et al. (1990; PCR Protocols: A guide to Methods and Application) 3i 5-322}
using primers NS1 and NS2 and P. parasitica EmWa DNA as templates. RNA was purfied from frozen tissue by phenotichioroform extraction following lithium chloride precipitation (t~grimini et al) 1987: PNAS, 84: 7542-7546). Samples (7.5 ug) were separated by electrophoresis through formaldehyde agarose gels and blotted to nylon membranes (Hybond-N+, Amersham) as described by Ausbel et al. ( 1987). Flybridizations and washing were according to Church and Gllbert (1984) PNAS, 81:1991-1995). Relative amounts of the transcript were determined using a Phosphor Imager ( Molecular Dynamics, Sunnyvale, CA } following manufacturers instructions.
Sample loading was normalized by probing stripped filter blots with the constitutively expressed b-tubulin Arabidopsis cDNA. The infestation of the untreated plants corresponded to 0 % fungal growth inhibition.
Application of metafaxyl alone, the "plant activator" BTH alone, or both metalaxyl and BTH to the cim3 mutants described above produced a greater-than-additive, i.e., synergistic, disease-resistant effect. This effect was determined as the synergy factor (SF), which is the ratio of observed (O) effect to expected (E) effect. The following results were obtained:
Table 33 Action Against Peronospora parasidca In Arabidopsis Component I: cim3 mutation Component II: metalaxyl Test Components Fungal Growth Synergy no. Inhibition %

Factor cim3 metalaxyl O (observed) O/E
E (expected) control wt - 0 1 cim3 ~ 12.5 2 wt 12.5 mg/I 52.7 3 wt 2.5 mg/l 0 4 wt 0.1 mg/) 0 wt 0.02 mg/I ND

6 cim3 12.5 mg/1 ND ND ND

7 cim3 2.5 mgll 82.2 12.5 6.6 8 cim3 0.1 mg/1 57.8 12.5 4.6 WO 98!29537 PCT/EP97/0~253 ~73~
cim3 0.02 mgll ~ 55.6 ~ ND j ND
wt = wild-type Col-0 ND = not determined Table 34 Action Against Peronospora paresitica In An~bidopsis Component I: cim3 mutation Component II: BTH
Test Components Fungal Growth Synergy no. Inhibition %

Factor cim3 BTH O (observed) O/E
E (expected) controlwt - 0 1 cim3 - 12.5 2 wt 0.1 mM 85.7 3 wt 0.03 mM 20.8 4 wt 0.01 mM 0 cim3 0.1 mM ND 96.2 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-type Col-0 ND = not determined Table 35 Action Against Peronospora parasitica In An3bidopsis Component I: cim3 mutation Component II: 8TH and metalaxyl (M) Test Components Fungal Growth Synergy no. Inhibition %

Factor cim3 BTH+M O (observed) O/E
E (expected) controlwt - 0 1 cim3 - 12.5 2 wt BTH 0.01 100 mM

+ M 0.5 mg/I

3 wt BTH 0.01 40.7 mM

+ M 0.1 mg/I

4 wt BTH 0.01 ND
mM

+ M 0.02 mg/l cim3 BTH 0.01 ND 100 ND
mM

+ M 0.5 mg/I

6 cim3 BTH 0.01 100 53.2 1.9 mM

+ M 0.1 mg/l 7 cim3 BTH 0.01 77.7 ND ND
mM

+M0.02mg/l wt = wild-type Col-0 ND = not determined As can be seen from the above tables, synergistic disease-resistant effects were demonstrated in the cim3 plants by apptication of metalaxyl alone, by application of BTH
alone, and by application of metalaxyl and BTH in combination. For example) in the untreated cim3 plant) 12.5% fungal growth inhibition was seen relative to the untreated wild-type plant; this demonstrates that the constitutive SAR gene expression in the cim3 mutant correlates with disease resistance. As shown in Table 30, however) by applying metalaxyl at 0.0001 gll (a concentration normally insufficient for efficacy) to the immunomodulated (SAR-on) cim3 plant, the observed level of fungal growth inhibition increased to 57.8%.
The synergy factor of 4.6 calculated from these data clearly demonstrates the synergistic effect achieved by applying a microbicide to an immunomodulated plant.
The data presented in Table 31 demonstrates that synergy is also achieved by applying a chemical inducer of systemic acquired resistance such as BTH to an immunomodulated (SAR-on) cim3 plant. For example, in wild-type plants, a 0.03 mM
concentration of BTH is normally insufficient to confer effective disease resistance, providing only 20.8% fungal growth inhibition. However, in cim3 plants, this normally inadequate concentration of BTH provided 73.1 % fungal growth inhibition, which was nearly as high as the level of inhibition provided by 0.1 mM BTH, the recommended concentration for efficacy. The synergy factor of 2.2 calculated from the data in Table 31 clearly demonstrates the synergistic effect achieved by applying BTH to a plant that is already immunomodulated through other means.

The effects on disease resistance were even more dramatic when both BTH and metalaxyl were applied to the cim3 plant. As set forth above in Example 13 (Table 29), in wild-type plants, no fungal growth inhibition is achieved by separately applying either 0.01 mM BTH or 0.0001 g/l metalaxyl, because these concentrations are normally insufficient for efficacy. However, by applying both of these compounds to the plants at these normally insufficient concentrations, 40.7% fungal growth inhibition was observed, which is a synergistic effect with respect to the wild-type plants. In the cim3 plants, the simultaneous application of 0.01 mM BTH and 0.0001 g/I metalaxyl resulted in 100% fungal growth inhibition, clearly demonstrating even further synergistic activity.
Thus, the combined use of of immunomodulated cim plants with low, normalcy ineffective concentrations of chemicals to achieve disease resistance provide advantages that should be apparent to those skilled in the agricultural arts. Normally toxic or otherwise undesirable concentrations of chemicals can be avoided by taking advantage of the synergies demonstrated herein. In addition) economic gains can be realized as a result of the decreased quantity of chemicals required to provide a given level of protection to plants.
III. Synergistic Disease Resistance Effects Achieved By Application Of Conventional Microbicides and/or Chemical Inducers of Systemic Acquired Resistance To Transgenic Plants Containing Forms of the NIMi Gene The NIMi gene is a key component of the systemic acquired resistance (SAR) pathway in plants (Ryals et aL,1996). The NIM1 gene is associated with the activation of SAR by chemical and biological inducers and, in conjunction with such inducers, is required for SAR and SAR gene expression. The location of the NIMi gene has been determined by molecular biological analysis of the genome of mutant plants known to carry the mutant nim t gene, which gives the host plants extreme sensitivity to a wide variety of pathogens and renders them unable to respond to pathogens and chemical inducers of SAR.
The wildtype NIM1 gene of Arapidopsis has been mapped and sequenced (SEQ ID N0:1 ). The wild-type NIM1 gene product (SEQ ID N0:2) is involved in the signal transduction cascade leading to both SAR and gene-for-gene disease resistance in Arabidopsis (Ryals et al.) 1997). Recombinant overexpression of the wild-type form of NIM1 gives rise to immunomodulated plants with a constitutive immunity (CIM) phenotype and therefore confers disease resistance in transgenic plants. Increased levels of the active NIM1 protein produce the same disease-resistance effect as chemical induction with inducing chemicals such as BTH, INA) and SA. See, co-pending U.S. Application Serial No.
08J880,179, incorporated herein by reference.
Furthermore, the NIM1 gene product has been shown to be a structural homologue of the mammalian signal transduction factor IxB subciass oc (Ryals et al., 1997).
Mutations of IxB have been described that act as super-repressors or dominant-negatives of the NF-xB/IxB regulation scheme. Thus, certain altered forms of NIMi act as dominant-negative regulators of the SAR signal transduction pathway. These altered forms of NIM1 confer the opposite phenotype in plants transformed therewith as the nim 1 mutant; i.e., immunomodulated plants transformed with altered forms of NlM1 exhibit constitutive SAR
gene expression and a CIM phenotype. See, co-pending PCT application "METHODS
OF
USING THE NlM1 GENE TO CONFER DISEASE RESISTANCE IN PLANTS" incorporated herein by reference.
Example 20: Transformation of Plants with Cosmid Clones Containing the Wild-Type NIM1 Gene Cosmid D7 (deposited with the ATCC on September 25, 1996, as ATCC 97736) was generated from a clone spanning the NIM1 gene region and therefore includes the wild-type NIM1 gene (SEA ID NO:i ). Cosmid E1 was also generated from a clone spanning the NIM1 gene region and therefore also includes the wild-type NIM1 gene (SECT ID
N0:1 ).
Cosmids D7 and E1 were moved into Agrobacferium tumefaciens AGL-1 through conjugative transfer in a tri-parental mating with helper strain HB101 (pRK2013) as described in the U.S. Patent Application No. 08/880,179. These cosmids were then used to transform a kanamycin-sensitive nim f mutant Arabidopsis line using vacuum infiltration (Mindrinos et al., 1994, Cell 78) 1089-1099). Seed from the infiltrated plants was harvested and allowed to germinate on GM agar plates containing 50 mg/ml kanamycin as a selection agent. Seedlings that survived the selection were transferred to soil approximately two weeks after plating.
Plants transferred to soil were grown in a phytotron for approximately one week after transfer. 300mM INA was applied as a fine mist to completely cover the plants using a chromister. After two days, leaves were harvested for RNA extraction and PR-1 expression analysis. The plants were then sprayed with Peronospora parasitica (isolate EmWa) and n grown under high humidity conditions in a growing chamber with 19°C
day/17°C night temperatures and 8h light/16h dark cycles. Eight to ten days following fungal infection) plants were evaluated and scored positive or negative for fungal growth. Ws and nim 1 plants were treated in the same way to serve as controls for each experiment.
Total RNA was extracted from the collected tissue using a LiCUphenol extraction buffer (Verwoerd et al., 1989, Nuc Acid Res, 2362). RNA samples were run on a formaldehyde agarose gal and blotted to GeneScreen Plus (DuPont) membranes.
Blots were hybridized with a ~P-labeled PR-1 cDNA probe. The resulting blots were exposed to film to determine which transformants were able to induce PR-1 expression after INA
treatment.
To see if any of the D7 and E1 transformants overexpressed NIM1 due to insertion site (position) effect, primary transformants containing the D7 or E1 cosmids were selfed and the T2 seed collected. Seeds from one E1 line and 95 D7 lines were sown on soil and grown as described above. When the T2 plants had obtained at least four true leaves) a single leaf was harvested separately for each plant. RNA was extracted from this tissue and analyzed for PR-1 and NIM1 expression. Plants were then inoculated with P.
parasitica (EmWa) and analyzed for fungal growth at 10 days following infection. A number of transformants showed less than normal fungal growth and four of them, namely, lines D7-2, D7-74, D7-89 and E1-1, showed no visible fungal growth at all. Plants showing higher than normal NIM1 and PR-1 expression and displaying fungal resistance demonstrate that overexpression of NIM1 confers disease resistance.
Example 21: NIMJ Overexpression Under Its Native Promoter Plants constitutively expressing the NIM1 gene were generated from transformation of Ws wild type plants with the BamHl-Hindlll NIM1 genomic fragment (SECT ID NO:
1 - bases 1249-5655) containing i .4 kb of promoter sequence. This fragment was cloned into pSGCG01 and transformed into the Agrobacterium strain GV3101 (pMP90) Koncz and Schell (1986) MoL Gen. Genet. 204:383-396). Ws plants were infiltrated as previously described. The resulting seed was harvested and plated on GM agar containing 50 Ngiml kanamycin. Surviving plantlets were transferred to soil and tested as described above for resistance to Peronospora parasitica isolate Emwa. Selected plants were selfed and selected for two subsequent generations to generate homozygous lines. Seeds from several of these lines were sown in soil and 15-18 plants per fine were grown for three weeks and tested again for Emwa resistance without any prior treatment with an inducing chemical. Approximately 24 hours, 48 hours, and five days after fungal treatment, tissue was harvested, pooled and frozen for each line. Plants remained in the growth chamber until ten days after inoculation when they were scored for resistance to Emwa.
RNA was prepared from aU of the collected samples and analyzed as previously described (Delaney et al, 1995). The blot was hybridized to the Arabidopsis gene probe PR-1 (Uknes et al) 1992). Five of the 13 transgenic lines analyzed showed early induction of PR1 gene expression. For these lines, PR-1 mRNA was evident by 24 or 48 hours following fungal treatment. These five lines also had no visible fungal growth. Leaves were stained with iactophenol blue as described (Dietrich et al., 1994) to verify the absence of fungal hyphae in the leaves. PR-1 gene expression was not induced in the other eight lines by 48 hours and these plants did not show resistance to Emwa.
A subset of the resistant lines were also tested for increased resistance to the bacteria! pathogen Pseudomonas syringae DC3000 to evaluate the spectrum of resistance evident as described by Uknes et al. (1993). Experiments were done essentially as described by Lawton et al. (1996). Bacterial growth was slower in those lines that also demonstrated constitutive resistance to Emwa. This shows that plants overexpressing the NIM1 gene under its native promoter have constitutive immunity against pathogens.
To assess additional characteristics of the CIM phenotype in these lines, unifected plants are evaluated for free and glucose-conjugated salicylic acid and leaves are stained with lactophenol blue to evaluate for the presence of microscopic lesions.
Resistance plants are sexually crossed with SAR mutants such as NahG and ndrl to establish the epistatic relationship of the resistance phenotype to other mutants and evaluate how these dominant negative mutants of NIM1 may influence the salicylic acid-dependent feedback loop.
Example 22: 35S Driven Overexpression of NIM1 The full-length NIMi cbNA (SEd ID NO: 6) was cloned into the EcoRl site of pCGN1761 ENX (Comai et al. (1990) Plant Mol. Biol. 15, 373-381 ). From the resulting plasmid, an Xbal fragment containing an enhanced CaMV 35S promoter, the NIM1 cDNA in the correct orientation for transcription, and a tml 3' terminator was obtained. This fragment was cloned into the binary vector pCIB200 and transformed into GV3101. Ws plants were infiltrated as previously described. The resulting seed was harvested and plated on GM
agar containing 50 Ng/ml kanamycin. Surviving ptantlets were transferred to soil and tested as described above. Selected plants were selfed and selected for two subsequent generations to generate homozygous lines. Nine of the 58 lines tested demonstrated resistance when they were treated with Emwa without prior chemical treatment.
Thus, overexpression of the NIM1 cDNA also resul.s in disease-resistant plants.
Example 23: NIM1 Is A Homolog Of IxBa A multiple sequence alignment between the protein gene products of NIM1 and IkB
was performed by which it was determined that the NIMI gene product is a homolog of IxBa (Figure 1 ). Sequence homology searches were performed using BLAST
(Altschul et al., J. MoI. Biol. 215) 403-410 (1990)). The multiple sequence alignment was constructed using Clustal V (Higgins et al., CABIOS 5,151-153 (1989)) as part of the Lasergene Biocomputing Software package from DNASTAR (Madison) WI). The sequences used in the alignment were NIM1 (SEA ID N0:2), mouse IxBa (SEQ ID N0:3, GenBank Accession #: 1022734), rat IxBa (SEQ ID N0:4, GenBank accession Nos. 57674 and X63594;
Tewari et al., Nucleic Acids Res. 20, 607 (1992)), and pig IxBa (SEA ID N0:5, 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)). Parameters used in the Clustal analysis were gap penalty of 10 and gap length penalty of 10. Evolutionary divergence distances were calculated using the PAM250 weight table (Dayhoff et al.) °A model of evolutionary change in proteins. Matrices for detecting distant relationships ° In Atlas of Protein Sequence and Structure, Vol. 5, Suppl. 3, M.O., Dayhoff, ed (National Biomedical Research Foundation, Washington, D.C.), pp. 345-358 (1978)). Residue similarity was calculated using a modified Dayhoff table (Schwartz and Dayhoff, "A model of evolutionary change in proteins." In Atlas of Protein Sequence and Structure, M.O. Dayhoff) ed (National Biomedical Research Foundation, Washington, D.C.) pp. 353-358 (1979); Gribskov and Burgess, Nucleic Acids Res. 14, 6745-6763 {1986)).
Homology searches indicate similarity of NIM1 to ankyrin domains of several proteins including: ankyrin, NF-xB and IxB. The best overall homology is to IxB and rotated molecules (Figure 1 ). NIM1 contains 2 serines at amino acid positions 55 and 59; the serine at position 59 is in a context (D/ExxxxS) and position (N-terminal) consistent with a role in phosphorylation-dependent, ubiquitin-mediated, inducible degradation.
All IxBa's have these N-terminal serines and they are required for inactivation of txB
and subsequent release of NF-xB. NIM1 has ankyrin domains (amino acids 262-290 and 323-371).
Ankyrin domains are believed to be involved in protein-protein interactions and are a ubiquitous feature for IxB and NF-xB molecules. The C-termini of ixB's can be dissimilar.
NIM1 has some homology to a QL-rich region (amino acids 491-499) found in the C-termini of some IxBs.
Example 24: Generation Of Altered Forms Of NIM1 Changes Of Serine Residues 55 and 59 To Alanine Residues Phosphorylation of serine residues in human IxBa is required for stimulus-activated degradation of IxBa thereby activating NF-xB. Mutagenesis of the serine residues (S32-S36} in human IxBa to alanine residues inhibits stimulus-induced phosphorylation thus blocking IxBa proteosome-mediated degradation (E. Britta-Mareen Traenckner et al., EMBO
J. 14: 2876-2883 (1995); Brown et al., Science 267:1485-1488 (1996); Brockman et al., Molecular and Cellular Biology 15: 2809-2818 (1995); Wang et al., Science 274:784-787 (1996)).
This altered form of IxBa functions as a dominant negative form by retaining NF-xB in the cytoplasm, thereby blocking downstream signaling events. Based on sequence comparisons between NIM1 and IxB) serines 55 (S55) and 59 (S59) of NIM1 are homologous to S32 and S36 in human IxBa. To construct dominant-negative forms of NIM1, the serines at amino acid positions 55 and 59 are mutagenized to alanine residues.
This can be done by any method known to those skilled in the art, such as) for example) by using the QuikChange Site Directed Mutagenesis Kit (#200518:Strategene).
Using a full length NIM1 cDNA (SEQ ID N0:6) including 42 by of 5' untranslated sequence (UTR) and 187 by of 3' UTR, the mutagenized construct can be made per the manufacturer's instructions using the following primers (SEQ ID N0:6) positions 192-226): 5'-CAA CAG CTT CGA AGC CGT CTT TGA CGC GCC GGA TG-3' (SEQ ID N0:25) and 5'-CAT CCG GCG _CGT CAA AGA CGG _CTT CGA AGC TGT TG-3' (SEQ 1D N0:26), where the underlined bases denote the mutations. The strategy is as follows: The NIM1 cDNA

cloned into vector pSE936 (Elledge et al., Proc. Nat Aced Sci. USA 88:1731-1735 (1991)) is denatured and the primers containing the altered bases are annealed. DNA
polymerase (Pfu) extends the primers by nonstrand-displacement resulting in nicked circular strands.
DNA is subjected to restriction endonuclease digestion with Dpnl, which only cuts methylated sites (nonmutagenized template DNA). The remaining circular dsDNA
is transformed into E.coli strain XL1-Biue. Plasmids from resulting colonies are extracted and sequenced to verify the presence of the mutated bases and to confirm that no other mutations occurred.
The mutagenized NIMi cDNA is digested with the restriction endonuclease EcoRt and cloned into pCGN1761 under the transcriptional regulation of the double 35S
promoter of the cauliflower mosaic virus. The transformation cassette including the 35S
promoter, NIM1 cDNA and tml terminator is released from pCGN1761 by partial restriction digestion with Xbal and ligated into the Xbal and ligated into the Xbal site of dephosphorylated pCiB200.
SEA ID NO's:7 and 8 show the DNA coding sequence and encoded amino acid sequence, respectively, of this altered form of the NIMI gene.
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.) of human IxBa, which includes K21, K22, S32 and S36) results in a dominant-negative ixBa phenotype in transfected human cell cultures. An N-terminal deletion of approximately the first 125 amino acids of the encoded product of the NIM1 cDNA removes eight lysine residues that may serve as potential ubiquitination sites and also removes putative phosphorylation sites at S55 and S59 (see Example 2). This altered gene construct may be produced by any means known to those skilled in the art. For example, using the method of Ho et al., Gene 77:51-59 (1989), a NlM1 form may be generated in which DNA
encoding approximately the first 125 amino acids is deleted. The following primers produce a 1612-bp PCR product (SEQ ID N0:6: 418 to 2011 ): 5'-gg oat tca-ATG GAT TCG GTT GTG
ACT
GTT TTG-3' (SEQ ID N0:27) and 5'-gga att cTA CAA ATC TGT ATA CCA TTG G-3' (SEQ
ID N0:28} in which the synthetic start cadon is underlined (ATG) and EcoRl linker sequence is in lower case. Amplification of fragments utilizes a reaction mixture comprising 0.1 to 100 ng of template DNA) 1 OmM Tris pH 8.3/50mM KCU2 mM MgCI~/0.001 % geiatiN0.25 mM
each dNTP/0.2 mM of each primer and t unit rTth DNA polymerase in a final volume of 50 mL and a Perkin Elmer Cetus 9600 PCR machine. PCR conditions are as follows:
94°C
3min: 35x (94°C 30 sec: 52°C 1 min: 72°C 2 min):
72°C 10 min. The PCR product is cloned directly into the pCR2.1 vector (Invitrogen). The PCR-generated insert in the PCR vector is released by restriction endonuclease digestion using EcoRl and ligated into the EcoRl site of dephosphorylated pCGN1761 ( under the transcriptional regulation of the double 35S
promoter. The construct is sequenced to verify the presence of the synthetic starting ATG
and to confirm that r~ other mutations occurred during PCR. The transformation cassette including the 35S promoter, modified N1M1 cDNA and tml terminator is released from pCGN1761 by partial restrictibn digestion with Xbal and ligated into the Xbal site of pCIB200. SEA ID NO's:9 and 10 show the DNA coding sequence and encoded amino acid sequence, respectively, of an altered form of the NlM1 gene having an N-terminal amino acid deletion.
Example 26: Generation Of Altered Forms Of NIM~ - C-terminal Deletion The deletion of amino acids 261-317 of human IxBa is believed to result in enhanced intrinsic stability by blocking the constitutive phosphorylation of serine and threonine residues in the C-terminus. A region rich in serine and threonine is present at amino acids 522-593 in the C-terminus of NIM1. The C-terminal coding region of the NIMi gene may be modified by deleting the nucleotide sequences which encode amino acids 522-593. Using the method of Ho et al. (1989), the C-terminal coding region and 3' UTR of the NIM1 cDNA (SEQ lD N0:6: 1606-2011 ) is deleted by PCR, generating a 1623 by fragment using the following primers: 5'-cggaattcGATCTCTTTAATTTGTGAATTT C-3' (SEQ
ID N0:29) and 5'-ggaattcTCAACAGTT CATAATCTGGTCG-3' (SEQ lD N0:30) in which a synthetic stop colon is underlined (TGA on complementary strand) and EcoRl linker sequences are in lower case. PCR reaction components are as previously described and cycling parameters are as follows: 94°C 3 min: 35x (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 restriction endonuciease digestion using EcoRl and ligated into the EcoRl site of dephosphorylated pCGN1761, which contains the double 35S promoter. The construct is sequenced to verify the presence of the synthetic in-frame stop colon and to confirm that no other mutations occurred during PCR. The transformation cassette including the promoter, modified NIMi cDNA, and tml terminator is released from pCGN1761 by partial restriction digestion with Xbal and ligated into the Xbal site of dephosphorylated pCIB200. SEA ID NO's:11 and 12 show the DNA
coding sequence and encoded amino acid sequence, respectively, of an altered form of the NIM1 gene having a C-terminal amino acid deletion.
Example 27: Generation Of Altered Forms Of NIM1- N-terminaUC-terminal Deletion Chimera An N-terminal and C-terminal deletion form of NIM1 is generated using a unique Kpnl restriction site at position 819 (SEQ ID N0:6). The N-terminal deletion form (Example 25) is restriction endonuclease digested with EcoRllKpnl and the 415 by fragment corresponding to the modified N-terminus is recovered by gel electrophoresis.
Likewise) the C-terminal deletion form (Example 26) is restriction endonuclease digested with EcoRllKpnl and the 790 by fragment corresponding to the modified C-terminus is recovered by gel electrophoresis. The fragments are ligated at 15°C, digested with EcoRl to eliminate EcoRl concatemers and cloned into the EcoRl site of dephosphorylated pCGN1761. The N/C-terminal deletion form of NIMi is under the transcriptional regulation of the double 35S
promoter. Similarly, a chimeric form of NIMl is generated which consists of the S55/S59 mutagenized putative phosphorylation sites (Example 24) fused to the C-terminal deletion (Example 26). The construct is generated as described above. The constructs are sequenced to verify the fidelity of the start and stop codons and to confirm that no mutations occurred during cloning. The respective transformation cassettes including the 35S promoter, NIMi chimera and tml terminator are released from pCGN1761 by partial restriction digestion with Xbal and ligated into the Xbal site of dephosphorylated pC1B200.
SEA ID NO's:13 and 14 show the DNA coding sequence and encoded amino acid sequence, respectively, of an altered form of the NIM1 gene having both N-terminal and C-terminal amino acid deletions.
Example 28: Generation Of Altered Forms Of NIMf - Ankyrin Domains NIM1 exhibits homology to ankyrin motifs at approximately amino acids 103-362.
Using the method of Ho et al. (1989), the DNA sequence encoding the putative ankyrin domains (SEQ ID N0:1: 3093-3951 ) is PCR amplified (conditions: 94°C 3 min:35x (94°C 30 sec: 62°C 30 sec: 72°C 2 min): 72°C 10 min) from the NIM1 cDNA (SEGO ID N0:6: 349-1128) using the following primers: 5'-ggaattcaATGGACTCCAACAACACCGCCGC-3' (SEQ
lD N0:31 ) and 5'-ggaattcTCAACCTTCCAAAGTTGCTTCTGATG-3' (SEQ ID N0:32). The resulting product is restriction endonuclease digested with EcoRl and then spliced into the EcoRl site of dephosphorylated pCGN1761 under the transcriptional regulation of the double 35S promoter. The construct is sequenced to verify the presence of the synthetic start codon (ATG), an in-frame stop codon (TGA) and to confirm that no other mutations occurred during PCR. The transformation cassette including the 35S promoter, ankyrin domains, and tml terminator is released from pCGN1761 by partial restriction digestion with Xbal and ligated into the Xbal site of dephosphorylated pCIB200. SEQ ID
NO's:15 and 16 show the DNA coding sequence and encoded amino acid sequence) respectively) of the ankyrin domain of NIMi.
Example 29: Construction Of Chimeric Genes To increase the likelihood of appropriate spatial and temporal expression of altered NIM1 forms, a 4407 by HindIII/BamHl fragment (SEQ ID NO:1: bases 1249-5655) andJor a 5655 by EcoRV/BamHl fragment (SEQ ID N0:1: bases 1-5655) containing the NIM1 promoter and gene is used for the creation of the altered NIM1 forms in Examples 24-28 above. Atthough the construction steps may differ, the concepts are comparable to the examples previously described herein. Strong overexpression of the altered forms may potentially be lethal. Therefore, the altered forms of the NIM1 gene described in Examples 24-28 may be placed under the regulation of promoters other than the endogenous NIM1 promoter, including but not limited to the nos promoter or small subuntt of Rubisco promoter. Likewise, the altered NIM1 forms may be expressed under the regulation of the pathogen-responsive promoter PR-1 (U.S. Pat. No. 5,614,395). Such expression permits strong expression of the altered NIM1 forms only under pathogen attack or other SAR-activating conditions. Furthermore, disease resistance may be evident in the transformants expressing altered NIM1 forms under PR-1 promoter regulation when treated with concentrations of SAR activator compounds (i.e.) BTH or INA) which normally do not activate SAR, thereby activating a feedback loop (Weymann et al.) (1995) Plant Cell 7:
2013-2022).

Example 30: Transformation Of Altered Forms Of The NIM1 Into Arabidopsis fhaliana The constructs generated (Examples 24-29) are moved into Agrobacterium iumefaciens by electroporation into strain GV3101. These constructs are used to transform Arabidopsis ecotypes Coi-0 and Ws-0 by vacuum infiltration (Mindrinos et al., Ce1178, 1089-1099 ( 1994)) or by standard root transformation. Seed from these plants is harvested and allowed to germinate on agar plates with kanamycin (or another appropriate antibiotic) as selection agent. Only plantiets that are transformed can detoxify the selection agent and survive. Seedlings that survive the selection ace transferred to soil and tested for a CIM
(constitutive immunity) phenotype. Plants are evaluated for observable phenotypic differences compared to wild type plants.
Example 31: Assessment of CIM Phenotype in Plants Transformed with the Wild-Type NIM1 Gene or an Altered Form of the NIM1 Gene A leaf from each primary transformant is harvested, RNA is isolated (Verwoerd et al., 1989) Nuc Acid Res) 2362) and tested for constitutive PR-1 expression by RNA
blot analysis (Uknes et al., i 992). Each transformant is evaluated for an enhanced disease resistance response indicative of constitutive SAR expression analysis (Uknes et al., 1992).
Conidial suspensions of 5-10x10° spores/ml from two compatible P.
parasitica isolates, Emwa and Noco (i.e. these fungal strains cause disease on wildtype Ws-O and Col-0 plants) respectively)) are prepared, and transformants are sprayed with the appropriate isolate depending on the ecotype of the transformant. Inoculated plants are incubated under high humidity for 7 days. Plants are disease rated at day 7 and a single leaf is harvested for RNA blot analysis utilizing a probe which provides a means to measure fungal infection.
Transformants that exhibit a CIM phenotype are taken to the T1 generation and homozygous plants are identified. Transformants are subjected to a battery of disease resistance tests as described below. Fungal infection with Noco and Emwa is repeated and leaves are stained with lactophenol blue to identify the presence of fungal hyphae as described in Dietrich et al., (1994). Transformants are infected with the bacterial pathogen Pseudomonas syringae DC3000 to evaluate the spectrum of resistance evident as described in Uknes et al. (1993). Uninfected plants are evaluated for both free and glucose-conjugated SA and leaves are stained with lactophenol blue to evaluate for the presence of microscopic lesions. Resistant plants are sexually crossed with SAR mutants such as NahG (U.S. Pat. No. 5,614,395) and ndri to establish the epistatic relationship of the resistance phenotype to other mutants and evaluate how these dominant-negative mutants of NIMi msy influsnce the SA-dependent feedback loop.
Example 32: Isolation Of NlM1 Homologs NIMJ homologs are obtainable that hybridize under moderately stringent conditions either to the entire NIMt gene from Arabidopsis or, preferably) to an oligonucleotide probe derived from the Arabidopsis NIM1 gene that comprises a contiguous portion of its coding sequence at least approximately 10 nucleotides in length. Factors that affect the stability of hybrids determine the stringency of the hybridization. One such factor is the melting temperature Tm, which can be easily calculated according to the formula provided in DNA
PROBES, George H. Keller and Mark M. Manak ( Macmillan Publishers Ltd, 1993, Section one: Molecular Hybridization Technology; page 8 ff. The preferred hybridization temperature is in the range of about 25°C below the calculated melting temperature Tm, preferably in the range of about 12-15°C below the calculated melting temperature Tm) and, in the case of oligonucfeotides) in the range of about 5-10°C below the melting temperature Tm.
Using the NIMi cDNA (SEA ID N0:6) as a probe, homologs of Arabidopsis NIM1 are identified through screening genomic or cDNA libraries from different crops such as, but not limited to those listed below in Example 33. Standard techniques for accomplishing this include hybridization screening of plated DNA libraries (either plaques or colonies; see, e.g.
Sambrook et al., Molecular Cloning , eds., Cold Spring Harbor Laboratory Press. (1989)) and amplification by PCR using oligonucleotide primers (see, e.g. tnnis et al.) PCR
Protocols, a Guide to Methods and Applications eds., Academic Press (1990)).
Homofogs identified are genetically engineered into the expression vectors herein and transformed into the above listed crops. Transformants are evaluated for enhanced disease resistance using relevant pathogens of the crop plant being tested.

NlM1 homologs in the genomes of cucumber, tomato, tobacco, maize, wheat and barley have been detected by DNA blot analysis. Genomic DNA was isolated from cucumber) tomato, tobacco, maize, wheat and barley) restriction digested with the enzymes BamHl) Hindlll, Xbal, or Sall) electrophoretically separated on 0.8% agarose gels and transferred to nylon membrane by capillary blotting. Following UV-crosslinking to affix the DNA) the membrane was hybridized under low stringency conditions [(1 %BSA;
520mM
NaP04, pH7.2; 7°~0 lauryl sulfate, sodium salt; 1 mM EDTA; 250 mM
sodium chloride) at 55°C for 18-24h] with ~P-radiolabelled Arabidopsis thaliana NIMi cDNA.
Following hybridization the blots were washed under low stringency conditions [6XSSC for 15 min.
(X3) 3XSSC for 15 min. (X1 ) at 55°C; 1 XSSC is 0.15M NaCI, lSmM Na-citrate (pH7.0)] and exposed to X-ray film to visualize bands that correspond to NIM1.
In addition, expressed sequence tags (EST) identified with similarity to the NIM1 gene can be used to isolate homologues. For example, several rice expressed sequence tags (ESTs) have been identified with similarity to the NIM1 gene. A multiple sequence alignment was constructed using Clustal V (Higgins, Desmond G. and Paul M.
Sharp (1989)) Fast and sensitive multiple sequence alignments on a microcomputer, AC
BIOS
5:151-153) as part of the DNA' (1228 South Park Street, Madison Wisconsin, 53715) Lasergene Biocomputing Software package for the Macintosh (1994). Certain regions of the NIM~ protein are homologous in amino acid sequence to 4 different rice cDNA protein products. The homologies were identified using the NIM1 sequences in a GenBank BLAST
search. Comparisons of the regions of homology in NIMi and the rice cDNA
products are shown in Figure 2 (See also, SEQ ID N0:2 and SEQ ID NO's:17-24). The NIM1 protein fragments show from 36 to 48% identical amino acid sequences with the 4 rice products.
These rice ESTs may be especially useful for isolation of NIMi homologues from other monocots.
Homologues may also be obtained by PCR. In this method, comparisons are made between known homologues (e.g., rice and Arabidopsis). Regions of high amino acid and DNA similarity or identity are then used to make PCR primers. Regions rich in amino acid residues M and W are best followed by regions rich in amino acid residues F) Y, C, H, Q, K
and E because these amino acids are encoded by a limited number of codons.
Once a suitable region is identified) primers for that region are made with a diversity of substitutions in the 3'~ codon position. This diversify of substitution in the third position may be constrained depending on the species that is being targeted. For example) because maize is GC rich, primers are designed that utilize a G or a C in the 3'~ position) if possible. The PCR reaction is performed from cDNA or genomic DNA under a variety of standard conditions. When a band is apparent, it is cloned andlor sequenced to determine if it is a NIM1 homologue.
Example 33: Expression of a Form of NIM1 In Crop Plants Those constructs conferring a CIM phenotype in Col-0 or Ws-0 are transformed into crop plants for evaluation. Alternatively, altered native NIMI genes isolated from crops in the preceding example are put back into the respective crops. Although the NIM1 gene can be inserted into any plant cell falling within these broad classes) it is particularly useful in crop plant cells, such as rice, wheat, barley, rye) corn) potato) carrot, sweet potato) sugar beet, bean, pea) chicory) lettuce, cabbage) cauliflower) broccoli) turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery) carrot) squash) pumpkin) zucchini) cucumber) apple) pear, quince, melon, plum, cherry, peach, nectarine) apricot) strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane. Transformants are evaluated for enhanced disease resistance. In a preferred embodiment of the invention) the expression of the NIM1 gene is at a level which is at least two-fold above the expression level of the native NIM1 gene in wild type plants and is preferably ten-fold above the wild type expression level.
Example 34: Synergistic Disease Resistance Attained by Applying A Conventional Microbicide to Transgenic Plants Overexpressing NIM1 The plant lines used in this example (6E and 7C) were generated from transformation of wild-type Arabidopsis thaliana plants (ecotype Ws) with the BamHl-HindJll NIM1 genomic fragment (SEQ ID N0:1 - bases 1249-5655)) as described above in Example 21.
The fungicides metalaxyl, fosetyl) and copper hydroxide, formulated as 25%, 80%, and 70%
active ingredient (ai), respectively, with a wettable powder carrier, were applied as fine mist to leaves of three week-ofd transgenic Ws plants constitutively expressing the NlM1 gene.
The wettable powder alone was applied as a control. Three days later, plants were inocutated with a Peronospora parasitica isolate Emwa oonidial suspension (i-2 x 105 sporesJml), as described in Delaney et al. (1995). Following inoculation) plants were covered to maintain high humidity and were placed in a Percival growth chamber at i 7°C with a 14-hr day/10-hr night cycle (Uknes et al.,1993). Tissue was harvested 8 days after inoculation.
Fungal infection progression was followed for 12 days by viewing under a dissecting microscope to score development of conidiophores (Delaney, et al. (1994);
Dietrich, et al.
(1994)). Lactophenoltrypan blue staining of individual leaves was carried out to observe fungal growth within leaf tissue. Fungal growth was quantified using a rRNA fungal probe obtained by PCR according to White et al. (1990; PCR Protocols: A guide to Methods and Application, 315-322) using primers NS1 and NS2 and P. parasitica EmWa DNA as templates. RNA
was purled from frozen tissue by phenoUchloroform extraction following lithium chloride precipitation (Lagrimini et al,1987: PNAS, 84: 7542-7546). Samples (7.5 pg) were separated by electrophoresis through formaldehyde agarose gels and blotted to nylon membranes (Hybond-N+, Amersham) as described by Ausbel et al. (1987). Hybridizations and washing were according to Church and Gilbert (1984) PNAS, 81: 1991-1995). Relative amounts of the transcript were determined using a Phosphor Imager ( Molecular Dynamics, Sunnyvale, CA ) following manufacturers instructions. Sample loading was normalized by probing stripped fitter blots with the constitutively expressed b-tubulin Argbidopsis cDNA. The infestation of the untreated plants corresponded to 0 % fungal growth inhibition.
Application of metalaxyl, fosetyl) or copper hydroxide to plant lines overexpressing NIM1 produced a greater-than-additive) i.e., synergistic, disease-resistant effect. This effect was determined as the synergy factor (SF), which is the ratio of observed (O) effect to expected (E) effect. The following results were obtained:
Table 36 Action Against Peronospora parasitica In Arabidopsis Component I: NIM1 overexpression (line 6E) Component I1: metalaxyl Test Components Fungal Growth Synergy no. Inhibition Factor NIM1 metalaxyl % O/E

O (observed) E (expected) controlwt -- 0 2 wt 0.0125 g/I 59 3 wt 0.0012 g/I 27 4 NIM1 0.0125 g/l 76 69 1.1 NIM) 0.0012 gll 56 37 1.5 wt = wild-type Ws Table 37 Action Against Peronospora parasitica In Arabidopsis Component I: NIM1 overexpression (line 6E) Component It: fosetyl Test Components Fungal Growth Synergy no. Inhibition %

Factor NIM1 fosetyl O (observed) O/E
E (expected) control wt -- 0 2 wt 5.0 g/I 7 3 wt 0.5 g/I 2 4 wt 0.05 g/I 0 5 NIM1 5.0 g/I 93 17 5.5 6 NIM1 0.5 g/I 83 12 6.9 7 NIM1 0.05 g/I 42 10 4.2 wt = wild-type Ws Table 38 Action Against Peronospora parasitica In Arabidopsis Component I: NIM! overexpression (line 7C) Component II: fosetyl Test Components Fungal Growth Synergy no. Inhibition %

Factor NIM1 fosetyl O (observed) O/E
E (expected) control wt - 0 2 wt 5.0 g/I 7 3 wt 0.5 g/I 2 4 NIMl 5.0 g/I 80 21 3.8 5 0.5 g/I 56 16 3.5 wt = wild-type Ws Table 39 Action Against Peronospora parasitica In Arabidopsis Component I: NIM1 overexpression (line 6E) Component II: copper hydroxide Test Components Fungal Growth Synergy no. Inhibition %

Factor NIMi Cu(OH)2 O (observed) O/E
E (expected) control wt --2 wt 2.0 g/I 0 3 wt 0.2 g/I 0 4 wt 0.02 g/I 0 NIMi 2.0 g/I 66 10 6.6 6 NIM1 0.2 g/I 14 10 1.4 7 NIM1 0.02 g/I 20 10 2.0 wt = wild-type Ws Table 40 Action Against Peronospora parasitica In Arabidopsis Component I: NIMi overexpression (line 7C) Component ll: copper hydroxide Test Components Fungal Growth Synergy no. Inhibition %

Factor NIM1 Cu(OH)2 O (observed) O/E
E (expected) control wt -- 0 1 NIMi - 14 2 wt 2.0 g/I 0 3 wt 0.2 g/I 0 4 ~wt 0.02 g/I 0 NIMi 2.0 g/I 77 14 5.5 6 NIMi 0.2 g/l 51 14 3.6 7 NIMi 0.02 g/l 55 14 3.9 wt = wild-type Ws As can be seen from the above tables,. synergistic disease-resistant effects were demonstrated in plants overexpressing NiM1 by application of metat«xyt, fosetyl, and copper hydroxide. For example, in the untreated NIM1 plant (line 6E), 10%
fungal growth inhibition was seen relative to the untreated wild-type plant; this demonstrates that the constitutive SAR gene expression in this NIM1 overexpressor correlates with disease resistance. As shown above in Table 37, however, by applying fosetyl at 5.0 g/I (a concentration normally insufficient for efficacy) to the immunomodulated (SAR-on) NIM1 overexpressing plant, the observed level of fungal growth inhibition increased to 93%. The synergy factor of 5.5 calculated from these data clearly demonstrates the synergistic effect achieved by applying a microbicide to an immunomodutated (SAR-on) plant. In another example, in the untreated NIM1 plant (fine 7C), 14% fungal growth inhibition was seen relative to the untreated wild-type plant) demonstrating that the constitutive SAR gene expression in this NIM1 overexpressor correlates with disease resistance. As shown above in Table 40, however, by applying copper hydroxide at 2.0 g/I (a concentration normally insufficient for efficacy) to the immunomodulated (SAR-on) NIM1 overexpressing plant, the observed level of fungal growth inhibition increased to 77%. The synergy factor of 5.5 calculated from these data further demonstrates the synergistic effect achieved by applying a microbicide to an immunomodulated (SAR-on) plant.
Thus, the combined use of of immunomodulated plants overexpressing NIMi with low, normally ineffective concentrations of microbicides to achieve disease resistance provides advantages that should be apparent to those skilled in the agricultural arts.
Normally toxic or otherwise undesirable concentrations of microbicides can be avoided by taking advantage of the synergies demonstrated herein. In addition) economic gains can be realized as a result of the decreased quantity of microbicides required to provide a given level of protection to plants.
Example 35: Synergistic Disease Resistance Attained by Applying A Chemical Inducer Of SAR to Transgenic Plants Overexpressing NIMi Transgentc plants containing the NIM1 genomic DNA fragment under its own promoter (Example 21 } were also analyzed for response to different concentrations of BTH
relative to the wild-type Ws fine. Seeds from each line were sown and grown as previously described. At approximately three weeks post-planting, leaf samples were harvested from each line (day 0 controls), and the remaining plants were treated with H20, 10NM BTH) or 100NM BTH. Additional samples were ha:vested at days 1, 3) and 5 following treatment.
After harvesting the day 3 samples) a subset of plants for each line was removed and treated with Peronospora parasitica isolate Emwa as described above. RNA was prepared from the harvested tissue and Northern analysis was performed using the Arabidopsis PR-1 gene probe. Plants were scored for fungal resistance 8 days following infection.
The results of Northern analysis for Ws and four of the NIIVEoverexpressing tines (3A, 5B, 6E) and 7C) are shown in Figure 3. PR-1 gene expression in the wild-type Ws line was barely detectable after the low level 10NM BTH treatment (a BTH concentration of 100-300 uM is normally required for efficacy). Ws plants from this treatment were also still susceptible to the fungal pathogen P. parasitica (Emwa). in all of the NIMi-overexpressing fines) however, there was a much stronger response for PR-1 gene expression following the low-level BTH treatment. In addition) all of the NlM1-overexpressing lines treated with 10NM BTH showed complete or almost complete resistance to P. parasitica.
Leaves stained with lactophenol blue to identify the presence of fungal hyphae (Dietrich et al.
(1994)) confirmed the absence of fungal growth in the NIM1-overexpressing lines. PR-1 gene expression in leaf tissue following the 100NM BTH treatment was also much stronger and quicker in the NIM1-overexpressing lines relative to wild-type. Thus, immunomodulated plants are able to respond much faster and to much lower doses of BTH, as shown by PR-1 gene expression and resistance to P. parasitica, than wild-type plants. This data demonstrates that synergistic disease resistance is achieved by applying a chemical inducer of systemic acquired resistance such as BTH to an immunomodulated (SAR-on) plant such as a NIM1-overexpressing plant.
Thus, the combined use of immunomodulated plants overexpressing NIMi with low, normally ineffective concentrations of SAR-inducing chemicals such as BTH to achieve disease resistance provides advantages that should be apparent to those skilled in the agricultural arts. Normally toxic or otherwise undesirable concentrations of SAR-inducing chemicals can be avoided by taking advantage of the synergies demonstrated herein. In addition) economic gains can be realized as a result of the decreased quantity of .SAR-inducing chemicals required to provide a given level of protection to plants.

WO 98!29537 PCT/EP97/07253 SDQUE~E LISTING
( 1 ) GEL~AL INF~FtI~TICH~1:
(i) APPLICANT:
(A) NAME: Novartis AG
(B) STREET: Sct~warzWaldallee 215 (C) CITY: Basel ( E ) CC~f.~ITRY : Switzerland (F) POSTAL CODE (ZIP): 4002 (G) TELEPf~IC~: +41 61 69 11 11 (H) TELEF'AX: + 41 61 696 79 76 (I) TELEK: 962 991 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (ii) TITLE OF INVENTION: METHOD FOR PROTECTING PLANTS
(iii) NUMBER OF SEQUENCES: 32 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5655 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(ix) FEATURE:
(A) NAME/KEY: exon (B) LOCATION: 2787..3347 (D) OTHER INFORMATION: /product= "1st exon of NIM1"
(ix) FEATURE:
(A) NAME/KEY: exon (B) LOCATION: 3427..4162 (D) OTHER INFORMATION: /product= "2nd exon of NIM1"
(ix) FEATURE:
(A) NAME/KEY: exon (B) LOCATION: 4271..4474 (D) OTHER INFORMATION: /product= "3rd exon of NIM1"
(ix) FEATURE:
(A) NAME/KEY: exon (B) LOCATION: 4586..4866 (D) OTHER INFORMATION: /product= "4th exon of NIM1"
(ix) FEATURE:
{A) NAME/KEY: CDS
(B) LOCATION: join(2787..3347, 3427..4162, 4271..4474, 4586..4866) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

TGGGATATGT CATTGGGTTT AGCGGTAATC.GGATTGAACC CTTTCCGGTA TAAAATACAA 180 AGCAACGGGC CGACACTTTA P~~T AAP~AAAAATGGGCCGACAAA TGCAAACGTA840 CTATAATAGA TGGTAGAAGA TP~AAAAAATTATATCAGATTGATTCAATTA AATTTTATAA1440 TATATCATTT TAAP.AAATTA ATTAAAAGAAAACTATTTCATAAAATTGTT CAAAAGATAA1500 _98_ _ TCTTACGTTG AACCTATCTT

GTCTTTT'ATATGTATACAATAATTGTTTTTAAATCAAATCCTAATTAAAAAAATATATTC 2040 ATG ACC GGA TTC
GCC

Met Asp Thr Thr Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu Ala Ala Glu Gln Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gln Leu Leu Ser Asn Ser Phe Glu Ser Val Phe Asp Ser Pro Asp Asp Phe Tyr Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His Arg Cys Val Leu Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala Ala Ala Lys Lys Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln _~~_ Arg His Leu Leu Asp Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys GTATGGATTC CTTATCACAA
TCACCCACTT AAAACAAAAC

Thr TTAAACATGG TTTATCATCA
TTTTGTTACT
TGCTGTCTGA

TG
GAA
CTC
GGG
AAA
CGii TTC
TTC
CCG
CGC
TGT
TCG
GCA
GTG
CTC

Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu GAG CAA TGC

AspGlnIleMet Asn Cys AspLeu Thr Leu Ala Gly Glu Glu Gln Cys CGA AAG TAC

AspAspThrAla Glu Lys LeuGln Lys Gln Arg Met Glu Arg Lys Tyr AAG GAG TTG

IleGlnGluThr Leu Lys AlaPhe Ser Asp Asn Glu Leu Lys Glu Leu GAT TCC AAA

GlyAsnSerSer Leu Thr SerThr Ser Thr Ser Ser Thr Asp Ser Lys Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg Arg ACACAGCAAG

(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 594 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Asp Thr Thr Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu Ala Ala Glu Gln Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gln Leu Leu Ser Asn Ser Phe Glu Ser Val Phe Asp Ser Pro Asp Asp Phe Tyr Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His (~~_ Arg Cys Val Leu Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala Ala Ala Lys Lys Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cars His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu Asp Gln Ile Met Asn Cys Glu Asp Leu Thr Gln Leu Ala Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gln Lys Lys Gln Arg Tyr Met Glu Ile Gln Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn Leu Glu Leu Gly Asn Ser Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg ~g (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 314 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Met Phe Gln Pro Ala Gly His Gly Gln Asp Trp Ala Met Glu Gly Pro Arg Asp Gly Leu Lys Lys Glu Arg Leu Val Asp Asp Arg His Asp Ser Gly Leu Asp Ser Met Lys Asp Glu Glu Tyr Glu Gln Met Val Lys Glu Leu Arg Glu Ile Arg Leu Gln Pro Gln Glu Ala Pro Leu Ala Ala Glu Pro Trp Lys Gln Gln Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu Ala Ile Ile His Glu Glu Lys Pro Leu Thr Met Glu Val Ile Gly Gln Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln Thr Pro Leu His Leu Ala Vai Ile Thr Asn Gln Pro Gly Ile Ala Glu Ala Leu Leu Lys Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly Asn Thr Pro Leu His Leu Ala Cys Glu Gln Gly Cys Leu Ala Ser Val Ala Val Leu Thr Gln Thr Cys Thr Pro Gln His Leu His Ser Val Leu Gln Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser Thr His Gly Tyr Leu Ala Ile Val Glu His Leu Val Thr Leu Gly Ala Asp Val Asn Ala Gln Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala Val Asp Leu Gln Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly Ala Asp Val Asn Arg Val Thr Tyr Gln Gly Tyr Ser Pro Tyr Gln Leu Thr Trp Gly Arg Pro Ser Thr Arg Ile Gln Gin Gln Leu Gly Gln Leu Thr Leu Glu Asn Leu Gln Met Leu Pro Glu Ser Glu Asp Glu Glu Ser Tyr Asp Thr Glu Ser Glu Phe Thr Glu Asp Glu Leu Pro Tyr Asp Asp Cys Val Phe GIy Gly Gln Arg Leu Thr Leu (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 314 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Phe Gln Pro Ala Gly His Gly Gln Asp Trp Ala Met Glu Gly Pro Arg Asp Gly Leu Lys Lys Glu Arg Leu Val Asp Asp Arg His Asp Ser Gly Leu Asp Ser Met Lys Asp Glu Asp Tyr Glu Gln Met Val Lys Glu Leu Arg Glu Ile Arg Leu Gln Pro Gln Glu Ala Pro Leu Ala Ala Glu Pro Trp Lys Gln Gln Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu Ala Ile Ile His Glu Glu Lys Thr Leu Thr Met Glu Val Ile Gly Gln Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln Thr Pro Leu His Leu Ala Val Ile Thr Asn Gln Pro Gly Ile Ala Glu Ala Leu Leu Lys Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly Asn Thr Pro Leu His Leu Ala Cys Glu Gln Gly Cys Leu Ala Ser Val Ala Val Leu Thr Gln Thr Cys Thr Pro Gln His Leu His Ser Val Leu 165 1?0 175 Gln Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser Ile His Gly Tyr Leu Gly Ile Val Glu His Leu Val Thr Leu Gly Ala Asp Val Asn Ala Gln Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala Val Asp Leu Gln Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly Ala Asp Val Asn Arg Val Thr Tyr Gln Gly Tyr Ser Pro Tyr Gln Leu Thr Trp Gly Arg Pro Ser Thr Arg Ile Gln Gln Gln Leu Gly Gln Leu Thr Leu Glu Asn Leu Gln Thr Leu Pro Glu Ser Glu Asp Glu Glu Ser 2?5 280 285 Tyr Asp Thr Glu Ser Glu Phe Thr Glu Asp Glu Leu Pro Tyr Asp Asp Cys Val Phe Gly Gly Gln Arg Leu Thr Leu (2) INFORMATION FOR SEQ ID N0:5:
ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 314 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ZD N0:5:
Met Phe Gln Pro Ala Glu Pro Gly Gln Glu Trp Ala Met Glu Gly Pro Arg Asp Ala Leu Lys Lys Glu Arg Leu Leu Asp Asp Arg His Asp Ser Gly Leu Asp Ser Met Lys Asp Glu Glu Tyr Glu Gln Met Val Lys Glu Leu Arg Glu Ile Arg Leu Glu Pro Gln Glu Ala Pro Arg Gly Ala Glu Pro Trp Lys Gln Gln Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu Ala Ile Ile His Glu Glu Lys Ala Leu Thr Met Glu Val Val Arg Gln VaI Lys Gly Asp Leu Ala Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln Thr Pro Leu His Leu Ala Val Ile Thr Asn Gln Pro Glu Ile Ala Glu Ala Leu Leu Glu Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly Asn Thr Pro Leu His Leu Ala Cys Glu Gln Gly Cys Leu Ala Ser Val Gly VaI Leu Thr Gln Pro Arg Gly Thr Gln His Leu His Ser Ile Leu -111_.
Gln Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser Ile His Gly Tyr Leu Gly Ile Val Glu Leu Leu Val Ser Leu Gly Ala Asp Val Asn Ala Gln Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala Val Asp Leu Gln Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly Ala Asp Val Asn Arg Val Thr Tyr Gln Gly Tyr Ser Pro Tyr Gln Leu Thr Trp Gly Arg Pro Ser Thr Arg Ile Gln Gln Gln Leu Gly Gln Leu Thr Leu Glu Asn Leu Gln Met Leu Pro Glu Ser Glu Asp Glu Glu Ser Tyr Asp Thr Glu Ser Glu Phe Thr Glu Asp Glu Leu Pro Tyr Asp Asp Cys Val Leu Gly Gly Gln Arg Leu Thr Leu (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2011 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Arabidopsis thaliana (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 1..2011 (D) OTHER INFORMATION: /note= "NIM1 cDNA sequence' (ix) FEATURE:
( A ) NAME / KEY : CDS
(B) LOCATION: 43..1824 (D) OTHER INFORMATION: /product= 'NIM1 protein"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:

Met Asp Thr Thr Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu Ala Ala Glu Gln Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gln Leu Leu Ser Asn Ser Phe Glu Ser Val Phe Asp Ser Pro Asp Asp Phe Tyr Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His Arg Cys Val Leu Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala Ala Ala Lys Lys GAG AAA GAC TCC AAC AAC ACC GCC GCC GTG AAG CTC GAG eTT AAG GAG 390 Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Thr Leu Glu Asn Leu Gln Met Le Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu Glu Pro Asp Arg ACT GTA

Leu GlyThr LysArg ThrSer Pro Gly LysIle AlaPro Phe Thr Val AGA CTAGAA GAGCAT CAAAGT AGA CTA GCG~CTT TCTAAA ACC 1542 ATC AAA

Arg LeuGlu GluHis GlnSer Arg Leu AlaLeu SerLys Thr Ile Lys GAA TGT

Val LeuGly LysArg PhePhe Pro Arg SerAla ValLeu Asp Glu Cys ATT CTG

Gln MetAsn CysGlu AspLeu Thr Gln AlaCys GlyGlu Asp Ile Leu ACT CAA

Asp AlaGlu LysArg LeuGln Lys Lys ArgTyr MetGlu Ile Thr Gln GAG GAC

Gln ThrLeu LysLys AlaPhe Ser Glu AsnLeu GluLeu Gly Glu Asp TTG ACA

Asn SerLeu ThrAsp SerThr Set Ser SerLys SerThr Gly Leu Thr AAG CGT

Gly ArgSer AsnArg LysLeu Ser His ArgArg Lys Arg ATTTTTGCTG
TACCATATAA

TGGCG TAGTTTCGCT

TCAAACAAAT
GTTGTAACAA

(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2011 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 43..1824 (D) OTHER INFORMATION: /product= 'altered form of NIM1"
/note= "Serine residues at amino acid positions 55 and 59 in wild-type NIM1 gene product have been changed to Alanine residues."
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 205..217 (D) OTHER INFORMATION: /note= "nucleotides 205 and 217 changed from T's to G's compared to wild-type sequence."
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:

Met Asp Thr Thr Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu Ala Ala Glu Gln Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gln Leu Leu Ser Asn Ser Phe Glu Ala Val Phe Asp Ala Pro Asp Asp Phe Tyr Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His Arg Cys Val Leu Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala Ala Ala Lys Lys g5 90 95 100 Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Va1 His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gin Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg Va1 Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu Asp Gln Ile Met Asn Cys Glu Asp Leu Thr Gln Leu Ala Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gln Lys Lys Gln Arg Tyr Met Glu Ile Gln Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn Leu Glu Leu Gly ACC GGT

Asn Leu Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg Arg 585 ~ 590 ATGATGACTG

CATCCTGTGT

TGGTATACAG

(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 594 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Met Asp Thr Thr Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu Ala Ala Glu Gln Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gln Leu Leu Ser Asn Ser Phe Glu Ala Val Phe Asp Ala Pro Asp Asp Phe Tyr Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His Arg Cys Val Leu Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala Ala Ala Lys Lys Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys Val VaI Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala WO 98129537 PCT/EP9?/07253 -i~-Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala 5er Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Giu Asn Arg Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu Asp Gln Ile Met Asn Cys Glu Asp Leu Thr Gln Leu Ala Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gln Lys Lys Gln Arg Tyr Met Glu Ile Gln Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn Leu Glu Leu Gly Asn Leu Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg Arg (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1597 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLCSGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
A ) NAME / KEY : CDS
(B) LOCATION: 1..1410 (D) OTHER INFORMATION: /product= "Altered form of NIM1"
/note= "N-terminal deletion compared to wild-type NIM1 sequence."
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:

Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys Val Val Ile Glu Asp Trr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala VaI Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val WO 9$/29537 PCT/EP97/07253 Leu His Val Ala Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys Ile Ala Pro Phe Arg IIe Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe -~2s-Pro Arg Cys Ser Ala Val Leu Asp Gln Ile Met Asn Cys Glu Asp Leu Thr Gln Leu Ala Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gln Lys Lys Gln Arg Tyr Met Glu Ile Gln Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn Leu Glu Leu Gly Asn Leu Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg Arg (2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 470 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

-127- _ Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu VaI Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu Asp Gln Ile Met Asn Cys Glu Asp Leu Thr Gln Leu Ala Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gln Lys Lys Gln Arg Tyr Met Glu Ile Gln Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn Leu Glu Leu Gly Asn Leu Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg Arg 4 6.5 4? 0 (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1608 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 43..1608 (D) OTHER INFORMATION: /product= "Altered form of NIMl"
/note= "C-terminal deletion compared to wild-type NIM1."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Met Asp Thr Thr Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu Ala Ala Glu Gln Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gln Leu Leu Ser Asn Ser Phe Glu Ser Val Phe Asp Ser Pro Asp Asp Phe Tyr Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His Arg Cys Val Leu Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala Ala Ala Lys Lys Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu Asp Gln Ile Met Asn Cys (2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 522 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:

Met Asp Thr Thr Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu Ala Ala Glu Gln Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gln Leu Leu Ser Asn Ser Phe Glu Ser Val Phe Asp Ser Pro Asp Asp Phe Tyr Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His Arg Cys Val Leu Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala g5 90 95 Ala Ala Lys Lys Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys VaI Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser _1 Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His VaI Ser Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu Asp Gln Ile Met Asn Cys (2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1194 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1194 (D) OTHER INFORMATION: /product= ~Altered form of NIM1~
/note= ~N-terminal/C-terminal chimera.~
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:

Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu ATA CTA GAG CAA GAA GAC APrA CGA GAA CAA ATT CCT AGA GAT GTT CCT 864 Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys Ile Ala Pro Phe Arg Ile Leu Glu Glu His,Gln Ser Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu Asp Gln Ile Met Asn Cys (2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 398 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg VaI

Arg Pro Pro Pro Lys Gly VaI Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu Asp Gln Ile Met Asn Cys (2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 786 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..786 (D) OTHER INFORMATION: /product= "Altered form of NIM1"
/note= "Ankyrin domains of NIM1."
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:

Met Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys Val GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT~AAT ATA TGT GGT 336 Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyz Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly (2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 262 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Met Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys Val VaI Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly (2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:

_144_ Ile Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile Glu Leu Val Lys Leu Met Val Met Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala Val His Tyr Ala Val Gln His Cys Asn (2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Pro Thr Gly Lys Thr Ala Leu His Leu Ala Ala Glu Met Val Ser Pro Asp Met Val Ser Val Leu Leu Asp His His Ala Asp Xaa Asn Phe Arg Thr Xaa Asp Gly Val Thr (2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
Ile Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile Glu Leu Val Lys Leu Met Val Met Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala Val His Tyr Ala Val Gln His Cys Asn (2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
Arg Arg Pro Asp Ser Lys Thr Ala Leu His Leu Ala Ala Glu Met Val Ser Pro Asp Met Val Ser Val Leu Leu Asp Gln (2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant -1as-(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
Ile Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile Glu Leu Val Lys Leu Met Val Met Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala Val His Tyr Ala Val Gln His Cys Asn {2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Arg Arg Pro Asp Ser Lys Thr Ala Leu His Leu Ala Ala Glu Met Val Ser Pro Asp Met Val Ser Val Leu Leu Asp Gln (2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
Ile Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile Glu Leu Val Lys Leu Met Val Met Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala Val His Tyr Ala Val Gln His Cys Asn (2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
Pro Thr Gly Lys Thr Ala Leu His Leu Ala Ala Glu Met Val Ser Pro Asp Met Val (2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:

f2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (Dy TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:

(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = 'oligonucleotide' (xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:

(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:

(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:

- i 50 -(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide' (xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:

(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:

(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = 'oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:

Claims (31)

What Is Claimed Is:

1. A method for protecting a plant from pathogen attack through synergistic disease resistance, comprising the steps of:
(a) providing an immunomodulated plant having a first level of disease resistance; and (b) applying to said immunomodulated plant at least one microbicide that confers a second level of disease resistance;
(c) whereby application of said microbicide to said immunomodulated plant confers a synergistically enhanced third level of disease resistance that is greater than the sum of the first and second levels of disease resistance.

2. A method according to claim 1, wherein said immunomodulated plant is (a) a constitutive immunity (cim) mutant plant;
(b) a lesion mimic mutant plant;
(c) obtained by recombinant expression in a plant of an SAR gene;
(d) obtained by applying to a plant a chemical capable of inducing SAR.

3. A method according to claim 2, wherein said cim mutant plant is selected from a population of plants according to the following steps:
(a) evaluating the expression of SAR genes in uninfected plants that are phenotypically normal in that said uninfected plants lack a lesion mimic phenotype; and (b) selecting uninfected plants that constitutively express SAR genes in the absence of viral, bacterial, or fungal infection.

4. A method according to claim 3, wherein said lesion mimic mutant plant is selected from a population of plants according to the following steps:
(a) evaluating the expression of SAR genes in uninfected plants that have a lesion mimic phenotype; and (b) selecting uninfected plants that constitutively express SAR genes in the absence of viral, bacterial, or fungal infection.

5. A method according to claim 3, wherein said SAR gene is a functional form of a NIM1 gene encoding a NIM1 protein involved in the signal transduction cascade leading to systemic acquired resistance in plants.

6. A method according to claim 5, wherein said NIM1 protein comprises the amino acid sequence set forth in SEQ ID NO:2.

7. A method according to claim 5, wherein said NIM1 gene comprises the coding sequence set forth in SEQ ID NO:1.

8. A method according to claim 3, wherein said SAR gene encodes an altered form of a NIM1 protein that acts as a dominant-negative regulator of the SAR signal transduction pathway.

9. A method according to claim 8, wherein said altered form of the NIM1 protein has alanines instead of serines in amino acid positions corresponding to positions 55 and 59 of SEQ ID NO:2.

10. A method according to claim 9, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO:8.

11. A method according to claim 9, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:7.

12. A method according to claim 8, wherein said altered form of the NIM1 protein has an N-terminal truncation of amino acids corresponding approximately to amino acid positions 1-125 of SEQ ID NO:2.

13. A method according to claim 13, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO:10.

14. A method according to claim 13, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:9.

15. A method according to claim 8, wherein said altered form of the NIM1 protein has a C-terminal truncation of amino acids corresponding approximately to amino acid positions 522-593 of SEQ ID NO:2.

16. A method according to claim 15, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEQ ID NO:12.

17. A method according to claim 15, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:11.

18. A method according to claim 8, wherein said altered form of the NIM1 protein has an N-terminal truncation of amino acids corresponding approximately to amino acid positions 1-125 of SEQ ID NO:2 and a C-terminal truncation of amino acids corresponding approximately to amino acid positions 522-593 of SEQ ID NO:2.

19. A method according to claim 18, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEA ID NO:14.

20. A method according to claim 18, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:13.

21. A method according to claim 8, wherein said altered form of the NIM1 protein consists essentially of ankyrin motifs corresponding approximately to amino acid positions 103-362 of SEQ ID NO:2.

22. A method according to claim 21, wherein said altered form of the NIM1 protein comprises the amino acid sequence shown in SEO ID NO:16.

23. A method according to claim 21, wherein said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:15.

24. A method according to any one of claims 7, 11, 14, 17, 20 and 23, wherein said DNA molecule hybridizes under the following conditions to the nucleotide sequence set forth in SEQ ID Nos: 1, 7, 9, 11, 13 or 15: hybridization in 1%BSA; 520mM
NaPO4, pH7.2;
7% lauryl sulfate, sodium salt; 1mM EDTA; 250 mM sodium chloride at 55°C for 18-24h, and wash in 6XSSC for 15 min. (X3) 3XSSC for 15 min. (X1) at 55°C.

25. A method according to any of claims 1 to 24, wherein said microbicide is a fungicide selected from the following group:
4-[3-(4-chlorophenyl)-3-(3,4-dimethoxyphenyl)acryloyljmorpholine ("dimethomorph");
5-methyl-1,2,4-triazolo[3,4-b][1,3]benzothiazole ("tricyclazole");
3-allyloxy-1,2-benzothiazole-1,1-dioxide ("probonazole");
.alpha.-[2-(4-chlorophenyl)ethyl]-.alpha.-(1,1-dimethyiethyl)-1H-1,2,4-triazole-1-ethanol, ("tebuconazole");
1-[[3-(2-chlorophenyl)-2--(4-fluorophenyl)oxiran-2-yl]methyl]-1H-1,2,4-triazole, ("epoxyconazole");
.alpha.-(4-chlorophenyl)--.alpha.-(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-methoximino--2-[.alpha.-{o-tolyloxy)--o-tolyl]acetate, ("BAS 490 F", "cresoxime methyl");
2-(2-phenoxyphenyl}-(E)-2-methoximino--N-methylacetamide);
[2-(2,5-dimethylphenoxymethyl)-phenyl]-{E)--2-methoximino-N-methylacetamide);
(1R,3S/1S,3R)-2,2-dichloro-N-[(R)-1-(4-chlorophenyl)ethyl]-1-ethyl-3-methylcyclopropanecarboxamide, ("KTU 3616");

manganese ethylenebis(dithiocarbamate)polymer-zinc complex, ("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-ylmethy I)-1,2,4--triazole, ("difenoconazole");
1-[2-(2,4-dichlorophenyl)pentyl--1H-1,2,4-triazole,("penconazole");
cis-4-[3-(4-tart-butylphenyl)--2-methylpropyl}--2,6-dimethylmorpholine, ("fenpropimorph");
1-[3-(4-tart-butylphenyl)--2-methylpropyl]-piperidine, ("fenpropidin"); 4-cyclopropyl-6-methyl-N-phenyl-2-pyrimidinamine ("cyprodinil");
(RS)-N-(2,6-dimethylphenyl--N-(methoxyacetyl)-alanine methyl ester ("metalaxyl");
(R)-N-(2,6-dimethylphenyl--N-(methoxyacetyl)-alanine methyl ester ("R-metalaxyl");
1,2,5,6-tetrahydro--4H-pyrrolo[3,2,1-ij]quinolin-4-one("pyroquilon"); and ethyl hydrogen phosphonate ("fosetyl°).

26. A method according to claim 25, wherein said fungicide is metalaxyl.

27. A method according to any of claims 2 to 26, wherein said chemical capable of inducing SAR is either a benzothiadiazole compound, an isonicotinic acid compound, or a salicylic acid compound.

28. A method according to any of claims 1 to 26, wherein said microbicide is either a benzothiadiazole compound, an isonicotinic acid compound, or a salicylic acid compound.

29.. A method according to any of claims 1 to 28, wherein two microbicides are concurrently applied to said immunomodulated plant.

30. A method according to claim 29, wherein one of said microbicides is a fungicide selected from the following group:
4-[3-(4-chlorophenyl)-3-(3,4-dimethoxyphenyl)acryloyl]morpholine ("dimethomorph");
5-methyl-1,2,4-triazolo[3,4-b][1,3jbenzothiazole ("tricyclazole");
3-allyloxy-1,2-benzothiazole-1,1-dioxide ("probonazole");

.alpha.-[2-{4-chlorophenyl)ethyl]--.alpha.e-{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, ("epoxyconazol");
.alpha.(4-chlorophenyl)--.alpha.e-(1-cyclopropylethyl)--1H-1,2,4-triazote--1-ethanol, ("cyproconazol");
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, ("tetraconazote");
methyl-{E)-2-{2-[6-(2-cyanophenoxy)pyrimidin--4-yloxy]phenyl]-3-methoxyacrylate) ("ICI A 5504", "azoxystrobin");
methyl-(E)--2-methoximino--2-[.alpha.-(o-tolyloxy)--o-tolyl]acetate, ("BAS 490 F", "cresoxime methyl");
2-(2-phenoxyphenyl)-(E)-2-methoximino--N-methylacetamide);
[2-(2,5-dimethylphenoxymethyl)-phenyl]-(E)--2-methoximino-N-methylacetamide);
(1R,3S/1S,3R)-2,2-dichloro--N-[(R)-1-(4-chlorophenyl)ethyl]--1-ethyl-3-methyfcyclopropanecarboxamide, ("KTU 3616");
manganese ethylenebis(dithiocarbamate)polymer-zinc complex) ("mancozeb");
1-[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxoian--2-ylmethyl]--1H-1,2,4--triazole, ("propiconazole");
1-{2-[2-chloro-4-{4-chlorophenoxy)phenyl]-4-methyl--1,3-dioxolan--2-ylmethy l)--1H-1,2,4--triazole, ("difenoconazole");
1-[2-(2,4-dichlorophenyl)pentyl--1H-1,2,4-triazole, ("penconazole");
cis-4-[3-(4-tent-butytphenyl)--2-methylpropyl]--2,6-dimethylmorpholine, ("fenpropimorph");
1-[3-(4-tart-butyiphenyl)--2-methylpropyl]-piperidine, ("fenpropidin"); 4-cyclopropyl-6-methyl-N-phenyl-2-pyrimidinamine ("cyprodinil');
(RS)-N-(2,6-dimethylphenyl--N-(methoxyacetyl)-alanine methyl ester ("metalaxyl");
(R)-N-(2,6-dimethylphenyl--N-(methoxyacetyl)-atanine methyl ester ("R-metalaxyl");
1,2,5,6-tetrahydro--4H-pyrrolo[3,2,1-lj]quinoiin-4-one ("pyroquiton"); and ethyl hydrogen phosphonate ("fosetyl");

and the other microbicide is is either a benzothiadiazole compound, an isonicotinic acid compound, or a salicylic acid compound.

31. A method according to claim 30, wherein the fungicide is metalaxyl and the other microbicide is a benzothiadiazole compound.