UA73714C2 - A method of 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 microbictde 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 NIM1 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

Description of the invention

This invention relates to a method of protecting plants from pathogenic influence by creating synergistic 2 resistance to diseases, which is created by introducing a bactericide into an immunomodulated plant.

Plants are constantly infected by a variety of pathogenic organisms, including viruses, bacteria, fungal organisms, and nematodes. Cultivated plants are particularly vulnerable because they are usually grown as genetically homogeneous monocultures; losses in case of occurrence of such diseases are very large. However, most plants have their own natural defense mechanisms against pathogenic organisms. 70 Natural varieties of resistance to plant pathogens have been identified by plant breeders and pathologists and bred in many cultivated plants. These natural disease resistance genes often provide quite high levels of resistance or immunity to pathogens.

Systemic acquired resistance (SAC) is one of the components of a complex system of using the plants themselves for self-defense against pathogens (publication Nipi apa Kuaiv, publication Stii. Kem. ip Riapi Zsi. 15, 583-606 (1996), 72 references to which are given in this description in its entirety; Kuaiv ei ai!., Riapi Seyi 8, 1809-1819 (1996), which is referenced in this description in its entirety). See (see also US Patent No. 5,614,395, which is incorporated herein by reference in its entirety). SAC is a particularly important aspect of plant immune responses to pathogens because it represents pathogen-induced systemic resistance to a wide range of infectious agents, including viruses, bacteria, and fungal organisms. When the signal transduction pathway

AB is blocked, plants become more susceptible to pathogens that normally cause disease, and also become susceptible to some infectious agents that would not normally cause disease. to which is given in this description in its entirety; Oeiapeu ei aiYi., Zsiepse 266, 1247-1250 (1994), which is referenced in this description in its entirety; YuOeiapeu ei aiYi., Rgos. May Asad All together. OBA 92, 6602-6606 (1995), which is referenced in this description in its entirety; Oeiapeu, Riapi Rpuz. 113, 5-12 (1997), which is referenced in this Ge) description in its entirety; Vi ei ai., Riapi U. 8, 235-245 (1995), which is referenced in this description in its entirety; Mashsy-Mapi apa Ziyzagepko, Riapi Seiyi 8, 203-212 (1996), which is referenced in this description in its entirety). These observational results indicate that the signal transduction pathway

ZAC is critical from the point of view of maintaining plant health. WITH

Conceptually, the immune response to ZAC can be divided into two phases. In the initial phase, a pathogenic infection "is recognized and a signal is released that passes through the phloem to distant tissues. This systemic signal is perceived by target cells, which respond by expressing both the genes of ZAC and resistance to the disease. The phase of preservation of ZAK lasts for a certain period of time, from a few weeks to the entire life period of the plant, during which the plant is in a semi-stable state, and resistance to the disease is preserved.

It turns out that the accumulation of salicylic acid (SA) is necessary for the transduction of the ZAK signal. Plants that cannot accumulate ZA due to treatment with specific inhibitors, epigenetic repression of phenylalanine ammonia lyase, or transgenic expression of salicylate hydroxylase, which specifically cleaves 5A, "also cannot induce either the expression of ZAK genes or resistance to the disease | SaiPpeu et al., 1993; WITH

Oeiapeu ei aiI., 1994; Maisp-Mapi apa Biyzagepko 1996; Mapyeg eyi ai., Rgos. May Asad All together. OBA 91, pp. 7802-7806 (1994), which is referenced in this description in its entirety; RaiYiaz ei ai., Riapi 9. 10, 281-293

From" (1996), which is referenced in this description in its entirety). Although it has been suggested that ZA may play a role as a systemic signal, this is currently a controversial issue, and at this point in time, all that is known for sure is that if 5A cannot accumulate, BAC signal transduction is blocked (|RaiYaz ei aiYi. , 1996; ZpschShiaiem ei ai., Riapi Seili 1, 1691-1701 (1995), the reference to which is given in this description in its entirety; Megpoi| ei ai., Riapi Seili II b, 959-965 (1994), reference which is given in this description in its entirety).

Recently, it has been found that Agabridorziz plays the role of a modeling system for the study of ZAK |OKpevs and ei ai., Riapi Seyi 4, 645-656 (1992), the reference to which is given in this description in its entirety; OKpez her - 20 a), My. Riapi-Misgore Ipiegashi 6, 692-698 (1993), which is incorporated herein by reference in its entirety; Sategop et al., Riapi U. 5, 715-725 (1994), which is referenced in this description in its entirety; Maisy-Mapi apa biyzagepko, Moi. Riapi-Misgore Ipiegasi 7, 378-383 (1994), which is incorporated herein by reference in its entirety; JuOetrzeu apa Kiezzid, WiPeip de I" pozvojtsi Ravieig93, 167-186 (1995), which is referenced in this description in its entirety! ZAC has been shown to be activated in 52 Agaridorsis by both pathogens and chemical reagents such as 5A, 2,6-dichloroisonicotinic acid (IMA) and

HF) benzo|1,2,3Hiadiazol-7-carbothioic acid-5-methyl ester (VTN) (OKpez ei ai., 1992; Megpoi| ei ai., Moi.

Riapi-Misgore ipiegasi. 8, 228-234 (1995), which is incorporated herein by reference in its entirety; about Iaulop ei ai., Riapi U. 10, 71-82 (1996), reference to which is given in this description in its entirety).

After treatment with IMA, or VTN, or infection with a pathogen, at least three protein genes associated with pathogenesis, namely 60 RA-1, RK-2 and RK-5, respectively, are induced simultaneously with the beginning of the creation |OKpez ei ai., 1992, 1993).

For tobacco (the most characterized species), treatment with a pathogen or immunizing compound induces the expression of at least nine sets of genes (Maga et al., Journal of Medicine 3, 1085-1094 (1991), which is incorporated herein by reference in its entirety). Transgenic disease-resistant plants have been created by transforming plants with various ZAC genes (US Patent No. 5,614,395). Because several Agaridors mutants were isolated that modified the BAC signal transduction (|Oeiiapeu, 1997). The first of these mutants were the so-called Iza mutants (Ieviop zyticiaipod disease - a disease that modulates lesions) and asa2 (asseiegaieai sei! deafy - accelerated cell death ) |Oieigisy ei ai., Se! 11, 565-577 (1994), which is incorporated herein by reference in its entirety; Sgeepreguo ei aiYi., Se! 11, 551-563 (1994), which is incorporated herein by reference in its entirety). For all these mutants, there is some formation of spontaneous necrotic lesions on leaves, an increase in the concentration of ZA, accumulation of mRNA for ZAK genes, as well as a significant increase in resistance to the disease. At least seven different mutants of Iza were isolated and described. MUeuthapp et al., Journal 1, 2013-2022 (1995), which is incorporated herein by reference in its entirety. Another interesting class of mutants is sit mutants (sopziytshime /0 ittipnu - constitutive immunity) (amp ei aY,, "Te toiesciag Biooodu oi zuzietis asadtsigei geviviapse" in

Mespapiztz oi Yuegepvze Kezropvez ip Riapiv, V. Rgyid apa M. I edgapa, edz (Oogagesni, Te MeiPegiapav: Kishumeg

Asadetis Rybiizpegv), pp. 422-432 (1993), which is referenced in this description in its entirety). See as well as the International PCT application UUO 94/16077, the reference to which is given in this description in its entirety).

Similar to Iza and as42 mutants, sit mutants increased the level of ZA and the expression of 5AK genes, as well as resistance, /5 but, unlike Iza or asa2, were not marked by visible lesions on leaves. srg1 (the constitutive expressor of RK genes) can represent a type of mutant sit; however, since the presence of microscopic lesions on srg1 leaves is not excluded, srg1 may also be an Iza-type mutant

IVomiipa ei a/!., Riapi Sei! 6, 1845-1857 (1994), which is incorporated herein by reference in its entirety).

Mutants that are blocked in the path of transmission of the ZAK signal have also been identified. pag1 (race (strain) non-specific resistance to the disease) is a mutant that causes the growth of both Rzeidotopaz zugipdae, which contains various avirulence genes, and the usual avirulent strains of Rhegopozrog ragaziis |Sepiigu ei ai.,

Rgos. May Asai.Zsi. OBA 92, 6597-6601 (1995), which is incorporated herein by reference in its entirety. Apparently, this mutant is blocked at the very beginning of the ZAC signaling pathway. prg1 (popehrgezzeg oi РV depez - a non-expressor of RK genes) is a mutant that cannot induce the expression of the BAC signal transduction pathway after treatment with IMA ISao ei ai., Riapi SeiI 6, 1583-1592 (1994), the reference to which is given here description in its entirety). The edz (increased susceptibility to disease) mutants were isolated on the basis of i) their ability to support bacterial infection after inoculation of bacteria at a low concentration |CiIagebgook ei a!., Sepiysz 143, 973-982 (1996), which is referenced in this description in all its integrity; RagKeg et al., Journal of Scientific Research 8, 2033-2046 (1996), which is referenced in this description throughout

From || integrity). Some AIDS mutants are phenotypically very similar to pri1, and more recently avids5 and aids53 have been found to be allelic to prg! |SiagergooK ei ai., 1996). sweat! (popipdisibie ittipyu - -- non-induced immunity) is a mutant that supports the growth of R. ragavziis (that is, the agent that causes the disease M false powdery mildew) after treatment with IMA |Oeiyapeu ei ai., 1995; M/094/16077|. Although sweat! can accumulate ZA after infection with a pathogen, it cannot induce the expression of ZAK genes or resistance to the disease, on the basis of which it can be assumed that the mutation blocks the pathway "downstream" of 5A. sweat! і- is also characterized by a weakened property of reacting to IMA or VTN, on the basis of which it can be assumed that the block exists "below" the place of influence of these chemical agents (Oeiapeu ei ai., 1995; I audop egai., 1996).

Recently, two allelic Agaridorziz genes were isolated and described, the mutants of which are responsible for "sweat phenotypes!" and pri1, respectively |Quaiz ei ai., Riapi Seii! 9, 425-439 (1997), which is referenced in this description in its entirety; Sao ei ai!., Se! 88, 57-63 (1997), referenced herein. in its entirety). The natural MIM1 gene product is included in the classical signal transduction pathway, what is it? leads both to 5AK and to resistance to the disease "gene-on-gene" in Agaridorziz (Kuaiv ei ai., 1997)

Kuaiv et al., 1997 also report the isolation of five additional alleles of sweat, characterized by a range of phenotypes - from slightly weakened due to chemically induced gene expression of RK-1 and resistance to the fungus - to -I strongly blocked. The transformation of the natural MRK1 gene into prg1 mutants not only complemented the mutations that restore the reactivity of ZAC induction with respect to the expression of the RK gene and resistance to the disease, but also made the transgenic plants more resistant to infection by R. zugipdae due to the absence of ZAC induction |Sao ei a/!. , 1997). -I ME-kV/kV signal transduction pathways have been used to create resistance responses to disease in a range of organisms from Ogozorpia to mammals. In mammals, ME-kCkV/CV signal transduction can be induced - with the help of certain stimulating agents, including exposure of cells to lipopolysaccharide, tumor necrosis factor, interleukin 1 (1-1) or viral infection. 87, 13-20 (1996); Waidmjip, Appy. To whom And so on. 14, 649-681 (1996)). Activation of the pathway leads to the synthesis of several factors involved in inflammatory and immune reactions, such as 1-2, I-6, I/-8 and stimulating factor

Colonies of granulocytes/macrophages (deMapip et al., Sepe 152, 253-255 (1995)). In experiments with transgenic mice, the destruction of ME-KkV/KV signal transduction leads to a defective immune response, including increased susceptibility to bacterial and viral pathogens |Ved apa Vatoge, Zsiepse 274, 782-784 (1996); Map of Apizhegr ei ai,, Zsiepse 274, 787-789 (1996); M/apd ei aiYi., Science 274, 784-787 (1996); Waetsege apa Waiitoge (1996)). For Agaridorziz, BAC is functionally analogous to inflammation in those normal processes of resistance that become possible after activation of ZAK, which leads to increased resistance to the disease | Vi ei ai., 1995;

Sao ei ai., 1994; Oeiapeu ei ai., 1995; YuOeiapeu ei ai., 1994; SaiPpeu ei ai., 1993; Maisi-Mapi apa Biyzagepko 1996; OeiIapeu, 19971. In addition, inactivation of the pathway leads to increased susceptibility to bacterial, viral, and fungal pathogens. Incidentally, it has been reported that ZA blocks the activation of ME-kV in mammalian cells (Corr apa SPpov5y, Zsiepse 265, 956-959 (1994)), while 5A activates signal transduction in 65 Agaridors. Bacterial infection of Ogozorpia activates the classical signal transduction pathway, which leads to the synthesis of several antifungal proteins, such as cercropin B, defensin, diptericin and drozomycin (Ir ei ai.,

Sci 75, 753-763 (1993); I etaige ei aiI., Se// 86, 973-983 (1996)). This induction depends on the dogga gene product! and ai, two ME-KV homologues, and is repressed by sasiiv5, an IKV homologue, in flies. Mutants with reduced synthesis of antifungal and antibacterial proteins are characterized by critically reduced resistance to infection.

Despite a lot of research and the use of quite complex and intensive plant protection measures, including genetic transformation of plants, the losses due to diseases remain very significant and amount to billions of dollars annually. Thus, there is a continuing need to design and develop new means of protection that are based on the ever-increasing understanding of the genetic basis of resistance to 7/0 disease in plants.

In view of the above, in the main aspect, this invention relates to a new method of protecting plants from the harmful effects of pathogens through synergistic disease resistance, which is created by introducing a bactericide into immunomodulated plants. Immunomodulated plants are those in which they are activated

ZAK, which are usually characterized by a higher expression of ZAK genes than in natural varieties and, thus, 75 are considered as plants "with established ZAK" ("ZAK-op"). Immunomodulated plants, which are used for the implementation of the method proposed in this invention, are obtained in at least three different ways: by introducing into the plants a chemical inducer of BAC, such as VTN, IMA and BA; by using a selective breeding program, according to which plants are selected on the basis of constitutive expression of ZAC genes and/or a disease-resistant phenotype; or through the use of genetic engineering of plants by transforming them with one or more ZAK genes, such as the functional form of the MIM1 gene.

The bactericide administered to the immunomodulated plants can be either a standard bactericide such as the fungicide metalaxyl, or - if applied to immunomodulated plants obtained by selective breeding or genetic engineering - a chemical inducer of SAC such as VTN, IMA or 5A .

Immunomodulation provides a certain level of disease resistance in the plant. Likewise, the introduction of a bactericide into the plant provides a certain level of resistance to the disease. The expected result of combining immunomodulation with the introduction of a bactericide will be the level of control that reflects the additional levels of control that are provided by specific methods of creating resistance to the disease.

However, with the help of parallel introduction of a bactericide into an immunomodulated plant, the resistance to the disease unexpectedly increases synergistically; therefore, the level of resistance to the disease appears to be 'And higher than the expected additional levels of resistance to the disease.

Thus, this invention relates to the cultivation of immunomodulated plants and the introduction into them of the appropriate amount of a standard bactericide. The best embodiments of the invention relate to plants that are processed using genetic engineering methods in order to make them contain and express the functional form of the MIM1 gene or its homologue or variant. and.

The method of the invention makes it possible to fight pathogens more effectively than can be achieved by the introduction of immunomodulation or a bactericide separately. Immunomodulation provides a certain level of disease resistance in the plant. Likewise, the introduction of a bactericide into the plant provides a certain level of resistance to the disease. The expected result of combining immunomodulation with the introduction of a bactericide will be the level of control that reflects the additional levels of control that are provided by specific methods of "creating resistance to the disease." However, with the help of parallel introduction of a bactericide in 8 s immunomodulated plant, the effectiveness of combating pathogenic diseases is unexpectedly synergistically enhanced; therefore, the level of disease control appears to be higher than the expected additional levels of "" disease resistance.

In addition to increased resistance to the disease, another advantage of the present invention is that the method of the present invention to achieve the level of resistance to the disease requires a smaller amount of bactericide than in the case of using -I ordinary natural plants. As a result, both the economic costs of using a bactericide are reduced, and the probability of harmful consequences for the environment, the cause of which is the toxicity of some bactericides, is reduced. In addition, the method of plant protection according to this invention - by combining the effects of -I immunomodulation and the introduction of a bactericide - leads to an extension of the period of antipathogenic action and, together with this, an increase in the yield of agricultural crops. Another advantage of the proposed method is that these two - combined types of pathogen control are completely different, and because of this, the threat of developing resistance is practically

Ta" is excluded.

Thus, this invention relates to a method of protecting a plant from the harmful effects of pathogens through synergistic disease resistance, including steps such as: (a) obtaining an immunomodulated plant having a first level of disease resistance; and (b) introducing into said immunomodulated plants at least one bactericide that creates a second iF) level of disease resistance; co (c) corresponding introduction of said bactericide into said immunomodulated plant creates a synergistically enhanced third level of disease resistance which is greater than the sum of the first and second levels of disease resistance.

The method according to the present invention is optimal, according to which said immunomodulated plant is a mutant plant with constitutive immunity (sieve).

In particular, the method according to the present invention is optimal, according to which said mutant plant plant is selected from a population of plants using the following steps: 65 (a) Evaluation of BAC gene expression in uninfected plants that are phenotypically normal in said uninfected deficient plants phenotype of the lesion mimic gene; and

(b) Selection of uninfected plants that constitutively express ZAC genes in the absence of viral, bacterial, or fungal infection.

Also optimal is the method according to the present invention, according to which said immunomodulated plant is a mutant plant with a lesion mimic gene.

In particular, the method according to the present invention is optimal, according to which said mutant plant with a lesion mimic gene is selected from a population of plants using the following stages: (a) Evaluation of BAC gene expression in uninfected plants having a phenotype of the lesion mimic gene; and (b) Selection of uninfected plants that constitutively express ZAC genes in the absence of viral, 7/0 bacterial, or fungal infection.

Also optimal is the method according to the present invention, according to which the said immunomodulated plant is obtained by recombinant expression of the 5AK gene in the plant.

In particular, the method according to this invention, according to which the mentioned ZAK gene is a functional form of the MIM1 gene, is optimal.

A preferred method according to the present invention is that said MIMI gene encodes a MIMI protein involved in a classical signal transduction pathway leading to systemic acquired resistance in plants.

Most preferably, the method according to the present invention, according to which said MIMI protein contains the amino acid sequence designated as SEQ. Mo ID: 2.

The method according to the present invention, according to which the said MIMI gene is hybridized to the 20 coding sequence of SEQ ID NO: 1, is preferred. ID Mo:/1 under the following conditions: hybridization in 19685A; 520 mM MaPO, pH 7.2; 7965 lauryl sulfate, sodium salt; 1MM EDTC; 250 mM sodium chloride at 5593 for 18-24 hours, and washing in 6X55C (sodium chloride and citrate solution) for 15 minutes. (Х3) 3ЗХ55С for 15Хmin. (X1) at 5520.

Most preferred is the method of the present invention, wherein said MIM1 gene comprises a coding sequence

POST ID MOo/1, and during the hybridization of all DNA molecules with it, moderately strict conditions are used. with

In particular, the method according to the present invention is optimal, according to which the mentioned BAK gene encodes an altered form of the protein

MIMI, which acts as a dominant-negative regulator of the BAC signal transduction pathway. and)

A preferred method according to the present invention is that said altered form of the MIMI1 protein has alanines instead of Augusts at amino acid positions corresponding to positions 55 and 59 of SEQ ID NO: Mo ID: 2.

Most preferably, the method according to the present invention, according to which said modified form of the MIMI protein contains "I amino acid sequence, which is designated as SEQ. Mo ID: 8.

The best is the method according to the present invention, according to which said DNA molecule contains a nucleotide sequence, which is designated as SEQ. ID Mo: 7, and for hybridization with it, all DNA molecules are used - moderately strict conditions.

The method according to the present invention is best, according to which the said DNA molecule is hybridized with the nucleotide sequence of POSL ID Mo:7 under the following conditions: hybridization in 19085A; 520 mM MaPO»4, pH7.2; 790 lauryl sulfate, sodium salt; 1MM EDTC; 250mM sodium chloride at 5523 for 18-24 hours, and washing in BH55C for 15mM. (Х3) 3ЖХ55С for 15 minutes. (X1) at 5526.

Most preferred is the method according to the present invention, according to which said altered form of the MIM1 protein has an M-terminal truncation of amino acids approximately corresponding to the position of amino acids 1-125 in SEQ. Mo ID: 2.

The best is the method according to the present invention, according to which said modified form of the MIMI protein contains - c amino acid sequence, which is designated as SEQ. Mo ID: 10. Most preferably, the method according to the present invention, according to which said DNA molecule contains a nucleotide sequence, which is designated as SEQ. ID Mo:9, and for hybridization with it all DNA molecules use moderately strict conditions.

The method according to this invention is best, according to which the mentioned DNA molecule is hybridized with the nucleotide sequence of POSL ID Mo:9 under the following conditions: hybridization in 19085A; 520 mM MaPo, pH7.2; 795 lauryl sulfate, sodium salt; 1MM EDTC; 250mM sodium chloride at 5593 for 18-24 hours, and washing in BH55C for 158 hours. (Х3) ХХ55С within 15 minutes. (X1) at 5520. - The best is the method according to the present invention, according to which said modified form of the MIMI1 protein has a C-terminal --oUu 20 truncation of amino acids, which approximately corresponds to the position of amino acids 522-593 in SEQ. Mo ID: 2.

Most preferably, the method according to the present invention, according to which said modified form of the MIMI protein contains the amino acid sequence designated as SEQ. Mo ID: 12.

Most preferred is the method according to the present invention, according to which said DNA molecule contains a nucleotide sequence designated as SEQ. ID Mo: 11, and for the hybridization with it of all DNA molecules, moderately strict conditions are used.

GF! The method according to this invention is best, according to which the mentioned DNA molecule is hybridized with the nucleotide sequence of POSL ID Mo:11 under the following conditions: hybridization in 19085A; 520 mM MaPo, pH7.2; 796 lauryl sulfate, o sodium salt; 1 mM EDTC; 250 mM sodium chloride at 55923 for 18-24 hours; and washing at 6X55C for 15 minutes. (Х3) ХХ5ЗС within 15 minutes. (X1) at 5596. 60 The method according to the present invention is preferred, according to which said altered form of the MIMI1 protein has an M-terminal truncation of amino acids approximately corresponding to the position of amino acids 1-125 SEQ. ID Mo:2 and C-terminal truncation of amino acids, which approximately corresponds to the position of amino acids 522-593 SEQ. Mo ID: 2.

Most preferably, the method according to the present invention, according to which said modified form of the MIMI protein contains the amino acid sequence designated as SEQ. Mo ID: 14. because the method according to the present invention, according to which said DNA molecule contains a nucleotide sequence designated as SEQ., is the best. ID Mo:13 and for the hybridization of all DNA molecules with it, moderately strict conditions are used.

The method according to this invention is best, according to which the mentioned DNA molecule is hybridized with the nucleotide sequence of POSL ID Mo:13 under the following conditions: hybridization in 19085A; 520 mM MaPo, pH7.2; 796 lauryl sulfate, sodium salt; 1MM EDTC; 250mM sodium chloride at 5523 for 18-24 hours, and washing in BH55C for 15 minutes. (Х3) ХХ5ЗС within 15 minutes. (X1) at 5596.

Most preferred is the method according to the present invention, according to which said altered form of the MIM1 protein consists mainly of ankyrin motifs approximately corresponding to the position of amino acids 103-362 in SEQ. ID 70 Moe:2.

Most preferably, the method according to the present invention, according to which said modified form of the MIMI protein contains the amino acid sequence designated as SEQ. Mo ID: 16.

Most preferred is the method according to the present invention, according to which said DNA molecule contains a nucleotide sequence designated as SEQ. ID Mo: 15, and for hybridization with it all DNA molecules use 75 moderately strict conditions.

The method according to this invention is best, according to which the mentioned DNA molecule is hybridized with the nucleotide sequence of POSL ID Mo:15 under the following conditions: hybridization in 19085A; 520 mM MaPo, pH7.2; 796 lauryl sulfate, sodium salt; 1MM EDTC; 250mM sodium chloride at 5523 for 18-24 hours, and washing in BH55C for 15 minutes. (Х3) ХХ5ЗС within 15 minutes. (X1) at 5596.

Examples of target crops for the specified areas of use disclosed herein include, but are not limited to, the following plant species: cereals (corn, wheat, barley, rye, oats, rice, sorghum and related crops); beet (sugar and fodder); pomegranates, stone and juicy fruits (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and black currants); legumes (beans, lentils, peas, soybeans); oil plants (rape, mustard, poppy, olives, sunflowers, coconut, castor oil source plants, cocoa beans, ground nuts); sa cucumber plants (zucchini, cucumbers, melons); fibrous plants (cotton, flax, hemp, jute); citrus fruits (5) (oranges, lemons, grapefruits, tangerines); vegetables (spinach, lettuce, asparagus, cabbage, carrots, onions, tomatoes, potatoes, red bell pepper); laurel (avocado, cinnamon, camphor); or plants such as tobacco, nuts, coffee, sugar cane, tea, grapes, hops, bananas and natural rubber plants, as well as ornamental plants (flowers, shrubs, broad-leaved trees and evergreens such as conifers). The same list of target plants is not limited. -

The method of the present invention is used to create resistance to a wide variety of plant pathogens, which include, among others: viruses or viroids, such as tobacco or cucumber mosaic virus, ring-spot virus or necrosis virus, geranium leaf curl virus, meadow clover mottle virus, with delayed growth of tomato bushes and other viruses; ascomycete fungi, such as the genera Mepischia, Rhodozrnaega,

Eguzirpe, Mopoiipia, Musozrpaegeia and Opsipsia; basidiomycete fungi, such as representatives of the genera Nepiigia, -

KPigosiopia and Ryssipia; Erypdi andrepesia (imperfect fungi), such as the genera Voiguiiz, NeiItipiiozrogiit,

KApupspozropisht, Bizagct (i.e. B. topoiyogte), Zerioga, Segsozrog, APMetaga, Rugisshaga and

Rzetsydosegsozrogheia (ie R. Pegroihispoidev); oomycete fungi, such as representatives of the genera Rhnuorpinoga " (i.e. R. ragaziysa), Regopozroga (i.e. R. (arasipa), Vgetia, Ruiisht and Riaztoraga; as well as other fungi such as Zsiegoriyoga tasgozroga, Zsiegorp(yoga gauizziae, Zsiegozroga dgatipisoia, Regopozziegozroga зогопі, - с Регопозсіегозрога рПійірріпепвзів, Регопозсіегозрога засспагі та Регопозсіегозрога таудів, РПпузорейа 7еае, и Сегсозрога геае-тауадів, СоПефігіспит дгатіпісоіа, СірбегеМПйа геае, Ехвегопішт (Шгсісит, Кабаїйей 2едае та "» Віроїан5 тауаїів; бактерії, такі як Рзепдотопаз зугіпдае, Рзепдотопаз іарасі та Egpmipia ziemagii, insects such as aphids such as Mulis regsisae, Lepidoptera such as Neioiis spp., nematodes such as

Meioiidodupe ipsodpi(a. -i Obtaining immunomodulated plants syu All three of the following general ways of obtaining immunomodulated plants are interconnected by the fact that all of them are adapted to the model of the BAK signal transduction pathway described in Ikuaev ei ai., (1996)). During the activation of the BAC signal transduction pathway in order to achieve resistance to the disease, the same set of ZAC genes is "turned on" and resistance to the disease is created regardless of which of the three pathways described below is chosen. The difference between these three paths is only which point of the BAC path is included; The end result is the same for all three ways. Thus, the analyzes and research results of immunomodulated plants obtained by one route can be extrapolated and applied to immunomodulated plants obtained by another route.

A. Application of a chemical inducer of systemic acquired resistance (SAC) o The first way of obtaining immunomodulated plants involves the introduction of a chemical agent capable of inducing AAC into the plant. In particular, benzothiadiazoles, such as benzo[1,2,3]hiadiazole-7-carbothioic acid-3-methyl ester (VTN), are powerful chemical inducers of ZAC. Derivatives of benzothiadiazoles, which are further used as regulators, are described in U.S. Patent Nos. 5,523,311 and 5,614,395, references to which are given in this description! described in (Egeiagisp ei ai!., Riapiv doigpa! 10(1), 61-70 (1996); amp ei ai., Riapiv dotsgpa! 10(1), 71-82 (1996); and Sopaspi ei ai., , Riapi Se! 8, 629-643 (1996, and references to each of these publications are given in the text of this description). Other chemical inducers of BAK, which are used to obtain immunomodulated plants according to the method of the present invention, 65 include isonicotinic acid compounds , such as 2,6-dichloroisonicotinic acid (IMA) and its lower alkyl esters, as well as salicylic acid compounds (5A). Examples of relevant compounds of IMA and 5A are described in (Pat.

USAMO 5,614,3951I.

B. Selection of mutant plants with constitutive immunity (SIM).

The second way of obtaining immunomodulated plants is a program of selective selection based on such features as constitutive expression of ZAC genes and/or a disease-resistant phenotype. Numerous data indicate close correlative relations between the expression of the genes of ZAK and the actual systemic acquired resistance (MMaga ei a. (1991); OKpez ei aYi. (1992); OKpez ei aiYi. (1993); Gamop, ei aiYi. (1993) ); and AiIehapdeg ei aiYi. (1993) РМА5

BA 90, 7327-7331, which are referenced in this description). Agaridorziz has examples of well-studied genes

The ZACs are RK-1, RK-2, and RK-5, with RK-1 being expressed at maximal levels with minimal background. 70 In order to identify and select plants that constitutively express BAC genes, Northern blotting analysis is performed, which allows to detect the expression of BAC genes. Known DNA sequences of the ZAK are used in experiments with cross-hybridization, as described in |(OKpez ei ai. (1992)). Methods of hybridization and cloning of nucleic acid sequences are well known to specialists. See, for example,

Moiiesshiag Siopipu, A Iarogayugu Mapia)Y, 2pa Eayyop, Moi. 1-3, ZatbrgooK ei ai. (Edv.) SoYi Zrgipd Nagrog /5 Garogaigu Rgezz (1989), as well as the references given in them). At least two classes of ZAC signal transduction mutants have been identified that constitutively express ZAC genes. One of the classes was called "Iva" mutants (Iva - Ieviop vitshciaeipu disease - a disease with imitation of a lesion), which they are also called "Class I sit" mutants. | See UMO 94/16077). Iza mutants (Class I site) form spontaneous lesions on leaves, contribute to the accumulation of high concentrations of ZA, high levels of RA-1, RK-2 and RK-5 mRNA, and are also resistant to fungal and bacterial pathogens. 1994; M/eutapp eyi ai., 1995). The second class was called "siet" mutants (siet - mutants with constitutive immunity), which they are also called "Class II sit" mutants. | See UUO 94/16077)|. sit mutants have all the characteristic features of is mutants, except for spontaneous lesions. That is, sit mutants are visually normal in terms of phenotype.

Since plants that constitutively express BAC genes are selected, they are used in plant breeding programs to introduce constitutive expression of BAC genes and resistance to pathogens into plant cell lines.

The offspring for further crossing are selected based on the evaluation of BAC gene expression and resistance to i) the disease, as well as other characteristics that are essential for the creation of mutants and the quality of this process, according to methods well known to specialists in the field of plant breeding. For example, due to the fact that Iza mutants are characterized by the formation of lesions and necrosis, sit mutants with their normal "g zo phenotypes are preferred for use in such selection programs and in the implementation of the method of the present invention, although if desired, Iva mutants can also be used. -

S. Plant transformation by BAK genes M

The third way of obtaining immunomodulated plants is the transformation of plants with the 5AK gene, in the optimal version, the functional form of the MIM1 gene. me) 1. Recombinant expression of the natural MIM1 gene

Recombinant overexpression of the natural form of MIMI1 (SEQ ID Mo:1) leads to the formation of transgenic plants with a disease-resistant phenotype. | See joint US patent application, registration number 08/880,179, which is incorporated herein by reference). Increased levels of active protein

MIMIs create the same disease resistance effect as chemical induction involving such "

Chemical agents such as VTN, IMA and ZA. In the optimal version, the expression of the MIM1 gene is maintained at a level that is at least two times higher than the level of expression of the MIMI gene in natural plants, and in the best version - at least ten times higher than the level of natural expression. In the section below, titled ;" "Recombinant DNA technology" describes protocols used to recombinantly express the native MIM1 gene in transgenic plants at higher than natural levels. IN

In another variant of the implementation of the invention, plants are transformed with the natural MRK1 gene in order to obtain resistant -I to plant diseases, as described in (Sabo, ei a/. (1997)). 2. Recombinant expression of the altered form of the MIM1 gene. o Immunomodulated plants, which are used according to the method of the present invention, are also created with the help of recombinant expression of a modified form of the MIM' gene, in connection with which the change of the MIM1 gene implies as a 5th recognition that the ZAK pathway in plants is characterized by functional parallels with regulation scheme - ME-KkV/IV in mammals and flies, as well as the discovery that the MIM1 gene product is a structural homologue of the subclass p. "from" factor IKV signal transduction in mammals. | See joint application of PCT "METNOYUZ ON THE VOLUME OF TNE MIMI SEME TO

SOMEEK OIZEAZE KEZIZTAMSE IM RI AMT5", the reference to which is given in this description.)

The sequence of the MIM1 gene (SEQ ID No:1) was used in studies of VI A5T, and candidates were identified on the basis of such features as the homology of one fairly highly stabilized domain in the sequence of the MIM1 gene with domains that were found in a number of proteins such as spectrins , ankyrins, ME-I kV and KV |Misnaeiu apa

Veppei, Tgepaz Sei| Vioi 2, 127-129 (1992)). A pairwise visual analysis of the MIM1 protein (SEQ ID Mo:2) was performed with 70 known ankyrin-containing proteins, due to which a striking similarity to members of the I kVo class was revealed. transcription regulators |Vaetsegie apa Vaiiitoge 1996; VaiYidm/p 1996). As can be seen in Figure 1, the MIM1 protein (SEQ ID 60 Mo:2) is highly homologous to IkVo proteins from tissues of mice, rats and pigs (SEQ ID MomMo: 3, 4 and 5, respectively). MIM1 contains several important IqVo structural domains throughout the length of the protein, including ankyrin domains (indicated by dashed lines in Fig. 1), 2 amino-terminal serines (amino acids 55 and 59 of MIM'I), a pair of lysines (amino acids 99 and 100 in MIM1) and acidic C-terminal. In general, MIM1 and Yik Wo. are identical at 3095 residues and have conservative substitutions at 5095 residues. Thus, homology with a total similarity ratio of 8090 is observed between the I kVo and MIM1 proteins.

One of the ways in which the IkVo protein functions in signal transduction is binding to the cytosolic transcription factor ME-kV and preventing it from entering the nucleus and changing the transcription of target genes.

Waiitoge, 1996; Vaidm/p, 1996). ME-KV target genes regulate (stimulate or inhibit) several cellular processes, including antiviral, antimicrobial, and cell-killing responses |Vaecepe apa Waitoge, 19961.

When the signal transduction pathway is activated, IkKVo is phosphorylated at two serine residues (amino acids 32 and 36 of mouse IKVo). This programs proliferation on a double lysine (amino acids 21 and 22 of mouse

Ikva). After spreading, the ME-kV/KV complex is passed through the proteosome, where IkVo is cleaved, and ME-IkV is released into the nucleus.

Phosphorylated serine residues, which are important for the function of IkKVo, are conserved in MIM1 within a large contiguous block of conserved sequence from amino acid C5 to 84 (Fig. 1). In contrast to IkVo, where the double lysine occupies approximately 15 amino acids towards the M-terminus of the protein, in MIM1 the double lysine occupies approximately 40 amino acids towards the C-terminal end. In addition, a high degree of homology exists between MIM!1 and IkVo, in the serine/threonine-enriched carboxy-terminal region, which turned out to be important in the main circulation of the substance |Zyp ei ai., Moi. SeiI. Vioi 16, 1058-1065 (1996)). According to the present invention based on structural homology analysis and the presence of elements known to be important for the function | kVo, MIM1 is expected to function as an IkVo, exerting a similar effect on plant gene regulation.

It is predicted that plants containing the natural MIM/1 gene, when treated with chemical inducers, will have more of the MIM1 gene product (IKB homolog) or less phosphorylation of the MIM'1 gene product (IKB homolog).

According to this model, the result is that the plant homolog of ME-KV is kept outside the nucleus, and the possibility of expression of genes of ZAC and resistance reactions appears. Sweat in mutant plants! a non-functional MIM1 gene product is present. Thus, according to this model, the ME-kV homolog can freely enter the nucleus and repress BAC resistance and gene expression. with

In complete agreement with this idea, cells of animals treated with salicylic acid are marked by an increased resistance/relative content of KV and a decrease in active ME-kV in the nucleus (Corr apa Spozp, 1994). at

IKV mutations acting as super-repressors or dominant-negatives are known |VgKa-Mageep TgaepsKpeg ei ai.,

EMVO 14: 2876-2883 (1995); Vol. VgosKtap ei aii., MoiIesshag apa

SeiIshag Vioodu 15: 2809-2818 (1995); MUapd ei aiYi.,, Zsiepse 274: 784-787 (19963). These mutant forms of IKV h;E bind to ME-KV, but are not phosphorylated or ubiquitinated and, thus, not cleaved. ME-kV - remains bound to IKV and cannot move into the nucleus.

In view of the above, it is possible to create modified forms of MiM' that act as dominant-negative in. regulators of the BAC signal transduction pathway. Plants transformed with these dominant-negative forms of MIM1 have the opposite phenotype to mutant MIM1 plants in the sense that plants transformed with altered forms of MIM'1 are characterized by constitutive expression of the ZAV genes and, thus, the CIM phenotype, that is, transgenic plants are immunomodulated. Due to the position that the MIMI1 gene occupies in the ZAK signal transduction channel, it can be assumed that a certain number of gene changes, in addition to those specifically disclosed in this description, will lead to the constitutive expression of BAK genes and, thus, to the formation of the SIM phenotype. The following section entitled "Recombinant DNA Technology" describes the protocols used to recombinantly express altered forms of the MIMI1 gene in transgenic plants at higher than natural levels. Further, the text describes several altered forms of the MIMI gene, which act as ""dominant-negative regulators of the ZAK signal transduction pathway." a. Change of serine residues 55 and 59 to alanine residues:

Phosphorylation of serine residues in IkVo from the human body is necessary for stimulus-stimulated cleavage of IkVo, which in turn activates ME-kV. Mutagenesis of serine residues (532 and 536) in IkVo z

Che of the human body with the formation of alanine residues inhibits stimulus-induced phosphorylation, blocking y

Ge) this method is mediated by the proteosome of the cleavage of I kVO (PgaepsKpeg ei ai, 1995; Vgomup ei ai., 1996;

VgosKtap ei a/., 1995; Mapa eyi a!., 1996). This modified form of IkKVo can function as a dominant-negative yiv. form due to the preservation of ME-kV in the cytoplasm, and thus, blocking the events of the signal transmission path -about 70 "from below". Based on the comparison of amino acid sequences between MIM1 and IKV shown in Fig. 1, it is possible

T" to determine that serines 55 (555) and 59 (559) in MIM1 (POSL. ID Mo:2) are homologous to 532 and 5Z3b in IkKVo from the human body. In order to create dominant-negative forms of MIM1, serines at positions of amino acids 55 and 59 are subjected to mutagenesis with the formation of alanine residues. Thus, in the optimal implementation variant 5 of the invention, the MIMI1 gene is changed in such a way that the coded product has alanines instead of Augusts in amino acid positions corresponding to positions 55 and 59 of the amino acid sequence of MIMI (SEQ ID No. Mo:2) in (F. Agaridorviv. ko b. M-terminal deletion:

Deletion of amino acids 1-36 |VhosKktap ei a!., 1995; Zy!p ei a!., 1996) or 1-72 IZytsp ei ai., 19961 in IkVo, from the human organism, which includes the lysine residues of the spread of K21 and K22, as well as phosphorylation sites 532 and 536, leads to the formation of a dominant negative phenotype of I kVO in transfected cell cultures from the human body. M-terminal deletion of the first 125 amino acids of the MIMI gene product will remove eight lysine residues that can serve as spreading sites, as well as putative phosphorylation sites in the 555 and 559 considered above. Therefore, in the optimal embodiment of the invention, the MIM1 65 gene is changed so that in the encoded the product omitted approximately the first 125 amino acids compared to the native amino acid sequence of MIM1 from Agaridorvzi (SEQ ID No. Mo:2).

in. C-terminal deletion:

Deletion of amino acids 261-317 in IkVo from the human body leads to increased self-resistance due to the blocking of constitutive phosphorylation of serine and threonine residues in the C-terminal. It is assumed that this altered form of I kVo will function as a dominant-negative form. A serine- and threonine-rich region is present at amino acids 522-593 in the C-terminus of MIMI. Thus, in the optimal embodiment of the invention, the MIM1 gene is changed in such a way that the coded product omits a region that roughly corresponds to its C-terminal part, including amino acids 522-593, in comparison with the native amino acid sequence of MIM'1 in Agaridorziz (POSL ID Mo:2). d. M-terminal/C-terminal deletion chimera and ankyrin domains.

Altered forms of the MIMI gene product are also obtained as a result of C-terminal and M-terminal segmental deletions or chimeras. In another embodiment of the present invention, constructs containing ankyrin domains from the MIM1 gene are obtained. 3. Recombinant expression of other ZAK genes

Immunomodulated plants, which are used according to the method of the present invention, are also created with the help of recombinant expression of various ZAK genes, such as described in (MuUaga et al. (1991)). See, for example, (US Patent No. 5,614,395), which describes disease-resistant plants created using overexpression of one or more RE protein genes. Although it is specifically about recombinant expression of gene forms

MIMI, the section below entitled "Recombinant DNA Technology" describes protocols that are also used to recombinantly express other 5AK genes, such as

PE protein in transgenic plants, at levels higher than natural.

Technology using recombinant DNA

The natural or modified form of the MIM1 gene, which provides plants with resistance to diseases by increasing the expression of the ZAK genes, is introduced into plant cells using standard recombinant DNA technology. Ha

This typically involves inserting a DNA molecule encoding a selected form of the MIM'I described above into an expression system with which the DNA molecule is heterologous (ie, not normally present), using (i) standard procedures known in the art cloning The vector contains elements that are necessary for the transcription and translation of the inserted protein-coding sequences. A large number of vector systems known in the field are used, such as plasmids, bacteriophage viruses and other modified viruses. "And Suitable vectors include, among others, viral vectors such as lambda vector systems 7KhoM1, KhoNO and

Spagp 4; plasmid vectors, such as rVI121, rVK322, pRASUS177, rASUS184, a series of pAK, pKkK223-3, pOsS8, pOso, - ris18, pOos19, ri 5339, pKK290, pKkO37, pKS101, pSOMAII and other similar systems. Components of the expression system are also modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications are used. The expression systems described in this text are used to transform the cells of almost any cultivated plant under appropriate conditions. Transformed y cells are regenerated into whole plants, so that the selected form of the MIM1 gene activates ZAK in transgenic plants.

A. Structure of plant expression cassettes

Gene sequences intended for expression in transgenic plants are first combined in expression cassettes under the appropriate promoter suitable for expression in plants. The expression cassettes may also contain any additional sequences required or selected for expression of the transgene. Such sequences include, but are not limited to, transcription terminators, external sequences to enhance expression, such as introns, vital sequences, and sequences designed to direct the gene product to specific organelles and cell compartments. These expression cassettes are easily transferred further into the plant transformation vectors described above. Below is a description of the various components of a typical expression cassette 475. -i 1. Promoters c The choice of promoter used in the expression cassettes will determine the spatial and temporal pattern of transgene expression in the transgenic plants. The selected promoters will express the transgenes in specific - I cell types (such as epidermal cells of leaves, mesophyll cells, cells of root cortex), or in shu 20 specific tissues or organs (for example, in roots, leaves, or flowers), and the choice will reflect the desired site of accumulation of the gene product. Alternatively, the selected promoter directs gene expression by

ChT" of various induction conditions. Promoters differ in the strength of their influence, that is, the ability to stimulate transcription. Any of several suitable promoters are used depending on the host cell system used. Possible examples (to which the invention is not limited) of promoters used in expression cassettes are given later in the text. and. Constitutive expression, promoter Samm 355: o The structure of plasmid pOOM1761 is described in (published patent application EP 0 392 225 (Example 23), to which reference is made in this text)! pCOM1761 contains a "dual" Samm 355 promoter and a yti transcriptional terminator, with a single EsoKI!I site between the promoter and terminator, and has a ruC-type backbone. 60 A derivative of RSOM1761 is being constructed, which has a modified polylinker that includes My! and Khpoi in addition to the existing EsoKI website. This derivative is denoted as pСОМ1761ЕМХ. pCOM1761EMX is used for cloning sequences to DNA or gene sequences (including sequences of OKE bacteria) inside its polylinker in order to carry out their expression under the control of promoter 355 in transgenic plants. The entire terminator cassette (those sequences of the 355 gene promoter with this structure are excised using the 5' 65 sites of Nipai, ry, ZaiYi and Khwai (promoter) and Zi Khvrai, Watni and Waoi! sites (terminator) for transfer into transformation vectors similar to those described further in the text.In addition, the dual promoter fragment 355 is removed by excision 5 using Nipai, Era, ZaiY, Chrai, or Pv and by excision 3 using any of the polylinker restriction sites (EsoKiI, Myi, or XpoiY), in order to replace it with another promoter. Optionally, modifications are made around the cloning sites by introducing sequences that can enhance translation. This is particularly useful if overexpression is desired. For example, pCOM1761 EMX is modified by optimizing the translation initiation site as described in ( Example 37 of US Pat. No. 5,639,949, which is referenced herein.b Expression using a chemically/pathogen-regulated promoter ra:

The dual promoter 355 in pCOM1761 EMH is replaced by any other selected promoter, which 7/0 leads to the creation of correspondingly high levels of expression. As an example, one of the chemically regulated promoters described in (US Pat. No. 5,614,395) can replace the dual promoter 355. The selected promoter is optimally excised at its source using restriction enzymes, but alternatively can be amplified by polymerase chain reaction ( RSK) using primers that have restriction sites at the corresponding terminals. If PCR amplification is used, the /5 promoter is resequenced to check for amplification errors after cloning the amplified promoter into the target vector. Regulated by a chemical method/pathogen, the PE-ia promoter of tobacco is cleaved (hydrolyzed) from the plasmid pSIV1004 | structure see in example 21 of the EP application 0 332 104, to which reference is given in this text) and transferred to the plasmid pSOM1761 EMH |OKpez ei ai., 19921. pSIV1004 is cleaved using MsoI, and the resulting "protrusion" from the linearized fragment is "blunted" by processing 2o DNA polymerase T4. The fragment is then cleaved by Nipagyl, resulting in a promoter-containing fragment

RA-1a, purified by gel and cloned into RSOM1761 EMH, from which the double promoter 355 was removed. This is done by cleavage using Hpo! and "blunting" with T4 polymerase, followed by cleavage with NipaiI and isolation of a larger fragment containing a terminator vector into which the pSIV1004 promoter fragment is cloned. This leads to the formation of a PCOM1761EMX derivative with a promoter of sdv RE-a and a terminator (ti, and an intermediate polylinker with unique sites Esoki! and MoiY. The selected coding sequence is inserted into this vector, and the fusion products (i.e., promoter-gene-terminator) are further transferred to i) any selected transformation vector, including those described above. To induce the expression of the selected coding sequence in plants transformed according to the present invention, a variety of chemical regulators are used, including benzothiadiazole, isonicotinic acid, and salicylic acid compounds.

Of the Acids that are described in (US Patent Nos. 5,523,311 and 5,614,395). in. Constitutive expression, actin promoter: -

It is known that in the vast majority of cell types, several isoforms of actin are expressed, and accordingly, the M actin promoter is a good variant of the constitutive promoter. In particular, the promoter of the Asi rice gene was cloned and described! |MsEiYigsu ei ai. Riapi SciI 2: 163-171 (1990)). It was found that a promoter fragment with a length of 1.3 kb contains all regulatory elements required for expression in rice protoplasts. In addition, numerous expression vectors based on the Asi promoter! was created specifically for use in monocots

IMsEiYigou ey a). My. Sept. Sepoys 231: 150-160 (1991)) These include Asi-intron 1, flanking sequence Aa 5 and Aapi-intron 1 (from the corn alcohol dehydrogenase gene), as well as the sequence from the Samm 355 promoter. The vectors with the highest expression were fusion of 355 and the ANI intron, or the flanking sequence of AapiIb5 and the AsI intron. Optimization of the sequences around the initiating ATO with c (reporter gene bI5) also increased expression. The promoter expression cassettes described in |MesEigou ei ai. (Moi. Sep. Sepei. 231: 150-160 (1991)) are easily modified for the purposes of gene expression and are, in particular, suitable for use in monocot host plants. For example, promoter-containing fragments are removed from MeEigo structures and used to replace the dual promoter 355 in pCOM1761EMX, which

It is then available for insertion of specific gene sequences. The fusion genes created in this way are then transferred to the corresponding transformation vectors. A separate report mentions that rice promoter Asi! with its first intron also causes a high level of expression in cultured barley cells |Spirbag ei a!. Riapi Sei! Ker. 12: 506-509 (1993). -I g. Constitutive expression, ubiquitin promoter:

Ubiquitin is known to be another gene product that accumulates in various cell types, and its promoter has been cloned from several species for use in transgenic plants (eg, sunflower - (|Vipei ei aiI. Riapi Zsiepse 79: 87- 94 (1991)) and corn - (SPgivieepzep ei aiYi. Riapi Moyes.

Vioi 12: 619-632 (1989). The maize ubiquitin promoter has been developed and obtained in transgenic monocot systems, and its sequence and vectors designed to transform monocots are described in the patent publication (EP 0 342 926 (issued I cbgi20oI!)) which is referenced in this description.

Tauiog ei ai. |Riapi Se! Ker. 12: 491-495 (1993)) describe a vector (pRANS25) containing the ubiquitin promoter (F, maize and the first intron, and its high activity in cell suspensions of numerous monocotyledonous plants when introduced by microparticle bombardment. The ubiquitin promoter is suitable for gene expression in transgenic plants, especially in monocots. Suitable vectors are derivatives of vRANS2g5 or any of the transformation vectors described in this application, modified by introducing a suitable ubiquitin promoter and/or intronic sequence. d. Specific expression in the root:

Another characteristic of gene expression is expression in the root. The corresponding root promoter is described in (de

Egatopa (PEV 5 290 103-106 (1991)), as well as in the published patent application (EP 0 452 2691, to which reference is made in this specification. This promoter is carried into an appropriate vector, such as pPCOM1761EMX, in order to insert the selected gene and subsequent transfer of the entire promoter-gene-terminator cassette into the desired transformation vector. e. Wound-induced promoters:

Wound-inducible promoters are also suitable for gene expression. Numerous similar promoters have been described previously (for example, Khei a). Riapi Moyes. Vio 22: 573-588 (1993), Iodetapp et al. Ripley Journal 1: 151-158 (1989), Copege 5: Ieppie, Ripley Moyes. Vioi 22: 783-792 (1993), Rigek et al. Riapi Moyes. Vio 22: 129-142 (1993), M/ateg ei ai. Riapi 9. 3: 191-201 (1993))| and suitable for use according to the method of the present invention.

Iodetapp ey and describe the sequences located above 5 dicotyledonous potato gene uipi. Hee hee hee hee claim that the wound-inducible promoter from dicotyledonous potato (rip2) is active in monocotyledonous rice. 7/0 In addition, Coppteig 8 Hepie describe the cloning of a wound-induced UMiri cKDDNA of maize and used to isolate a female-related promoter using standard technology. Similarly, Rigek ei a/, apa M/agpeg ei a/, described a wound-induced gene from the monocot Azragadiz opyPisipaiiz5, which is expressed in the wounded place and in the areas of pathogen invasion. Using cloning techniques well known to those skilled in the art, these promoters are transferred into appropriate vectors fused to the genes used 7/5 According to the present invention and used to express these genes in wounded plant parts. same Selective expression in the core of the plant:

The patent application (M/O 93/072781, to which reference is made in this description) describes the isolation of the ygrA gene of maize, which is selectively expressed in pith cells. The gene sequence and the promoter extending to -1726 bp from the start of transcription are presented . Using standard molecular biology techniques, this promoter or parts thereof are transferred into a vector such as pCOM1761, where it can replace the 355 promoter and is used to drive selective expression of a foreign gene from the core of the plant. In practice, fragments containing the core-selected promoter or its parts, transferred into any vector and modified for use in transgenic plants. z. Specific expression in leaves:

The maize gene encoding phosphoenolcarboxylase (PERS) is described in (Niedzrefj 85 Sgshzia (Riapi Moyes Vio! 12: 579-589 (1989). Using standard techniques of molecular biology, the promoter of this gene and) is used to stimulate the specific expression of any gene in the leaves of transgenic plants 2. Transcription terminators

Various transcription terminators are used in expression cassettes. They are responsible for the termination of transcription outside the boundaries of the transgene and its correct polyadenylation. Suitable transcription terminators are those known to function in plants and include the SamMm o-355 terminator, the iti terminator, the nopaline synthase terminator, and the pea grs5 E9U terminator. they are used both for M monocotyledonous plants and for dicotyledonous plants.

C. Sequences for enhancing or regulating the expression of me)

Numerous sequences that enhance gene expression from the middle of the transcription unit have been identified, and these sequences are used together with the genes of the present invention to increase their expression in transgenic plants.

Various intronic sequences have been shown to enhance expression, particularly in monocot cells. For example, it was found that the introns of the Aapi gene of corn significantly increase the expression of the natural gene under the conditions of introduction of a promoter related to the female line into corn cells. It was proved, in particular, that intron 1 with c was effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene

ISaiiv ei a/.,, Sepez YuOemeIoyuor. 1: 1183-1200 (1987). In the same experimental scheme, the intron from the gene ;" rgopge1 of maize had a similar effect in increasing expression. Intronic sequences were introduced into plant transformation vectors in a standard way, usually within the untranslated leader sequence. -And certain untranslated leader sequences derived from viruses are also known to enhance expression and are particularly effective in dicot cells. More specifically, the leader sequences from the tobacco mosaic virus (TMM, "M/-sequence"), the chlorotic corn speck virus (MSMU) and the alfalfa mosaic virus (AMU) proved to be effective in increasing expression, for example, Saiiis ei a/.

capes Asiaz Kev. 15: 8693-8711 (1987); ZKyg2ezkKi ei a/. Riapi Moyes. Vioi 15: 65-79 (1990). - 4. Targeting of the gene product in the cell It is known that in plants there are various mechanisms for the targeting of gene products, and the sequences that control the functioning of these mechanisms are described in sufficient detail. For example, the direction of gene products into the chloroplast is controlled by a signal sequence found at the two amino-terminal end of various proteins, which is cleaved during the transfer of the chloroplast to the mature protein |for example, Sotai ei a/. 9. Vioi. Spent 263: 15104-15109 (1988)). These signal sequences are fused (F, with heterologous gene products to carry out the transfer of heterologous products into the chloroplast (map ka dep VgoeskK, ei ai. Mayte 313: 358-363 (1985)). DNA encoding the corresponding signal sequences is isolated from the end of 5 ' cDNA acids encoding the protein COVIZSO, the protein SAV, the enzyme ERBZR synthase, the protein (352, and many other proteins known to localize to the chloroplast. See also the section entitled "Chloroplast Targeted Expression" in Example 37 of US Patent No. 5,639,949).

Other gene products are localized in other organelles, such as mitochondria and peroxisomes. For example,

Opdeg ei ai. Riapi Moyes. VioI. 13: 411-418 (1989)). The cDNA acids encoding these products can also be manipulated to direct heterologous gene products to these organelles. Examples of such 65 sequences are nuclear-encoded ATPases and mitochondria-specific aspartate aminotransferase isoforms.

Targeting of cellular protein bodies is described in Codex ei aY. (Rhos. Mai. Asad. Zsi. OBA 82:

6512-6516 (1985).

In addition, sequences have been described that determine the direction of gene products to other compartments of the cell. Amino-terminal sequences are responsible for targeting in the EC (endoplasmic reticulum), apoplast, and extracellular secretion from aleurone cells |(Koeyig 5 No, Riapi Se 2: 769-783 (1990)). In addition, the amino-terminal sequences, together with the carboxy-terminal sequences, are responsible for the vacuolar targeting of the gene products |ЗПипепПи ей ай. Riapi Moyes. Vioi 14: 357-368 (1990)).

By fusing the appropriate targeting sequences described above with the desired transgene sequences, the transgene product can be directed to any organelle or 7/o cell division. For targeting to the chloroplast, for example, the chloroplast signal sequence from the KOVIZSO gene, the SAV gene, the EROBR synthase gene, or the 552 gene is fused in frame with the amino-terminal ATO of the transgene. The selected signal sequence should include a known cleavage site, and the generated fusion should include any amino acids after the cleavage site that are required for cleavage. In some cases, this requirement is fulfilled by adding a small number of amino acids between the cleavage site and the /5 ATO of the transgene, or, in another variant of the substitution, several amino acids within the transgene sequence.

The created fusions for chloroplast transfer are checked for efficiency of chloroplast uptake by translation of ip myo transcribed ip myo structures and further by analysis of chloroplast ip myo uptake, using techniques described in (Vagienk et al. Ip: Edeitapp et al. (Eds.) Meiyyoaz ip

SPiohoriazi Moiesshtsiag Vioiodu, Eivemieg rr 1081-1091 (1982) apa MUaztapp ei aI. My. Sept. Sepoys 205: 446-453 (1986)). These structural technologies are well known in the art and are equally applicable to mitochondria and peroxisomes.

The cellular targeting mechanisms described above are used not only with cognate promoters, but also with heterologous promoters in order to provide a specific cellular targeting target under the conditions of transcriptional regulation of a promoter having an expression characteristic that differs from that of the promoter. from which the target direction signal is obtained. and)

B. Structure of plant transformation vectors

Numerous transformation vectors suitable for plant transformation are known to those skilled in the art, and the genes produced by the method of the present invention are used with any such vectors. The choice of "K zo Vector" will depend on the choice of the optimal transformation technology and the target species that will undergo transformation. For certain target species, preference is given to certain selection markers of antibiotics or herbicides. The standard selection markers used for transformation include the pria gene, M, which creates resistance to kanamycin and related antibiotics |Mezvzipd 4: Miegga. Sepe 19: 259-268 (1982);

Vemap ei ai., Mayte 304:184-187 (1983)), gene rag, which creates resistance to the herbicide phosphinothricin (M/pe me) ei aI.,, MysiI. Asidz Kez 18: 1062 (1990), Zrepseg ei aI. Tpeog. Arri. Sepei 79: 625-631 (19903), a gene that confers resistance to the antibiotic hydromycin IViospipdeg 5: Oiddeitapp, Mo! Seyi Vio! 4: 2929-2931), the gene anttg, which creates resistance to methotrexate (Voshgotsiz et al., EMVO 4. 2(7): 1099-1104 (1983)| and the gene ERZR5, which creates resistance to glyphosate | (US patents MoMo 4,940,935 and 5,188,642).

Many vectors are suitable for transformation using Adhorasiegisht (Stetasiep5z). These usually have at least one T-DNA flanking sequence and include vectors such as rVIM19 |Vemap, Mysi. .Asiaz Kevs. (1984) and pXU/7. description of the structure of two typical vectors suitable for transformation using Adgobasiegiszt. a. RSIV200 and rSIV2001:

Double vectors pSIV200 and pSIV2001 are used for the structure of recombinant vectors intended for use with Adhorasiegits and obtained in the following way. pTOU575Kap is created by enzymatic hydrolysis of the Magi vector pTu575 |Zsptidpatsveg 8 Neijipeki, 9. Vasiegioi. 164: 446-455 (1985)|, allowing the excision of the tetracycline resistance gene to occur, then by inserting a fragment of Assi with -I pOsSyaAK having MRT (Mezzipd b Miega, Sepe 19: 259-268 (1982): Vemap ei aI ., Maite 304: 184-187 (1983): MevVgide ei ai., Riapi Moiescshiag Viodou 14: 266-276 (1990)). Henna linkers! ligate with a fragment of EsokM from RSIV7, - containing the left and right borders of T-DNA, the chimeric gene poz/priy selected in the plant and the polylinker pOsS i" (Koyiziveip ey a), Sepe 53: 153-161 (1987)), and hydrolyzed Whoops! the fragment is cloned into hydrolyzed with Za//rg)B/5Kap, creating pSIV200 |see also EP 0 332 104, example 19). pSIV200 contains the following unique polylinker restriction sites: EsoKI, Zv, Krpi, Va, KhraI and Za. pSIV2001 is a derivative of pSIV200, which was created by inserting additional restriction sites into the polylinker. The unique restriction sites in the pRSIV2001 polylinker are EsokiI, Zv, Krpi, Va, hbvai, zaiYi, Misha, VsyIiI, AmgiiIi, Arai, NraIi and BMSIi. rSIV2001,

F) in addition to containing these unique restriction sites, also has selection for plant and bacterial ca kanamycin, left and right T-DNA boundaries for Adgobasiegisht-mediated transformation, derived from

KK2 function (PA for mobilization between E. soybean and other hosts, as well as Ogia and Ohim functions with KK2. Polylinker bo VSIV2001 is suitable for cloning plant expression cassettes that have their own regulatory signals. b. RSIV10 and its derivatives as a result of selection by hydromycin: The pSIV10O dual vector contains a gene encoding kanamycin resistance for selection in plants and left and right T-DNA border sequences, and includes sequences from the pKK252 plasmid, which is found in a wide variety of hosts, allowing it to replicate both in B. soybean, as well as in Adhorasiegits. Its structure is described in (|Koiivieip eyii a. (Sepe 53: 153-161 (1987). 65 Create various derivatives of the pSIV10 vector, which include the gene for phosphotransferase hydromycin B, which is described in |Ogiya 2 ei aY. (Sepe 25: 179-188 (1983))). These derivatives make it possible to select cells of transgenic plants by hydromycin alone (pSIV743) or hydromycin and kanamycin (pSIV715, pSIV717). 2. Vectors suitable for transformation without using tanya Adgobasiegit.

Transformation without the use of Adhorasiegisht (Steiasiepe) allows you to bypass the requirement for the presence of T-DNA sequences in the selected transformation vector, and thus it is possible to use vectors that do not have these sequences in addition to those vectors described above that have sequences

T-DNA. Transformation techniques that do not use Adhorasiegist include transformation by particle bombardment, protoplast absorption (eg, RES (polyethylene glycol) and electroporation), and microinjection. The selection of the vector largely depends on the selection of the species to be transformed. Next, the text describes the structure of typical vectors suitable for transformation without using Adgobasiegists. and. pRSIVZOba: pSIV3064 is a pOS-derived vector suitable for direct gene transfer technology in combination with bast (or phosphinothricin) herbicide selection. Plasmid pSIV246 7/5 Contains the Samm 355 promoter functionally fused to the E. soybean 505 gene and the Samm c55 transcription terminator, and this plasmid is described in (published in PCT application MO 93/07278|. The 355 promoter of this vector contains two sequences of the initial 5 ATO site. These sites are mutated using standard PCR technology, thereby deleting the ATO genes and creating Zvri apa Ru restriction sites. The new restriction sites are 96 and 37 bp away from the unique ZaiY site and 101 and 42 bp away . from the actual start site. The resulting pSIV246 derivative is designated as pSIV3025. The 505 gene is then excised from pSIV3025 by enzymatic hydrolysis using ZaiY and Zasi, the terminals of which are blunted and religated to form the plasmid vector pSIV3060. Plasmid rdIi182 was obtained from Ippez Sepige et al. . p ei a. EMVO 3 6: 2519-2523 (1987)). Thanks to this, Ha pSIVZO64 is formed, containing the rag gene under the control of the Samm 355 promoter and the herbicide selection terminator, the ampicillin resistance gene (for selection in E soybeans) and a polylinker with unique sites ZrpiY, Ray, NipashSh and i9)

cotton wool This vector is suitable for cloning plant expression cassettes that contain their own regulatory signals. b. рОс19 and реос35: "Her розОО35 is a transformation vector in which the dihydrofolate reductase (OEK) gene of E. soybean is used as a selective marker conferring resistance to methotrexate. RSK is used to amplify promoter 355 -- (-800 bp), intron b from the corn gene ap! (-550 b.p.) and 18 b.p. of the non-transmitted leader sequence /-|cha you from r»OS10O. A 250-bp fragment encoding the type II dihydrofolate reductase gene (E. soybean) is also amplified by PCR, and these two PCR fragments are assembled into a single structure with the Zasi-Rvi fragment from pB1221 o (Siopiesi)) containing the main chain of the risC19 vector and the nopaline synthase terminator. The assembly of these fragments into an ordered structure leads to the formation of reeOos19, which contains the promoter 355 in fusion with the sequence of intron b, the leader sequence 5І5, the ONEK gene and the terminator of nopaline synthase. Replacement of the leader sequence 5ОО5 in реОС19 with the leader sequence from the spotted chlorotic corn virus (MSMM) leads to the formation of the vector реОс35. The pOs19 and pOs35 vectors contain the rus gene for ampicillin resistance 70 and have NipaiI, ZrpI, Ragi and EsokI sites suitable for cloning foreign substances. 8 s B. Transformation ц As the desired coding sequence is cloned into the expression system, it is transformed into a "" plant cell. Methods of plant transformation and regeneration are well known in the art. For example, plasmid vectors

They are used for delivery of foreign DNA, as well as direct absorption of DNA, liposomes, electroporation, microinjection and bombardment with microparticles. In addition, bacteria from the genus Adhorasiegisht are used to transform plant cells. Below is a description of the main technologies for the transformation of both dicotyledonous and monocotyledonous plants. o 1. Transformation of dicotyledonous plants -I The technology of transformation of dicotyledonous plants is well known to specialists and includes methods based on the use of Adhorasiegisht and methods in which Adhorasiegisht is not used. Technology without use

Adhorasiegisht involves the absorption of exogenous genetic material directly by protoplasts or

Chl" cells. This is done by RES or uptake with intermediate use of electroporation, particle bombardment delivery or microinjection. Examples of these technologies are described in

IRaz2KomuvKki ei ai,, EMVO 3 3: 2717-2722 (1984), RomgukKiv ei a/., MoY. Sep. Sepoys 199: 169-177 (1985), Keisp ey a/., Vioyesppoiodo 4: 1001-1004 (1986) and Kiep ei a/.,, Maishge 327: 70-73 (1987) In each case, the transformed cells are regenerated until complete recovery plants using standard technologies,

F, known to specialists in this field. co Transformation using Adhorasiegisht is an optimal technology for the transformation of dicotyledonous plants due to its high efficiency and wide application with many different species. because transformation using Aadgobrasiegisht usually involves transfer of a double vector having the desired foreign DNA (e.g., pSIV200 or pSIV2001) into the corresponding Adgobasiegis strain, which depends on the complement of mig genes carried by the Aadgobrasiegisht host strain, either on a co-resident Ti plasmid or via chromosomes (for example, strain SIV542 for rSIV200 for rSIV2001 |(OKpez ei a/. Riapi Seyi! 5: 159-169(1993)). The transfer of the recombinant double vector in Adhorasiegpist is carried out by means of the 65 triple crossing procedure using E. soybean, which has a recombinant double vector, a helper strain of E. soya, which has a plasmid such as pKK20O13, and which is capable of transferring the recombinant double vector into the target strain Aadhorasiegisht. Mysi. Asiadz Kez. 16: 9877 (1988).

Transformation of a target plant species with recombinant Adhorasiegitst usually involves the co-cultivation of Adhorasiegitst with explants from the plant and is performed according to protocols well known in the art. The transformed tissue is regenerated in a selective medium that has an antibiotic or herbicide resistance marker present between the T-DNA boundaries of the binary plasmid.

Another approach to the transformation of plant cells with a genome involves the active introduction of inert or biologically active particles into plant tissues and cells. This technology is described in (US Patent Nos. 4,945,050, 5,036,006, and 5,100,792), all of which are issued to Zapioga et al. Typically, this procedure involves stimulating inert or biologically active particles in cells under conditions effective for penetration through the outer surface of the cell and facilitating entry into its internal structure. In the conditions of using inert particles, the vector is introduced into the cell by covering the particles with a vector containing the desired gene. In another variant, the target cell is surrounded by a vector in such a way that the vector is carried into the cell by a stream of particles. 7/5 Biologically active particles (eg cells of dry yeast, dry bacteria or bacteriophage, each of which contains the DNA to be introduced) are also actively introduced into plant cell tissue. 2. Transformation of monocots.

Transformation of most species of monocots has also become a standard operation. The optimal technology involves direct gene transfer into protoplasts using RES or electroporation and particle bombardment into the tissue of the influx. Transformation is performed with a single type of DNA or multiple types of DNA (ie, co-transformation), and both of these technologies are suitable for use in accordance with the present invention. Co-transformation has the advantage of avoiding full vector structures and creating transgenic plants with unlinked loci for the desired gene and the selectable marker, allowing the selective marker to be removed in subsequent generations if needed. However, the disadvantage of using co-transformation is the less than 1009U6 frequency with which certain types of DNA are integrated into the genome. Biosciences 4: 1093-1096 (1986)). and)

Patent applications (EP 0 292 435, EP 0 392 225 and MO 93/07278) describe the technology of influx (callus) and protoplasts from an elite inbred line of corn, transformation of protoplasts using RES or electroporation, as well as regeneration of plant forms of corn from transformed protoplasts. «g zo Ibogdop-Katt ei ai. (Reapi Se 2: 603-618 (1990)) apa GRgott ei ai. (Violations 8: 833-839 (1990)) published a technology for transforming an A188-derived corn line using particle bombardment. In addition, (MUHO 93/07278 apa Koliyei ei ai. (Vioiesppoydu 11: 194-200 (1993)))) also describe Kk technology of transformation of elite inbred lines of corn by means of particle bombardment. In this technology, immature corn embryos with a length of 1.5-2.5 mm, which were cut from the corn cob 14-15 days after pollination, as well as a bombardment device РОБ-1000Не и-

Vioiiz (Isv.

Rice transformation is also performed using direct gene transfer technology using protoplasts or particle bombardment. Transformation using a protoplast has been described for Daropis types and Ipais types (2papd ei ai. Riapi Se Ker 7: 379-384 (1988); Zpitatoio ei a). Maishge 338: 274-277 (1989); Oatsa ei ai. Physiology 8: 736-740 (1990). Both types are also transformed from c in a standard way using particle bombardment (Sngizoioi ei ai. VioFesppoiodu 9: 957-962 (1991))).

The patent application (EP 0 332 581) describes the technology for the reproduction, transformation and regeneration of Rosideae protoplasts. This technology makes it possible to transform B. and wheat. In addition, the transformation of wheat is described in | 19923), using particle bombardment of callus cells with long-term regeneration of type C, as well as in |Maviai ei ai. -I (Viotesppoiodu 11: 1553-1558 (1993)) apa UUeekz ei ai. (Reapi Rnuvzioi. 102: 1077-1084 (1993))) using particle bombardment of immature embryos and immature embryo callus However, the optimal wheat transformation technology involves transformation of wheat by particle bombardment of immature embryos and includes a high sucrose or high maltose stage prior to gene delivery. Before bombardment, a certain number of embryos (length 0.75-1 mm) are placed in M medium with 390 sucrose | Mygazpida 5 5Kosd, RPuzioiodia Riapi agit 15: 473-497 (1962)) and Zmg/l i» 2,4-O for the induction of somatic embryos that can be processed in the dark. On the selected day of bombardment, the embryos are removed from the induction medium and placed on an osmotic medium (ie induction medium with sucrose or maltose added at the desired concentration, typically 1595). Two embryos are given the opportunity to plasmolyze for 2-3 hours, after which they are bombarded. Twenty embryos per target plate is the norm, although this is not critical. The appropriate gene-containing plasmid (such as pSIV3064 or rheos5) (F) is deposited on micron-sized gold particles using standard procedures. Each plate of embryos is bombarded with a YuRopi VioiiviisvF helium device using a burst pressure of -1000 rvzi (70.Zkg/cm2 ) using standard filtration with an 8 Ohm filter hole size. After bo bombardment, the embryos are again placed in the dark to recover for about 24 h (also on the osmotic medium). After 24 h, the embryos are removed from the osmotic medium and placed back on the induction medium, where they remain approximately one month before regeneration. Approximately one month later, the embryo explants from the developing embryogenic callus are transferred to the regeneration medium (M5 with 1Tmg/l MAA, bmg/l CA), which additionally contains the appropriate selection agent (1Omg/l basta - in the case of rSIVZ3064 , and 2mg/l 65 of Metatrexat - in the case of rBOS35).About a month later, the developed rings are transferred to sterile of larger containers, known as "ZA?", containing semi-concentrated M5, 290 sucrose, and the selection agent in the same concentration.

The transformation of monocots using Aadgobasiegisht was recently described. See (MO 94/00977 and US Patent Mo 5,591,616), which are referenced in this description.

Cultivation of varieties

Immunomodulated plants obtained by transformation with a ZAK gene, such as one form of the MIM1 gene, can belong to any of a wide range of plant species, including representatives of monocots and dicots; however, immunomodulated plants used according to the method of the present invention are preferably selected from the list of agronomically important target crops, which are called zirga. The expression of the selected form of the MIMI1 gene in combination with other characteristics that are essential for the creation of mutants and their quality is introduced into plant cell lines by means of selection. Breeding methods and technology are known in the art. See for example, (MUeiYivy .). K., Ripdatepia!5 oi Riapi Sepeiyisv apa Vgeedipod, dhoip Muyeu 5 Bopv, MU (1981);

Storr Vgeedipo, Uosia 0. K. (Ed.) Ategisap bosieiu oi Adgopotu Madizop, M/izsopvzip (1983); Mauo O., Te Tneogu oi Riapi Vgeedipd, Zesopa Edyiop, Siagepdop Rgezz, Ozhhioga (1987); Bipop, O.R., Vgeedipd iTog Keviviapse 7/5 Oiveazez apa Ipvzesi Reviv, Zrgipdeg-Mepad, MU (1986); and UUgiske apa UUereg, Omapiyaime Sepeisv apa

Zeiiesiop Riapi Vgeedipo, UUakeg de Ogtsuieg apa So., Vegiip (1986)).

The genetic properties introduced into the above-described transgenic seeds and plants undergo the stages of sexual reproduction or vegetative growth and thus are preserved and propagated in the offspring of plants. In general, the above-mentioned conservation and propagation is implemented using known agrotechnical methods, 2o developed depending on the specific purpose, such as germination, sowing or collecting the necessary cells from the culture. Specialized methods such as hydroponics or greenhouse technologies are also used. Since the crop plant is vulnerable to attack by organisms and damage by insects or infections during growth, as well as to severe competition with weeds, measures are taken to control such weeds, plant diseases, insects, nematodes and other harmful conditions. , which makes it possible to improve the quality of crops. These include measures of a mechanical nature, such as tillage or removal of weeds and infected plants, as well as the use of agrochemicals, such as herbicides, fungicides, gametocides, nematocides, i) growth regulators, ripening agents and insecticides.

The use of improved genetic properties of transgenic plants and seeds according to the present invention is continued during plant breeding, the purpose of which is to create plants with improved properties, such as tolerance to pesticides, herbicides or stress, improved nutritional value, increased yield or improved structure, which leads to a reduction in losses from lying or destruction of crops. At different stages of selection, artificial human intervention is clearly defined, such as the creation of lines for crossing, the direction of cross-pollination of parental lines, or the selection of appropriate plant offspring. Depending on the required properties, different measures are used for plant selection. Suitable techniques are well known to those skilled in the art and include, i) but are not limited to, crossbreeding, inbreeding, backcrossing, multilineage breeding, multiple hybridization, interspecific crossing, aneuploid technology, and the like. Crossbreeding technology also includes sterilization of plants to produce male or female sterile plants by mechanical, chemical or biochemical means. Cross-pollination of a male sterile plant with pollen of another line guarantees that the genome of a male sterile, but capable of reproducing offspring of a female plant, will have the properties of both parental lines to the same extent. Thus, the transgenic seeds and plants of the present invention are used to breed plant lines with improved properties,

And which, for example, increases the efficiency of standard methods, such as herbicide or pesticide treatment, or allows flexible manipulation of the mentioned methods thanks to their modified genetic properties. In another variant, new cultures with improved tolerance to stress are obtained, which, thanks to their optimized genetic "equipment", allow to harvest a product with better properties than those observed in products that were not characterized by adequate tolerance to comparable harmful growth conditions.

For seed production, the essential characteristics of the product are the quality of germination and uniformity of the seeds, while the quality of germination and uniformity of the seeds collected and sold by the farmer are of no importance. -I Since it is difficult to protect the crop from the seeds of other crops and weeds, in order to combat seed-related diseases and to create seeds with good germination characteristics, growers who have extensive experience in the field of cultivation, to establish the optimal regime and conditions for the sale of seeds, very effective and well-defined production technologies have been developed. Thus, a farmer will typically buy certified seed that meets specific quality standards, rather than using seed that he collects from his own field. Seeds used as propagating material are usually treated with a protective coating containing herbicides, insecticides, fungicides, bactericides, nematocides, molluscicides, or mixtures thereof. Usually to the used protective coatings

F) include such compounds as captan, carboxin, thiram (TMTO"), metalaxyl (Argop 4) and pirimiphos-methyl ka (AsiasF). If desired, these compounds are prepared together with additional carriers, surface-active substances or stimulants during the application of ad 'juvants, which are commonly used in the formulation industry to provide protection against damage caused by bacterial, fungal or animal pests. Protective coatings are applied by impregnating the propagation material with a liquid formulation or by coating with a combination of a wet or dry formulation. Other applications are also possible , such as treatments aimed directly at buds or fruits.

Another aspect of the present invention is to obtain new agrotechnical methods, such as the methods exemplified above, which are characterized by the use of transgenic plants, transgenic plant material or transgenic seeds according to the present invention.

The seeds are received in bags, containers or vessels made of suitable packaging material, and the bag or container can be closed. A bag, container or vessel is designed either for short-term or long-term storage of seeds, or for both. Examples of suitable packaging material are paper such as kraft paper, rigid or flexible plastic or other polymeric materials, glass or metal. Preferably, the bag, container or vessel consists of several layers of packaging materials of the same or different type. In one of the variants of the implementation of the invention, the bag, container or vessel is made in such a way as to exclude or limit the contact of the seeds with water and moist substances. In one example, the bag, container, or vessel is sealed, 7/0 for example, by heat sealing to prevent water or moisture from entering. In another embodiment, water-absorbing materials are placed between or in direct contact with layers of packaging material. In yet another embodiment, the bag, container, or vessel, or the packaging material from which they are composed, is treated to limit, inhibit, or prevent disease, contamination, or other adverse effects of seed storage or transportation. 7/5 An example of such treatment is decontamination, for example by chemical means or radiation treatment.

The present invention provides the use of a branded bag containing the seeds of a transgenic plant with one of the forms of the MIMI gene or the MIMI protein expressed in said transformed plant, at concentrations higher than in natural plants, together with a suitable carrier, together with branded instructions for their use, which guarantees the creation of resistance in plants to a wide range of diseases.

Introduction of a bactericide into immunomodulated plants As mentioned above, the method of protecting plants according to this invention involves two stages: the first, i.e. activation of the ZAR pathway. with the aim of obtaining an immunomodulated plant, and the second, that is, the introduction of a bactericide into such immunomodulated plants with the aim of acquiring synergistically increased resistance to the disease.

A. Standard bactericides. high school

According to the method of the present invention, any commercial or standard bactericide is used in immunomodulated plants, which are obtained by any of the three ways described above. Examples of i) suitable bactericides include, among others, the following fungicides: 4-(3-(4-chlorophenyl)-3-(3,4-dimethoxyphenyl)acryloyl|morpholine ("dimethomorph"), (reference: C Totiip (Editor ):

Tpe Reziisiae MapaaiY, 18 eayop, Eatnpap; OK, 1994, radevz 3351-3521; "g zo 5-methyl-1,2,4-triazolo|3,4-51(11,3|benzothiazole and ("tricyclazole"), (reference: S. Totiipyi (Editor); Te

Reziiside Mapia), 10 eayop, Batpat, ShK, 1994, pages 1017-1018); 3-allyloxy-1,2-benzothiazole-1,1-dioxide (7 ("probonazole"), |ref: C Totiip (Editor): Te Resisiside Mapiai, 10th Avenue, Ratnapath, OK, 1994, Radez M 831-8321 o-(2-(4-chlorophenyl)ethyl|--o-(1,1-dimethylethyl)-1H-1,2,4-triazole-1-ethanol, ("tebuconazole"), (reference

ER-A-40 345);1-((3-(2-chlorophenyl)-2-(4-fluorophenyl)oxiran-2-yl|methyl|-1H-1,2,4-triazole, Sepoxiconazole"), i. (reference: EP-A-196 0381; c-(4-chlorophenyl)-c-(1-cyclopropylethyl)-1H-1,2,4-triazole-1-ethanol, ("cyproconazole"), according to ( reference: 5-4 664 696); 5-(4-chlorobenzyl)-2,2-dimethyl-1-(1H-1,2,4-triazol-1-ylmethyl)-cyclopentanol, ("metconazole" ), (reference: EP-A-267 71781); 2-(2,4-dichlorophenyl)--3-(1H-1,2,4-triazol-1-yl)-propyl--1,1,2 ,2-tetrafluoroethyl ether, Stetraconazole"), " (reference: EP-A-234 2421; methyl-(E)-2-(2-(6-(2-cyanophenoxy)pyrimidin--4-yloxy|phenyl) -3-methoxyacrylate, 40. SS A 5504", "azoxystrobin"), (reference: EP-A-382 375); - c methyl-(E)-2-methoxyimino-2-(o-(o-tolyloxy) )--o-tolylfidecetate, (VAB 490 PE", "kresoxymethyl"), (reference: and EP-A-400 4171; 2-(2-phenoxyphenyl)-(E)-2-methoxyimino-M-methylacetamide, | reference: EP-A-398 6921; is" (2-(2,5-dimethylphenoxymethyl)-phenyl|-(E)--2-methoxyimino-MI-methylacetamide, |reference: EP-A-398 6921); ( 1kK,35/15,3K)-2,2-dichloro-M-((K)-1-(4-chlorophenyl) ethyl|-1-ethyl-3-methylcyclopropanecarboxamide, (KT 3616"), reference: EP-A-341 475); manganese ethylenebis(dithiocarbamate) polymer-zinc complex, ("mancozeb"), -i (reference: 5 2 974 1561); 1-(2-(24-dichlorophenyl)-4-propyl-1,3-dioxolan-2--ylmethyl|-1H-1,2,4-triazole, Spropiconazole"), (reference: ov-1522657); 1-2-(2-chloro-4-(4-chlorophenoxy)phenyl)|-4-methyl-1,3-dioxolan--2-ylmethyl)--1H-1,2,4-triazole, ("difenoconazole "), - and (reference: 8B-2098601|; 1-(2-(2,4-dichlorophenyl)pentyl--1H-1,2,4-triazole), ("penconazole"), (reference: -l 20 8-15898521; cis-4-I3-(4-tert-butylphenyl)-2-methylpropyl|-2,6-dimethylmorpholine, ("fenpropimorph"), (reference:

OE 27521351; 1-I(3-(4-tert-butylphenyl)-2-methylpropyl|-piperidine, ("fenpropidine"), (reference: OE27521351;

I" 4-cyclopropyl-b-methyl-M-phenyl-2-pyrimidinamine ("cyprodinil") (reference: EP-A-3105501; (k5)-M-(2,6-dimethylphenyl--M-(methoxyacetyl) -alanine methyl ester ("metalaxyl"), |reference: 5B-15005811; (k)-M-(2,6-dimethylphenyl-M-(methoxyacetyl)-alanine methyl ester ("K-metalaxyl"), |reference: ZV-15005811; 1,2,5,6-tetrahydro-4H-pyrrolo|Z,2,1-i))quinolin-4-one ("pyrochilone"), (reference: 8B-1394373); ethyl hydrophosphonate o ( "fosetyl"), |reference: S. Totiip (Editor): Te Reswiiside Mapiai!, 10 Eaiyop, Rathpap, OK, 1994, radez 5300-5321; and copper hydroxide (ref: S. Totiip (Editor): Te Resviiside Mapiai! i, 10 Eaiiop, Ratpap, OK, imo) 1994, radev 229-230).

The selected bactericide is optimally introduced into immunomodulated plants to be protected, because in the form of a composition with additional carriers, surface-active substances or other stimulating adjuvants during use, which are traditionally used in the technology of preparing compositions. Acceptable carriers and adjuvants can be solid or liquid and are substances that are commonly used in formulation technology, for example, natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, thickeners, binders or inseminators. 65 The optimal way to introduce a bactericidal composition is to introduce it into the above-ground parts of plants, especially into the leaves (introduction into the leaves). The frequency and volume of application depend on the biological and climatic conditions of the life of the pathogen. However, the bactericide can also enter the plant through the roots from the soil or from water (systemic action), if the locus of the plant is permeated with a liquid composition (for example, in rice culture), or if the bactericide is introduced into the soil in a solid form, for example, in the form of granules (introduction into the ground). For seed treatment, the bactericide is also applied to the seeds (coating), either by impregnating the tubers or grains with a liquid composition of the bactericide, or by covering them with an already combined wet or dry composition. In addition, in special cases, other methods of introduction into plants are possible, for example, treatment aimed directly at buds or clusters of fruits.

The bactericide is used in an unmodified form or, in the optimal version, together with adjuvants, which / o are usually used in the technology of preparing compositions and, therefore, are prepared in a known way, for example, added to concentrates converted into an emulsion, pastes for coating, directly sprayed or dilute solutions, diluted emulsions, wettable powders, soluble powders, powders, granules, or by encapsulation in, for example, polymeric substances. Depending on the nature of the compositions, the methods of application, such as spraying, spraying, sprinkling with powders, scattering, /5 coating or watering are chosen according to the intended tasks and real circumstances. The optimal amount of bactericide injection is usually from 5O0g to 2kg a.i/ha, in the best version from 100g to 1000g a.i/ha, best from 150g to 700g a.i/ha. In the case of seed treatment, the amount of input is from

Oh, 5g to 1000g, better from 5g to 100g a.i. per 1OOkg of seeds.

The compositions are prepared in a known manner, for example, by homogeneous mixing and/or grinding of the bactericide with diluents and fillers, for example, solvents, solid carriers and, if necessary, surface-active compounds (surfactants).

Suitable solvents are: aromatic hydrocarbons, preferably fractions containing from 8 to 12 carbon atoms, for example 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 esters and esters such as ethanol, ethylene glycol, ethylene glycol monomethyl or monoethyl ether, ketones such as cyclohexanone, highly polarized solvents such as M-methyl-2-pyrrolidone, dimethylsulfoxide or (8) dimethylformamide, and vegetable oils or epoxidized vegetable oils such as epoxidized coconut oil or soybean oil; or water

Solid carriers used, for example, for powders and dispersed powders, are usually natural "Ezo mineral fillers, such as calcite, talc, kaolin, montmorillonite or attapulgite. In order to improve the physical properties, it is also possible to add finely dispersed silicic acid or finely dispersed (387) absorbent polymers. Suitable granular absorbent media are pore-type media, such as pumice, broken brick, sepiolite or bentonite, and suitable non-absorbent media are, for example, calcite or sand. In addition, numerous pre-granulated substances of an inorganic or organic nature are used, in particular dolomite or ground and dispersed plant residues. uh-

Depending on the nature of the bactericide, suitable surface-active compounds are non-ionic, cationic and/or anionic surfactants that have the ability to easily emulsify, disperse and wet. The term "surfactants" should also be understood to include mixtures of surfactants "

In particular, natural or synthetic phospholipids of the cephalin or lecithin series, for example, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and lysolecithin, are also optimal stimulants when used as adjuvants. Agrochemical compositions usually contain from 0.1 to 9995, preferably from 0.1 to 9595 of an active bactericidal component, from 99.9 to 195, preferably from 99.9 to 595, of a solid or liquid adjuvant, and from 0 to 2595 , preferably from 0.1 to 2595, of surfactant. -I While for commercial purposes the products are mostly prepared in the form of concentrates, the end user usually uses dilute compositions. o B. Bactericides that activate plants -I In the case of introduction into immunomodulated plants, which are obtained by the second or third method described above (selective breeding or genetic construction), the bactericide can, as an option, be a chemical inducer of ZAK (bactericide that activates plants ), such as benzothiadiazole, isonicotinic acid, or salicylic acid, as described in US Pat. Nos. 5,523,311 and 5,614,395. So, two methods of immunomodulation are used together. With the help of the introduction of plant-activating bactericides into immunomodulated plants, obtained either by selective breeding or genetic engineering, two "extra-immunomodulation" and synergistic strengthening of resistance to diseases are achieved.

As described further in the text, transgenic immunomodulated plants overexpressing MIM1 responded significantly (F) faster and to significantly lower doses of VTN, as shown by expression of the RK-1 gene and resistance to R. ragavziis, than wild-type plants. See Example C5 and the results of northern blotting analysis in Fig. 3. Synergistic enhancement of resistance to the disease in MIM1 overexpressors is achieved by the introduction of VTN at a concentration of only 1Ωm, that is, at a concentration usually insufficient for any effect at all. Usually, phytotoxic or otherwise undesirable concentrations of chemical agents inducing ZAC can be avoided by using such synergy.

In addition, using the change in the time mode of activation of the ZAC, which occurs when the chemical inducers

ZACs are injected into already modulated plants, such as MIM1 overexpressors. In addition, it is possible to obtain an economic profit due to the reduction in the amount of chemical BAC inducers used to provide 65 a given level of plant protection.

A. Standard bactericides along with plant-activating bactericides.

In order to achieve the greatest possible resistance to diseases, both a standard bactericide and a plant-activating bactericide are injected into immunomodulated plants obtained either through selective breeding or through genetic engineering. Due to this, a significantly higher level of synergistic disease resistance is obtained compared to the level of disease resistance achieved only by immunomodulation, by immunomodulation in combination with only one type of bactericide, or by simultaneous administration of both types of bactericide (standard and plant-activating). See, for example,

Table 35 in Example 19.

Evaluation of resistance to diseases. 70 Assessment of resistance to diseases is carried out using methods known in the field. See (|OKpevs ei ai, (1993) MoiIesshiag Riapi Misgore Ipiegasiops b: 680-685; (Zopasp ei a), (1996) Riapi Sei! 8:629-643;

AIehapdeg ei aY.,, Rgos. May Asai. All together. BOTH 90: 7327-7331 (1993). For example, several characteristic experiments on the study of resistance to diseases are described further on in the text.

A. An experiment on the study of resistance to Rpuiorpiyoga ragaziis (blackening of the stem).

Experiments on the study of resistance to Rnuiorpiyoga ragavziis, an organism that causes blackening of the stem, are carried out on six-week-old plants, as described in |AIekhapadeg ei aI., Rgos. May). Asad si. OBA 90: 7327-1331 (1993)), Plants are soaked, allowed to dry well and then inoculated by introducing 1 Oml of sporangium suspension (300 sporangia/ml) into the soil. Inoculated plants are kept in a greenhouse at a daytime temperature of 23-252C and a night temperature of 20-2220C. The assessment of wilting, which is used for research, is as follows: O - no symptoms; 1-no symptoms; 1-some signs of wilting, with reduced swelling; 2-clear signs of wilting, but without decay or growth retardation; C-clear signs of wilting with stunted growth, but without clear signs of stem rot; 4-severe wilting, with visible rotting of the stem and some damage to the root system; 5-same as for 4, but the plants die or have already died, and significant degradation of the root system is observed. The results of all experiments are quantitatively evaluated in a "blind way" on the basis of a random selection of plants.

B. An experiment on the study of resistance to Rzeidotopazg zugipdae. at

Strain Rzeshydotopaz zugipdae ru. garasi 25551 is injected into the two lower leaves of several plants 6-7 weeks old at a concentration of 1097 or 3x109 per ml in HO. Each of the six specific plants is evaluated at a specific point in time.

Infected Rzepdotopaz (arasi) plants are evaluated on a five-point scale of disease severity, with h;E 5-10095-e tissue necrosis, O-no symptoms. T-test (I 50) is performed on the basis of evaluations for each day, and after evaluating the average of the maximum permissible value of disease severity, grouping of plants is carried out according to the mentioned indicator. Values followed by the same letter of the day of assessment are statistically little - different.

V. An experiment on the study of resistance to Segsozrog pisoyapae. at

The leaf surfaces are sprayed with a suspension of spores of Segsozrog pisoyapae (ATSS Mo18366) (100,000-150,000 spores per ml) using a continuously operating device. The plants are kept in conditions of 100905 humidity for five days. After that, the plants are moistened with water vapor 5-10 times a day. Each of the six specific plants is evaluated at a specific point in time. The degree of damage to plants by the organism Segsozroga pisoyapae is estimated based on the measurement of the surface area of the leaf on which the symptoms of the disease are detected (in 905). T-test 40. (50) is carried out on the basis of estimates for each day, and after estimating the average maximum permissible value - with the severity of the disease, grouping of plants is carried out according to the mentioned indicator. Values followed by one and the same letter of the evaluation day are statistically little different. ,» G. An experiment on the study of resistance to Regopozrog ragazvziiis.

Experiments on studying resistance to Regopozrog ragaziiis are carried out on plants in the following way, which is described in |(OKpez ei ai, (1993)). Plants are inoculated with an active isolate of R. ragaziiis by spraying with a conidium suspension (approximately 5x107 spores per milliliter). Inoculated plants are incubated in conditions of high humidity at 172C in a growth chamber with a light/dark cycle of 14 hours per day/10 hours per day. Plants are examined for 3-14 days, in the optimal version for 7-12 days, after inoculation in the presence of conidiophores. - In addition, several plants after each treatment are selected by the method of random selection and stained -lШ 20 with lactophenol-trypan blue (Keoodn ei ai., Tgape. Vg. Musoi. Zos. 74: 329-333 (1980)), which allows to perform microscopic research. iz" Brief description of the drawings

Figure 1 shows the alignment of the MIMI1 protein sequence with 1 kW from tissues of mice, rats and pigs.

Vertical strokes (I) above the sequences indicate the identity of amino acids between the sequences of MIM1 22 and IikVo (matrix score equal to 1.5); colons () above sequences indicate evaluation

F! similarities 20.5; single dots (- above the sequences indicate a similarity score of "0.5 but 20.0; a score of "0.0 indicates no similarity and is not marked above the sequences (see Examples). (de Magpip et al., Sep 152, 253-255 (1995)). Dots within the sequence indicate gaps between MIMI and IkVo proteins. The five ankyrin 60 repeats in IkVo are shown by dashed lines below the sequence. Amino acids are numbered depending on the protein MIM1 with gaps inserted where necessary Plus signs (u) are placed above the sequences every 10 amino acids.

Figure 2 shows a comparison of the amino acid sequences of parts of the MIM1 protein (numbers correspond to the positions of amino acids in SEQ ID No. Mo:2) and protein products of ET rice (SEQ ID No. MoMo: 17-24). 65 Figure 3 shows the results of northern blotting analysis, which reflect the duration of gene expression

RA-1 in natural lines and MIMI lines with overexpression after treatment with water or VTN. RNA was prepared from treated plants and analyzed as described in the Examples. "MUv" means the natural ecotype of Uuz

Agaridorziv INaiyapa. "ZA", "5B", "bE" and "7C" correspond to specific MIMI plant lines with overexpression generated according to Example 21. "0 VTN" means water treatment; "10 VTN" means treatment of 10 μM VTN; "T10OVTN" means treatment of 100 μM VTN. "0" means control samples on day zero; "1", "3" and "5" correspond to samples for 1, 3 and 5 days.

A brief description of sequences in the list of sequences.

POST ID Mo:1 is a genomic sequence with a length of 5655 bp, which contains the coding region 7/0 of the natural gene MIM1 Agabridorviz (Naiapa.

POST ID Mo2 is the amino acid sequence of the natural protein MIM'1 Agabidorziz (Naiiapa), encoded by the coding region SELF. ID Mo:1.

POST ID Mo:C is the amino acid sequence of IkVo, from the organism of the mouse from Fig.1.

POST ID Mo:4 is the amino acid sequence of IkVo, from the body of the rat from Fig. 1.

POST ID Mo:5 is the amino acid sequence of IkVo, from the organism of the pig from Fig.1.

POST ID Mo:b is the cDNA sequence of the MIM'1 gene from Agaridorvziz INaiiapa.

POST ID MoMo:7 and 8 represent the DNA coding sequence and the encoded amino acid sequence, respectively, of the dominant-negative form of the MIMI protein, which has alanine residues instead of serine residues at amino acid positions 55 and 59.

POST ID MoMo and 10 represent the DNA coding sequence and the encoded amino acid sequence, respectively, of the dominant-negative form of the MIM1 protein, which has an M-terminal deletion.

POST ID MoMo/11 and 12 represent the DNA coding sequence and the encoded amino acid sequence, respectively, of the dominant-negative form of the MIM1 protein, which has a C-terminal deletion.

POST ID MoMo-13 and 14 represent the DNA coding sequence and the encoded amino acid c sequence, respectively, of the modified form of the MIMI! gene, which has both M-terminal and C-terminal amino acid deletions.

POST ID MoMo:15 apa 16 represent the DNA coding sequence and the encoded amino acid sequence, respectively, of the MIM1 ankyrin domain.

POST ID Mo:17 is the sequence of AA 33-155 from Figure-1 of Fig.2. "AND

POST ID Mo:18 is the sequence of AA 215-328 from Figure-1 of Fig.2.

POST ID Mo:19 is the sequence of AA 33-155 from Figure-2 of Fig.2. -

POST ID Mo:20 is the sequence of AA 208-288 from Figure-2 of Fig.2. -

POST ID Mo:21 is the sequence of AA 33-155 from Figure-3 of Fig.2.

POST ID Mo:22 is the sequence of AA 208-288 from Figure-3 of Fig.2. at

POST ID Mo:23 is the sequence of AA 33-155 from Figure-4 of Fig.2. -

POST ID Mo:24 is the sequence of AA 215-271 from Figure-4 of Fig.2.

POST ID MoMo: 25 - 32 are oligonucleotide primers.

Definition of "

The following definitions will facilitate a better understanding of the present invention:

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

Plant tissue: A group of plant cells organized into a structural and functional unit, including -and any plant tissue in a plantation or culture. 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 connection with any specific type of plant, listed above, or without connection therewith, or other aspects covered by this definition, shall in no way limit its application to any other type of plant fabrics

Protoplast: A plant cell without a wall. c" Progeny plant: a sexually or asexually produced plant of a future generation, which includes, but is not limited to, progeny plants.

Transgenic plant: A plant that has permanently introduced recombinant DNA into its genome.

Recombinant DNA: Any DNA molecule that is formed by combining DNA segments from different sources and that is created using recombinant DNA technology. o Recombinant DNA technology: Technology by which recombinant DNA is created and transferred into cells where it is expressed or propagated. See Sopsize

Oisniopagu oi Viotedisile apa Moiesciag Vioiodu, Eg. dio, SKS Rgez5, Vosa Kaiop (1996)), for example, 60 transfer of DNA into the protoplast(s) or cell(s) in a variety of forms, including, for example, (1) unprotected DNA in circular, linear, or supercoiled forms, (2) DNA contained in nucleosomes or chromosomes, or in nuclei or parts thereof, (3) DNA complexed or bound to other molecules, (4)

DNA enclosed in liposomes, spheroplasts, cells, or protoplasts, or (5) DNA transferred from organisms other than the host organism (eg, Adgobasiegiit (steyiasiepv). These and various other methods of introducing 65 recombinant DNA into a cell are known in the art and are used in order to create transgenic cells or transgenic plants of the present invention.

Technology using recombinant DNA also includes methods of homologous recombination, which are described in (Tgeso ei ai, MUO 94/12650, and Tgeso ei ai,, M/O 95/31560), and which are used to increase the activity of peroxidase in a monocotyledonous plant. More specifically, regulatory regions (e.g., promoters) are introduced into the plant genome in order to increase the expression of endogenous peroxidase.

Together with recombinant DNA technology, insertion of a peroxidase coding sequence without selected expression signals into a monocot plant and testing of the transgenic monocot plant for increased peroxidase expression due to endogenous control sequences in the monocot plant is used. This should lead to an increase in the number of copies of sequences encoding 7/o peroxidase in the plant.

The initial steps of inserting recombinant DNA into the genome of a Ke plant are performed not so much by traditional methods of plant breeding, but rather by the technical methods described in this text. After the initial stages of insertion, the transgenic offspring are actually propagated using traditional breeding methods.

Chimeric gene: A DNA molecule containing at least two heterologous fragments, such as parts derived from pre-existing DNA sequences that do not bind in their pre-existing states, and these sequences are generated in advance by recombinant DNA technology.

Expression cassette: A DNA molecule containing a promoter and a terminator between which a coding sequence is inserted.

Coding sequence: A DNA molecule that, when transcribed and translated, causes the formation of a polypeptide or protein.

Gene: A discrete region of a chromosome containing a regulatory DNA sequence responsible for controlling expression, i.e., transcription and translation, as well as a coding sequence that is transcribed and translated to produce a specific polypeptide or protein. c asa: a mutant plant with an accelerated cell-killing function

АРИ R: Amplified fragment length polymorphism about amgcrid: Krig avirulence gene isolated from Rzeidotopaz zuhipdae

VAS: Artificial chromosome of bacteria

ВТН: benzo!|1,2,3)thiadiazole-7-carbothioacid-5-methyl ester "I

SIM: Phenotype with constitutive immunity (ZAC is activated constitutively) net: Mutant plant with constitutive immunity -

SM: Centimorgans ysri1: Mutant plant with constitutive expressor of RK genes

SoI-O: Agaridorziz, ecotype SoYishtbia and,

ES: Combinations of enzymes and

Etuma: Regopozroga ragaziis isolate, compatible in the Muz-0 Agaridorz ecotype

EM5: Ethyl methanesulfonate

IMA: 2,6-dichloroisonicotinic acid

I eg: I apazbrego egesia, ecotype of Agaridorvi "

Willow. Mutant plant with disease-modulating lesions 8 s panps: Rzeidotopaz ryida with salicylate hydroxylase that converts salicylic acid to catechol and Mano: Agaridorz line transformed by the gene papo "" pag. Mutant plant with variety-specific resistance to sweat disease: Mutant plant with non-induced immunity

MIM1: Natural gene involved in the classical signal transduction pathway of ZAK -i MIMI: Protein encoded by the natural gene MiM' pit!: Mutant allele of MIM', which gives the plant susceptibility to diseases; also refers to the mutant plant Agabridorz (Nayapa, which has a mutant allele MIM1 -I Moso: Regopozrog ragaziiis isolate, compatible in ecotype So!-0 from Agaridorz

OCR: Open reading frame - RSzv: Primer combinations

Ta» RE: Associated with pathogenesis

PROS: Salicylic acid

ZAK: Systemic acquired resistance

ZAC-op: Immunomodulated plants in which ZAC is activated, which are usually characterized by higher than natural expression of ZAC genes and have a disease-resistant phenotype iF) ZOI P: Simple sequence length polymorphism ko o: Universal disease-susceptible phenotype

UUeia: Regopozroga ragaziis isolate, compatible in the U/eipipdep Agaridorvy ecotype in MuUv-O: Agavidorviz, IzziieemuzKi)ia ecotype

M/T: Natural

UAS: Yeast Artificial Chromosome

Examples

The invention is further illustrated by the detailed description of procedures, preparations and 65 examples given below. The examples are provided for illustration purposes only and do not in any way limit the scope of the present invention.

The standard recombinant DNA and molecular cloning technology used in the present invention is well known to those skilled in the art and is described in (ZatryokK, et al., MoiIesshag Siopippo, et al., Soy Zrgipa

Nagbog I arogaiyuogu Rgezv, Soid Zrgipd Nagrog, New York (1989), and in T.). Zyiamu, M. Vegtap apa I Mu. Epatsiv,

Ehrigetepiv m/y Sepe Ryviop. Soyd brgipd Nagbrog I aroghaiogu, Soyy Zrgipda Nagrog, MM (1984) apa Bu A!yzyrbei,

E.M. egai., Sitepi Rgoiosoiv ip MoiIesshag Vioiodu, rib. Bu Sgeepe Rybiizpipud Azvos. apa UmMieu-Ipiegzsiepse (1987)).

I. The effects of synergistic resistance to the disease, which are achieved by the simultaneous introduction into plants of a chemical inducer of systemic acquired resistance and a standard bactericide

In this group of examples, ZAK was induced in plants by administration of a chemical ZAK inducer such as benzothiadiazole. In addition, standard bactericides were injected into the plants. The plants were then subjected to 7/0 disease-causing effects of various pathogens. The combination of both methods of combating pathogens (chemical inducer and bactericide) created greater than additional, i.e. synergistic, resistance to diseases. This was defined as the synergy coefficient (SE), i.e. the ratio of the studied effect (0) to the predicted effect (E).

The predicted effect (E) for a given combination of active components is described in the so-called "Colby formula" (Colby's formula) and it is calculated according to the method given in the publication |SoIru, 5.K. ". MUeeavs, Moi. 15, radez 20-22 (1967):

PP - the number of milligrams of the active component (- a.i.) per liter of the spray mixture,

X: activity (in 90), which is caused by the introduction of the active component I in the amount of p ppt,

In x9o, the activity (in Uo), caused by the introduction of the active component I in the amount of 4 ppt,

E-predicted effect of active components III as a result of their introduction in a number of ppm (additional

INFLUENCE).

Colby's (Soir) formula E-Hm-(X.y)/100.

Example 1: Effect on Eguzirpe dgatips on barley.

Residual protective effect: Barley sprouts with a height of about 8 cm were sprayed until droplets were formed with a water-based spray mixture (max. 0.029% of the active component) and dusted after 3-4 days with

Fungal conidium. Infected plants were kept in a greenhouse at 222C. Infection with the fungus was assessed after 10 days.

Systemic effect: Barley sprouts with a height of about 10 cm were impregnated with a water-based spraying mixture (max. 0.00295 of the active component, based on the volume of the soil). Special attention was paid to ensure that the spraying mixture does not come into contact with the above-ground parts of the plants. Plants were dusted with fungal conidia after 3-4 days. Infected plants were kept in a greenhouse at 2220. Infection with the fungus was assessed through "I 10 days. - cha

Effect on Eguvzirne dgatipiv on barley: component II: metconazole vv 9611 21211111 « yu v 16117171 11 Z is pli nyny ny no p o В 52712 177177yu 1 :» psy nyny ny i NO Po ov PO I

NE LY SYNY SHE NI PN PO OO in PNT NI NIH ET NE lNN NENNEYA - | 61231 81yu r, ov | 66815 o ov yo v00v50 - 51218381 z | 2 | oh lo! вв те о) а ю 13 2 20.10 79 12 th» component І!: benzothiadiazole-7-carboxylic acid component ІІ: tetraconazole and vyklin ШЙ 2 2 27 и выш ши ши 4 2 63 6 0.6 2 13 82 68 12 71 2 1 06 31) la | in c) b5

Table C

Effect of Eguvirne dgatipiv on barley component !: benzo|1,2,3Hiadiazole-7-carbothioic acid-3-methyl ester component II: metconazole 9 O (research) | E (forecast) O/E 1 0.6 (in) 2 2 Z

Z 6 17 4 20 Z bo BO 6 0.6 6 1710 Z 17 1.9 7 0.6 20 1:30 BO Z 1.5 8 0.6 bo 1:100 83 BO 1.7

Example 2: Influence on SoiIIefigispyt Iadepagicht on Sisyth occupied Yi.

After a cultivation period of 10 to 14 days, the cucumbers were sprayed with a spray mixture obtained from the composition of the moistened powder of the test compound. After 3-4 days, the plants were infected with a spore suspension (1.0x109 spores/ml) of the fungus and incubated for 30 hours in conditions of high humidity and a temperature of 232C. After that, incubation was continued under conditions of normal humidity and temperature from 222C to 2320C. The protective effect was evaluated after 7-10 days, based on the degree of infection with the fungus.

After a cultivation period of 10 to 14 days, cucumbers were treated - by injecting into the soil - with a spray mixture obtained from the composition of the moistened powder of the test compound. After 3-4 days, the plants were infected with a spore suspension (1.5xX10? spores/ml) of the fungus and incubated for 30 hours in conditions of high humidity and a temperature of 232C. After that, incubation was continued under conditions of normal humidity and temperature of 22 96 °C. The assessment of the protective effect was made after 7-10 days, based on the degree of infection with the fungus.

Table 4

Effect on Sopeifigisnit Iadepagisht on Sisytiv occupied I./Introduction into leaves component !: benzothiadiazole-7-carboxylic acid component II: azoxystrobin chI zo Test Me |mgai. per liter (ort) ve

О (research) / Е (forecast)

EI PE PO PO PO PO OH PO NA vv 15111 st-

16111701 1111615 2 891 1166000000500022 1166000000500022 1166000000500022 12 16 m | with 9 with p

Table 5 - I Impact on Sopeygisnit Iadepagisht on Sisytiv zaymiz I./Introduction into the soil of component I!: benzothiadiazole-7-carboxylic acid

OO component II: azoxystrobin test Mo |mg a. per liter (mouth) ve - ; O/E

About (research) | E (forecast) - byu | 11611 sh vv 0011011yu zv 01118111

POPI POS NI ONYA MON POLO MON NOYA

8 ny nn pis o Po i VN v vv 1181 o v yuyu p p Po PO NO ET KOH MAYA i bo | 02 | ride! mo o 9 1111 06 || with | in /zz) 121 voo a 1002 | 6 in 1802 with | Education 6 (that "ZE synergy coefficient is not calculated b5

Table 6

Effect on SoyIeigispit Iadepagisht on Sisytiv zaymiz I./Introduction into leaves component !: benzothiadiazole-7-carboxylic acid component II: kresoximmethyl

O (research) / E (forecast) O/E

METY GI PNYA POLYA PO KOH MAN av 111 b 81121961 il DE PO PO PO NO in | 02 | tho | mo

Table 7

Effect on Sopeifigisnit Iadepagisht on Sisytiv occupied I./Introduction into leaves component !: benzo|1,2,3)hiadiazole-7-carbothioacids-3-ethyl ester component II: azoxystrobin

Test Me Hmg ach. per liter (mouth) in comp. | computer II O (research. E (forecast. O/E

P rev 111611 aa 111211 vv

PI PI PE PO POLE TONYA PO MON sch pgt PI PI PO PO SONYA PO PAN o v rea rt « zo Table 8

Effect on Sopeygisnit Iadepagisht on Sysuits zaymiz I ./Introduction into the soil o component !: benzo|1,2,3)hiadiazole-7-carbothioacids-3-ethyl ester component II: azoxystrobin o

Test Mo mg a. per liter (rt) weight so

About (research) | E (forecast) oE zv byu | 011611 m 51115000 zv 01115111

PRI NE NI PO PO Z PO NOYA " du p t PI PU I POET PO AN - s pil ni PO No Po i VO 61100100 :" 1311 ev 18 o.0o6 o sh tlo y 5 ovo | viv, - SONGS SI NNYA NE NO ZI NO YAN TOWER"

Example 3: Effect on Segsozroga pisoyapae on tobacco Tobacco (6 weeks old) was sprayed with a solution of the tested compound prepared in the form of a composition (concentration: max. 0.0295 of the active component). Four days after treatment, the plants were inoculated with a suspension of sporangia of Segsozrog pisoyapae (150,000 spores/ml) and

GF) were kept in conditions of high humidity for 4 to 5 days, after which they continued to incubate in

HF of the normal sequence of light/dark cycles. The evaluation of symptoms in these tests was done on the basis of such a feature as the area of the part of the surface of the leaf infected with the fungus. because Table 9

Effect on Segsovrog pisopapaye on tobacco component !: benzo|1,2,3)hiadiazole-7-carbothioacids-3-ethyl ester component II: tebuconazole 6BE O (research) | E (forecast) O/E 1 02 (c)

Esh shi SHY Eh ZO PO OY vv |v yayu 111 ni PI 122 liu 1601 shi EN EE NE o POL S nene y shi y Eh ni zn s: LYNE yu yu 126 13 i vv. писии ни ни Пез Низ NN ЕЯ 51616 11ю0010вв6в m | lu | in 13119178 component !: benzo|1,2,3)hiadiazole-7-carbothioacid-3-ethyl ester component II: cyproconazole og | 11119611

EE li a Po Eh ZO PO PO vv sm shchi yayu 1111 o 51712119 11 pis i PO PO PO PO POIT ra tyu vvva |v tyu 1o 1 y

Sh va rayo 1tva - syu a |v 08 1tvv m mov rovy v 155 vro mm o 7 m 15 1561811 jul 16 vo t «

The effect on the Segsovroga pisopapae on tobacco with component II: fenpropimorph g" sleep inhalation diarrhea 111111 onov 1 15 Eng ny NI PO ZY NO - 6011131 s 31720181 41611 - write she shi shi shi shih i NI M -.5 rovi

NOT LY I PER BUT POLO I SWEAT IN t" in the corner language113ya 8 | 026 0610123 yur 12 zmrov m v 16 zhili |v (F, yu vo component I!: benzothiadiazole-7-carboxylic acid component II: difenoconazole o | 0105000 benthol 1 and in 2 b 73

31» 1 171 yu 1 1 write tires shi shi shi 61771761» 11 7515 3101, ev 16 | oye lo lo | in z)

Example 4: Effects on Rupschiagia gougare on rice. 70 Approximately 2-week-old rice sprouts were placed with the soil around the roots in a container filled with a spray mixture (max. 0.00695 active ingredient). After 96 hours, the rice was infected with a suspension of conidia of the fungus. Infection with the fungus was assessed after incubation of infected plants for 5 days at 95-10095 relative humidity and a temperature of about 2426. and component !: benzo|1,2,3)hiadiazole-7-carbothioacids-3-ethyl ester component II: KT 3616 at 20 17161 1 1 5 2 | |о021| | 0 | (in entered 4 | 101 | h" 51110619 with what 6 | 121 11 zv | o 71 1 71761 1 mk 816102 zom 420115 8 | 6 | person |) 76 | from 7 v yus 1o2 zov tv h 7 m | 6 | o6 || in | in | - 2 | 6 | 2 || liu | in with 161 la | vv | in iso

On a plot of land 12m? rice was sprayed with a spray mixture obtained from the moistened powder of 35 active components. The infection was natural. The assessment was carried out as follows: the area of the leaf surface infected with the fungus was measured 44 days after treatment. The following results were obtained:

Effect on Rugisshagia ogulae on rice in the open ground with component II: pyrochilone g" nen rel. Ok. 11710 here

POETS OF NGU ZY NI NOYA PO ZY POLYA NOYA

- pog NI NT No PO PO la PO A s 11111701 15111700 - vovi bj 38001058, - o v ov | let's leave! 851773 Fr

Thu» Rice sprouts about 2 weeks old were placed together with the soil around the roots in a container filled with a spray mixture. Infection with the fungus was assessed after 36 days. Infection of untreated plants corresponded to exposure to 0905. » ka component !: benzo|1,2,3)hiadiazole-7-carbothioacids-3-ethyl ester component II: tricyclazole ryu 10176111 a ov 1 1 zm 111811 ar 00111051 b5 5 1 74

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Example 5: Influence on SoiIIeyuigispyt zr. (Apipgaspose) and Segsozroga zr. (leaf spotting) on chili

Effect on yield: On a plot of land with an area of approximately 10m2 (testing place: Kikampek,

Java, Indonesia) chilies were sprayed only 7 times with intervals of approximately 7 days with 500-700 liters of the spray mixture per hectare. Three days after the first spraying, the plants were artificially infected with the fungus. determination of the degree of contamination of the surface of chili fruits after the fifth spraying component I!: benzo|1,2,3Hyadiazole-7-carbothioacid-5-ethyl ester component II: mancozeb vv 1 pis I POET I PO I VANYA sch (8) determination of the degree infection on leaves after the sixth spraying "I component!: benzo|1,2,3)hiadiazole-7-carbothioacid-3-ethyl ester component II: mancozeb "--

Sat oltinnii went pkt oyu. in 81117111 not 31611181 that "

Effect on yield: Chili fruits were harvested after the sixth spray. component II: mancozeb 80011101 iv PI NYA PEN NOYA POLYA PO PNYA - (95) Example 6: Effect on Rissipia hesopaia in wheat. - 7-day-old wheat was sprayed until droplets were formed with a spray mixture obtained from the composition of the active component or a combination of active components. After 4 days, the treated plants were infected with a fungal conidia suspension, after which the treated plants were incubated for 2 days in conditions of relative

I" atmospheric humidity 90-10095 and temperature 202C. After 10 days, fungal infection was assessed. 29 component !: benzo|1,2,3Hiadiazole-7-carbothioacid-5-ethyl ester component II: propiconazole 101 pain 01600000 22115111 65 Table 20

Effect on Rissipia hesopaia in wheat component I!: benzothiadiazole-7-carboxylic acid component II: fenpropidine 9 O (research) |E (prediction) O/E 1-11 1 (o sontol//// пе 171ю 11 2 1st|

Example 7: Effect on Eguzirpe dgatipiz in wheat In field experiments (10m?), winter wheat in the growth stage was sprayed with a spray mixture obtained from the moistened powder of the active component. The infection was natural. After 10 days, fungal infection was assessed. The following results were obtained:

Table 21

Effect on Rissipia hesopaia in wheat component !: benzo|1,2,3)hiadiazole-7-carbothioacid-3-ethyl ester component II: propiconazole

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PENYA NO THEY NO OLYA PO VO

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Table 22

Effect on Rissipia hesopaia in wheat i) component I!: benzo|1,2,3Hiadiazole-7-carbothioacid-5-ethyl ester

From component II: cyprodinil i-

Test Mo ve

About (research) | E (forecast) O/E i -1-0100000 oon « niks PO PI PO PO i PO NOI - 7 ayu 121 s 5511 ш- ввм 1 z p - 45 Example 8: Effect on Musozrpaigeiia Piepviz in bananas. banana plants on an area of 300m2 were sprayed at intervals of 17-19 days with a mixture for spraying (95), obtained with a moistened powder of the active component, a total of 6 times. Infection was -1 natural. For evaluation, the area of the part of the leaf surface infected with the fungus was measured. The following results were obtained: - 70

Table 23 "from" vol

Effect on MusozrNaegeiPa Piepviv in bananas component I!: benzo|1,2,3Hyadiazole-7-carbothioacid-5-ethyl ester component II: propiconazole o O (research) / E (prediction) O/E 11 oyu yu 01800000 11170101 for

Example 9: Effect on AGegpagia zoiapi in tomatoes.

Tomatoes on a plot of 7m? sprayed at intervals of 7 days with a spray mixture obtained from the moistened powder of the active component, a total of 9 times. The infection was natural. To estimate 65, the area of the part of the leaf surface infected with the fungus was measured. The following results were obtained:

component !: benzo|1,2,3)hiadiazole-7-carbothioacid-3-ethyl ester component II: cyprodinil

FULL PAYMENT NOYA PO ZY NOYA PA vv 1011111 sh 18115001 1» | wow! at the 17th o

Example 10: Effect on Rpuorpyoga ipTeviapz in tomatoes.

Tomatoes of the cultivar "Koieg Spot" were sprayed until the formation of drops with a spray mixture obtained from the composition of the active component or a combination of active components. After 4 days, the treated plants were sprayed with a sporangium suspension of the fungus, after which they were incubated in a chamber for 2 days at 18-209C and a relative atmospheric humidity of 90-10095. After 5 days, fungal infection was assessed. The following results were obtained: component !: benzo|1,2,3)hiadiazole-7-carbothioacids-3-ethyl ester component II: metalaxyl He 7 shitrenno | from Hepktnu 9. o 1 st vm 55117101 « zo 16111601 byu 10171781 - 51511800 m piti she I Pn o VO shryu 117v011000001 z 17770911 83011001 m vv vm) jus 20, « component 0 v |v 1 z0 yu 29 !: benzothiadiazole-7-carboxylic acid -1 component II: metalaxyl -o give

EE PI PO PO AND PO NA

"415 | in 1 o UNIVERSITY OF POETS PO PO EN PON MA yuyu 61 191 with 1 row 111 vo el | in 1 in rom yur v61zlv 65

Example 11: Effect on Rzeidoregopozrog sibrepviv in cucumbers.

Cucumbers aged 16-19 days ("M/izsopvzip") were sprayed until the formation of droplets with a spray mixture obtained from the composition of the active component or a combination of active components or a combination of active components. After 4 days, the treated plants were infected with sporangium Rzeidoregopozroga sirepezzhmaz (strain 365,

Grey; max. 5000 per ml), after which the treated plants were incubated for 1-2 days at 18-20 2С and relative atmospheric humidity of 70-9095. After 10 days, fungal infection was evaluated and compared with infection of untreated plants. The following results were obtained: component I!: benzothiadiazole-7-carboxylic acid component II: metalaxyl

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Example 12: Impact on Regopogzrog (arasip on tobacco. o

Tobacco (aged 6 weeks) was sprayed with a solution of the tested compound obtained in the form of a composition. Four days after treatment, the plants were inoculated with a sporangium suspension of the fungus, kept in conditions of high humidity for 4 to 5 days, after which they continued to incubate in the normal sequence of light/dark cycles. The evaluation of symptoms in these tests was done on the basis of such a feature as the area of the part of the surface of the leaf infected with the fungus. Infection of untreated plants corresponded to exposure to 090. M no

Effect on Regopovroga iabasip on tobacco component II: dimethomorphine NG ONY PI PO UUAYA POH M h a mm o s vv y vv g» POVY NOYA PNYA NOYA PO UA NOYA MOYA in o5 15) m01v in ih I HNY VIS ET NO NO PE - in ohm | Example 13: Effect on Rhegopozroga ragaziis in Agabridorvzis (Naiiapa. -I Fungicides metalaxyl, fosetyl and copper hydroxide, as well as the activator AK tsu benzo(1,2,3)-thiadiazole-7-carbothioic acid Z-methyl ester ( VTN), obtained with a concentration of 2595, 8095, 70905 and 2595 of the active component (ai), respectively, together with the carrier in the form of a moistened powder, were administered in the form

Chl" of finely dispersed aerosol in the leaves of a three-week-old plant. For control, only the moistened powder itself was injected. After three days, the plants were inoculated with a suspension of conidia of Regopozrog ragaziiis, as described in

IOeiapeu ei ai. (1995)). "Muz" plants were inoculated with a compatible isolate of R. ragaziis Etma (1-2x10 5 spores/ml);

"Soy" plants were inoculated with a compatible isolate of R. ragaziis Moso2 (0.5-1x10 5 spores/ml). After inoculation

Gee! plants were covered to maintain high humidity and placed in a Percival chamber for cultivation at 1720 with a light/dark cycle of 14 h/10 h (OKpez ei a!., 1993). The tissue was collected 8 days after inoculation. o The progression of the fungal infection was followed for 12 days by studying under an analytical microscope, quantitatively evaluating the development of conidiophores in this way. (1994); Oieyis, egai. 60 (1994)). To study the growth of the fungus in the leaf tissue, individual leaves were stained with lactophenol trypan blue. Fungal growth was quantified using a fungal rRNA probe, which was obtained by PCR according to (Mupye et al. (1990; RSK Rgoiosoyiv: A dicide juyu Meiyodz apa Ariisayop, 315-322)3), using primers M51 and M52 and DNA Etumua with R. ragaziis as matrices. RNA was isolated from frozen tissue by extraction using phenol/chloroform after precipitation with lithium chloride (I adgitip eiye ai, 1987: bo RMAB, 84: 7542-7546). Samples (7.5 μg) were separated by electrophoresis through formaldehyde-agarose gels and subjected to blotting analysis on nylon membranes (Nuropa-Mzh, Ategepat) as described in |A!gzBbeyi ei ai!. (1987)|.

Hybridization and washing procedures were performed according to Spigsy apa siireg (1984, РМА5, 81:1991-1995)).

The relative amounts of the transcript were determined using the RIozrpog Itadeg device (MoYiIesshag Bupatisv,

Sunnyvale, CA) according to the manufacturer's instructions. The sample loading was optimized by

Probing scraped from filter blots with constitutively expressed βB-tubulin cDNA from Agaridors.

Infection of untreated plants corresponded to 095 inhibition of fungal growth. The following results were obtained: component I!: benzo|1,2,3Hyadiazole-7-carbothioacid-5-ethyl ester component II: metalaxyl

Test Mo Synergy factor O/E those vimy 316011 and 1 I with boimmi ol | yum 110015 and component I!: benzo|1,2,3Hyadiazole-7-carbothioacid-5-ethyl ester component II: metalaxyl

Test Mo Synergy factor O/E calculated:

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Gee boozmm| vm | lu | 75 | | oh -

Effect on Etzh Regopozrog ragavziis in Agaridorvy INaiyapa (M/v): component II: fosetyl

Test Mo Synergy factor OB no) with all 10010960 " 0161 51 memory 0100000000000 8 1111oamou 111

B. Add more to the bottom of the bottle: 025 - 50 t" component !: benzo|1,2,3)hiadiazole-7-carbothioacid-3-ethyl ester component II: copper hydroxide

Test Mo Synergy factor O/E o ne ele Z NN yuyu t! 1161 forehead 11111 vo a frobotmya6 11

As can be seen from Table 29, the effect of synergistic resistance to diseases is observed in natural plants

Agaridorziz (SoiI-0). Inhibition of the growth of the fungus was not detected as a result of the separate introduction into plants of either 65 0.01 mM VTN or metalaxyl (0.0001 g/l), since such concentrations are usually insufficient for the desired effectiveness. However, as a result of the introduction of both of these compounds into plants in similar concentrations insufficient for separate standard treatment, 40,795 inhibition of the growth of the fungus was found, which is an obvious synergistic effect. Tables 30-32 show the effect of synergistic resistance to diseases in natural plants of Agabridorz ("M/v"). Only 20-30905 inhibition of the growth of the fungus was detected as a result of the introduction of 0.01 mM VTN into "Muv" plants. However, as a result of the simultaneous introduction of VTN into plants, as well as metalaxyl, fosetyl, or copper hydroxide, synergistic resistance to diseases was revealed. This combined antifungal effect, by which the effective concentration of fungicide and VTN required to control the pathogen can be reduced, reduces the dose of chemicals required to stop fungal growth and thus reduce the likelihood of leaf damage through normalization of chemical tolerance. 70 PIP. Synergistic effect of resistance to diseases, which is achieved by introducing standard bactericides and/or chemical inducers of systemic acquired resistance in mutant plants with constitutive immunity (CIM)

In this set of examples, a high-throughput northern blotting filter was used to identify mutant plants that contained high concentrations of PKA-1 mRNA during normal growth, despite the fact that these mutants also exhibit systemic acquired resistance. With the use of this filter, several mutants were isolated that accumulated not only PkE-1 mRNA, but also REK-2 and RI-5 mRNA (I. (1995)). Such mutants also have increased ZA concentrations and are resistant to pathogen infection, confirming that this approach can be applied to BAC signal transduction mutants.

With the use of this filter, two classes of mutants were distinguished regarding the signal transduction of ZAC. One of the classes was named "Iza mutants" (Iza-disease with lesion modeling). This class of mutants is also referred to as "Class I Sith" as described in (MO 94/16077), the descriptive part of which is referenced in its entirety herein. This class Iza (also known as Class I site) was characterized by the formation of spontaneous lesions on leaves, the accumulation of high concentrations of ZA, high levels of RE-1, RK-2 and RK-5 mRNA, and resistance to fungal and bacterial pathogens. ., 1994; Mueutapp eyi a!., 1995).

A second class, called sit (sit-constitutive immunity), is described later in the text and has all the features of i) Is mutants, except for spontaneous lesions. This second class (sit) corresponds to the mutants of "Class ІІ sit" considered in (М/О 94/16077)І. Mutant plant line sitZ, which is described further in the text, belongs to the mentioned class of sit (Class II sit) and is a dominant mutation with natural properties that allow "gzo" to express stable, increased concentrations of ЗА, mRNA of the 5AK gene and are characterized by resistance to a wide range of spectrum of diseases. --

Example 14: Isolation and characterization of sit mutants with constitutive expression of AK genes. M 1100 individual mutagenized M2 (EM5) Agabidorziz plants were grown in Agasop trays (I epie Zeeaz, Round

Rock, Texas) in groups of about 100. Plants were grown as described in (OKpez ei ai., 1993), zirga, me) z5, and special attention was paid to avoid oversaturation with water and infection with the pathogen. To describe it briefly, Meigo Mich 360 was saturated with water and sterilized in an autoclave three times for 70 minutes in portions of 10 liters. The treated mixture was well mixed after treatment in the autoclave of each subsequent portion. Seed surfaces were sterilized at 2095°C for 5 minutes and washed seven times with fresh distilled water before sowing. The planted seeds were germinated for 3-4 days, after which they were grown in chambers with a light/dark cycle of Ugod/15h at 222. When the plants reached the age of three to four weeks, one or two pt) with leaves weighing from 50 to 10Omg were selected, and total RNA was isolated using the express technology for the preparation of small amounts of RNA | Mepmoega ei ai. (1989) Cape. Asii Kev. 17, 2362). The expression of the RK-1 gene was studied using northern blotting analysis and its adgitype. (1987) Rhos. May Asad All together. OBA 84, 7542-7546; Magician. 1991). Each group of plants also contained untreated A. Naiapa (CoI-O) and 2-day-old control plants treated with IMA (0.25 mg/ml). All plants were stored as described in M/eutapnp ai!., (1995)).

80 possible mutants were identified, which accumulated increased concentrations of RA-1 mRNA. After testing the offspring, five of them were selected for further characterization. Increased expression of ZAC genes in the absence of a pathogen or treatment with inducers was determined in possible mutants of -I net. Pz testing of progeny of possible sit mutants confirmed that constitutive expression of RK-1 is inherited. We continued to determine the characteristics of two sit mutants, sit2 and sit3, with the highest and most stable RA-1 expression.

Ta» During backcrossing with SoYishtbria, the recessive trait of "smoothness" was used - as a marker for the identification of E1 descendants. Buds of flowers SoI-dIi! were desexed before pollen emergence, and pollen from the mutants was injected immediately and the next day. E1 plants were grown in soil, and plants with special characteristics resulting from crossing were identified by the presence of trichomes.

After crossing sit2 and sit3 with the SoI-O or I a-eg ecotype, a large proportion of E1 plants with (F, high expression of BAC genes) were found, confirming that these traits were dominant. In the case of sit2, some, but not all, E1 plants had constitutive expression of BAC genes. A similar result would be expected if the sit2 mutants were dominant and contained as a heterozygote in the parent plant. Further genetic testing of sit2 revealed persistent variant segregation in the E2 generation due to incomplete penetrance.

For sitZ, 1:1 segregation was found in the E1 generation, as a result of which two separate E1 plants expressing a high level of RA-1 mRNA self-pollinated to form an E2 population. Segregation of E2, which was quantified by counting accumulated RE-1 mRNA, was characterized by the fact that 93 b5 E2 plants had a high content of accumulated RE-1 mRNA, and 25 G2 plants had an insignificant content of accumulated

RA-1 mRNA, that is, the ratio was 3.7:1 (c2-1.77; 0.5»Р»2О 1), which is consistent with the hypothesis that sitZ is a dominant, single gene mutation. Further selection by crossing confirmed that sit3 was inherited as a dominant mutation.

In the case of sitZ3, primary M2 plants were detected during screening, and the MZ population was detected with a normal appearance. However, because the sit3 plants were self-pollinated, some of the high-expressing lines had poor progeny reproduction. After backcrossing with

SoI-d11 plants with normal external signs and the ability to reproduce, as well as with high expression of RK-1, were obtained.

At the time of initial identification, sitZ was also noted for some signs of stunting 7/0 (dwarfism) and thin coated leaves. However, G2 plants, obtained as a result of crossing with the Co1-ID1 ecotype, kept a high expression of ZAC genes and did not differ from natural plants. This confirms that the phenotype with signs of dwarfism and covered leaves was formed as a result of an independent mutation not related to the constitutive expression of ZAK genes. The sitZ3 mutant phenotype was also detected when the plants were grown under sterile conditions, and this fact confirms that the accumulation of PE-1 mRNA was not caused by the pathogen.

Example 15: AK gene expression.

In addition to RKE-1, two other BAK genes, REK-2 and RE-5, are also highly expressed in sit3. The expression levels of BAC genes in different progenies differed from each other, but were always at least 10-fold higher than the expression levels in control plants and were of the same order as the levels obtained in 2o treatment of natural plants with IMA (0, 25mg/ml) with induction of resistance.

Example 16: Analysis of salicylic acid content.

It was found that endogenous concentrations of ZA increase as a result of pathogen-induced necrosis

Agaridorziz (OKpez ei aiYi.,, 1993, zirga). Salicylic acid and its glucose conjugate were analyzed as described in (OKpez ei ai., 1993). Leaf tissue was collected from 10 sytZ plants and 10 control 4-week-old plants. Leaves from individual plants were collected and analyzed to determine the expression of the RK-1 gene. ZA levels were measured in plants expressing RK-1. The concentration of free 5A in sit3Z was 3.4 times i) higher than in uninfected natural Agaridorvziz (233-435 in comparison with 69-8ng/g of raw material weight, respectively). The content of glucose conjugate ZA (ZAS) was 13.1 times higher in sit3 than in uninfected natural

Agaridorziv (4519-4473 uv. 34458ng/g weight of raw substance, respectively). These increased levels of BA and ALS are of the same order of magnitude as levels known for either pathogen-infected tissue or the srg mutant.

Example 17: Resistance to disease. -- sitZ3 was evaluated for resistance to Regopozrog ragavziis (MoSog2), the agent that causes the disease of powdery mildew in Agaridorziz. Thirty seedlings (confirmed by RKE-1 RNA expression) and thirty control plants (ecotype Soyshtbia), each approximately 4 weeks old, were inoculated with R. ragaziis at 3-5 as described in (OKpev, ei aYi. 1992, zirga). Seven days later, the plants were analyzed for sporulation and stained with trypan blue to visualize fungal structures as described in Ikeop et al. (1980)

Thapv Hg. Moses wasps 74. Growth of hyphae, conidia, and oospores was observed in natural (CoI-0) plants, while in natural plants treated with "

IMA (0.25 mg/ml), and no fungal growth was detected in sitZ plants. Conditioned sIT resistance is usually manifested in the form of a small group of dead cells at the site of pathogen infection. This type of resistance is similar to that observed in mutants of Iza |Oieifispy ei ai., 1994, zirga; Mu'eutapp ei ts aI.,, 1995, zirga| or in natural plants in which ZAK was induced (OKpez ei ai., 1992, zirga). Phenotypes with intermediate resistance were occasionally observed, including terminal necrosis in the hyphal tip trace in sit3 plants. This terminal necrosis is similar to that observed in wild-type plants treated with low doses of ZA or IMA (OKpez et al., 1992, zirga ; OKpez ei ai., 1993, zirga|i. However, no sporulation was detected on sit3 -I plants at all, while sporulation was observed on control plants. During trypan blue staining, no spontaneous lesions were detected on the leaves of inoculated sit3.

Mn In addition to resistance to the fungal pathogen R. ragaziisa, sitZ3 also have resistance to infection with the -I bacterial pathogen Rzeidotopaz zuhipdae OS3000. Six-week-old wild-type plants (4 IMA treatment) shu 50 and sitZ plants were inoculated with a suspension of R. zuhipdae OSZ000, and disease progression was monitored by regulating the growth of bacteria extracted from infected leaves over time. Difference between

ЧТ» by bacterial titers in SoI-O, SoI-ОЖИМА and sit3 both on day 0 and on day 2 was statistically insignificant.

However, on the fourth day, a 31-fold decrease in the growth of the bacterial colony was observed in natural plants in relation to sitZ plants (Р«0.003; oka! apa Kopiai, 1981). Plants were also subjected to visual analysis for disease symptoms. Leaves from natural plants had a very pronounced chlorotic coloration, and the symptoms of the disease spread far beyond the initial zone of infection. In contrast, both wild-type plants pretreated with IMA and sit3 plants had virtually no disease symptoms.

For this example, the strain OSZ3000 tomato Rzeidotopaz zugipdae ru. were grown on King's B 60 medium (Kipd'z B teadia) (agar plates or liquid medium) plus rifampicin (5 Ωkg/ml) at 282C. (1991) Riapi Seii 3, 49-59). The culture kept for a day was diluted and re-transformed into a suspension in 10 mm of MdeYi, bringing the density to 2-BH102 cells per ml, and injected into Agabidorzi leaves. The introduction was carried out by creating a small hole with a special Mo28 needle in the middle part of the upper surface of the leaf, into which about 250 μl of a diluted solution with bacteria was actually injected using a syringe with a capacity of 1 cm3. At different points in time, 10 samples were taken randomly from 10 plants after each treatment, consisting of randomly selected pieces of leaf tissue punched out with a special Mo drill. The punched-out pieces of the leaf were placed in an Eppendorf tube (Errpaogy Schre) with ZOOmkl 10MM Mdsi" and rammed with a pestle. The resulting suspension of bacteria was appropriately diluted, placed on

King's B medium plus rifampicin (5 Ωkg/ml) and grown for 4 days at 282C. Bacterial colonies were quantitatively analyzed and the data were subjected to Student's statistical analysis (|Ziydepev her viaiviiisa! apaiuvziv) (oka! apa Kopii (1981), Vioteigu, 2p4 ed. Mem Mogk: MU.3. Egeetap apa Sotrapu).

Also for this example, 2,6-Dichloroisonicotinic acid (IMA) was transformed into a suspension in sterile distilled water with a content of 25956 of the active component, obtained as a composition in wetted powder 7/0 (0.25 mg/ml, 325 mM; (Kevzvtapp et al. ( (1994) App. Chem. RnuyuoraiiNoi. 32, 439-591). All plants were sprayed with water or IMA solutions with the obligatory achievement of such a state when the liquid began to flow.

Example 18: Role of 5A in BAC gene expression and disease resistance.

To study the relationship between ZA, expression of BAC genes and resistance in sit3, crosses were made with Agaridorziz plants expressing the salicylate hydroxylase (paps) gene (OeiYapeu, 1994). These "75 Mayo plants" were created by transforming gene-driven 355 papob into Agaridorziz. using mediated Adhorasiegisht transformation. See publication (Nyapo, 3. Ma, N. (1992) Riapi Moi. Vio1!.

Ker. 10, 372-383, which is referenced in this description; Saipeu, ey a). (1993) Zsiepse 261, 754-756) to which reference is made in this description; and |Oeiapeu, ei a). (1994) Zsiepse 266, 1247-1250), to which reference is made in this description. Agaridorrhiza with CoI-payo contains a dominant gene for resistance to kanamycin in addition to the dominant pans gene, so CoI-payo was used as a pollen donor. E1 seeds were hydrogenated in water for 30 minutes, then surface sterilized at 1095 Siogoh, 0.0590 Tmeep 20 for five minutes and rinsed well in sterile water. The seeds were placed on a medium for germination (SM medium, Migazpide and 5Kos09d, containing sucrose at a concentration of 10 g/l, buffered with 2-(M-morpholino) ethanesulfonic acid (0.5 g/l), pH 5.7 with KOH), which contains kanamycin (25 mg/ml), for the purpose of selecting plants E,. See |MayimeKepz ei ai. p. (1988) Rhos. May Asad Zsi, OBA 85, 5536-5540). E plants resistant to kanamycin were transferred to the ground after 18 days. The presence of the pai gene and the expression of RK-1 were confirmed in all experiments using Northern blot analysis.

Since both sit3 mutant and papo phenotypes are dominant, epistasis between these two genes is analyzed in E1 plants. Seventy E1 plants from the cross of sit3 X papio were analyzed for RKE-1 and the expression of the Izo papo gene. In the northern blotting analysis of mRNA expression, the presence of the paps gene was correlated with suppression of the expression of ZAC genes. The presence of cIT in each ET was confirmed by quantitative assessment of RAK-1 mRNA in the obtained E2 segregation products. uh-

In order to determine whether the sitZ mutation was epistatic to papio for disease resistance, 5 E1 plants from the sitZ3 X papb cross, which confirmed the presence of paro and the absence of RK-1 mRNA, i. self-pollinated and planted 20-30 G2 seeds. The expression of PAI and RE-1 mRNA was analyzed in individual R. plants from this E2 population, which were then subjected to control infection with R. ragavziis (MoCo2) in order to assess their susceptibility to the disease. Disease resistance created by sit3 was removed by the presence of the paps gene, on the basis of which it can be concluded that papo is epistatic to sit3Z with regard to the expression of ZAC genes and resistance to disease phenotypes. "

Example 19: Synergistic resistance to diseases, which is created with the help of the introduction of bactericide 8 s and/or VTN in mutants of c. y Three days before the inoculation of the pathogen, a chemical inducer of systemic acquired resistance (benzo!|1,2,3)hiadiazole-7-carbothioic acid-5-methyl ester) prepared in in the form of a composition of the active component (a.i) in a concentration of 2595 with a moistened powder carrier (Metgaikh et al., 1991) and/or - and the bactericide metalaxyl (СОА 48988), prepared in the form of a composition of the a.i. in a concentration of 2595, or only with moistened powder. Plants were inoculated with a suspension of conidia (1.8xX109 spores/ml) of a compatible pathogen on MoCo2 from Regopozrog ragaziiis. After inoculation, the plants were covered to maintain high humidity and placed in a Percival chamber (RegsimaI) for cultivation at 17 C with with a light/dark cycle of 14 h/10 h -l 20 (Okpez ei a/i., 1993).The tissue was collected 8 days after inoculation.

The growth of fungi was determined using a probe of fungi from rRNA, which was obtained by RSK according to (M/pie chz» ei aI. (1990; RSK Rgoiosoiiv: A dside yuyu Meijposdz apa Arriisayop, 315-322), using as a matrix primers M51 and M52 and Etum R. ragaziis DNA RNA was isolated from frozen tissue by phenol/chloroform extraction after lithium chloride precipitation (Aii, 1987: РМА5, 84: 7542-7546).

Samples (7.5 μg) were separated by electrophoresis through formaldehyde-agarose gels and subjected to blotting analysis on nylon membranes (Nubopa-mya, Ategepat) as described in IA!zrbei ei a. (1987)). Hybridization and washing procedures were carried out according to Spigsp apa sire (1984, RMAB5, 81:1991-1995). Relative ime) transcript amounts were determined using a Riozrpog Itadeg device (MoiIesshag Bupatisv, Sunnyvale, California) according to the manufacturer's instructions. Sample loading was optimized by probing the blots of constitutively expressed βB-tubulin cDNA scraped from the filter from Agabridorziz. Infection of untreated plants corresponded to 095 inhibition of fungal growth.

Application of only metalaxyl itself, only the "plant activator" VTN itself, or both metalaxyl and

VTN to the sit3 mutants described above created a greater than additive, i.e. synergistic, effect of disease resistance. This effect was defined as the synergy factor (SE), which is the ratio of the obtained effect 65 (90) to the predicted effect (E). The following results were obtained:

Table 33

Effect on Rhegopozrog ragavziis in Agaridorvy component І: mutation sіtС component ІІ: metalaxyl

Test Mo| Components | inhibition of the growth of the fungus 95 Synergy factor O/E

About (research) | E (forecast) 016110 and 19501

With m 12.5 mg/l

Femt b and vyv avmu va» 25000056 in eta om | eyelash 125 | 26

MIe-natural SoI-O

MO-undefined

Table 34

Effect on Regopozrog ragavziis in Agaridorvy component !: mutation sitZ with component II: VTN

And I

Test Mo) Components | Inhibition of fungal growth 95 Synergy factor O/E Ge)

About (research) | E (forecast) volume 33195101

ANNE EI NOYA POL ch zmu vi 10000 vomvvvimi i 000 -

Xia mm 901 shcha in vtvossmm| you 13000022 Fo and -

MIe-natural SoI-O

MO-undefined

Table 35 shsh s Impact on Regopozrog ragavziis in Agaridorvy component !: mutation sitZ :z» component II: VTN and metalaxyl (M)

Test Mo| Components Inhibition of fungal growth 95 Synergy factor O/E vtn-M O (research) | E (forecast) sh tim 0-06 woodpecker at 2. | m Ivtn boiMM i 100 - t M O, 5mg/l

With m |VTN b O1MM k 40.7 -oUu 70 i M b mg/l

A m |VTN b O1MM k IKIv) "z" k M 0.O2mg/l 5 sit3|VTN 0.01MM x IKIv) 100 IKIv) t M O.5mg/l sit3|VTN 0.01MM x 100 53.2 1.9

M b O1 mg/l o gm sogmti I

M 0О2mg/l ko

MIe-natural SoI-O 60 MO-undefined

As can be seen from the above tables, the effect of synergistic resistance to diseases was manifested in sitZ3 plants by introducing only metalaxyl itself, by introducing only VTN itself and by introducing metalaxyl and VTN in their combination. For example, 12.5905-th inhibition of fungal growth was found in an untreated sytZ plant compared to an untreated natural plant; from this, it can be concluded that the constitutive expression of BAC genes in the sit3 mutant is correlated with disease resistance. However, as can be seen from

Table 30, by introducing metalaxyl at a concentration of 0.0001 g/l (concentration usually insufficient to achieve the required efficiency) into an immunomodulated (with "on" ZAC) plant sitZ3, the investigated level of inhibition of fungal growth was increased to 57.895. A synergy factor of 4.6 calculated from these data clearly indicates a synergistic effect achieved by introducing the bactericide into the immunomodulated plant.

The data presented in Table 31 indicate that synergy is also achieved by introducing a chemical inducer of systemic acquired resistance, such as

VTN. For example, in natural plants, a concentration of 0.03MM VTH is usually insufficient to produce effective disease resistance, as it provided only 20.895 inhibition of fungal growth. However, in 7/0 plants, this normally inadequate concentration of BTN provided 73.19% inhibition of fungal growth, which is roughly equivalent to the level of inhibition provided by 0.1 mM BTN, i.e., the recommended concentration for efficacy. The synergy factor of 2.2, calculated from the data in Table 31, clearly indicates that a synergistic effect is achieved by introducing VTN into a plant that is already immunomodulated by other means.

The effect on disease resistance was even more critical when both VTN and metalaxyl were injected into the sit3 plant. As can be seen from Example 13 (Table 29), by separate introduction of either 0.01mM VTN or metalaxyl (0.0001g/l), inhibition of the growth of the fungus in natural plants is not achieved, since such concentrations are usually insufficient to ensure effectiveness. However, by introducing both of these compounds into plants together in a usually insufficient concentration, 40.79% inhibition of fungal growth was achieved, which is a synergistic effect with respect to natural plants. In sitZ plants, the simultaneous introduction of 0.01 mM VTN and metalaxyl at a concentration of 0.0001 g/l leads to 10095th inhibition of the growth of the fungus, which clearly indicates even additional (total) synergistic activity.

Thus, the combined use of immunomodulated plants with low, usually ineffective, concentrations of chemical agents to achieve disease resistance provides numerous advantages that are obvious to agronomists. Typically, toxic or otherwise undesirable concentrations of chemical agents can be avoided by taking advantage of the synergistic effects disclosed in this i) description. In addition, thanks to the reduction of the consumption of chemical agents necessary to ensure a given level of plant protection, an economic effect can be obtained.

Sh. Synergistic effects of resistance to the disease, which are achieved by introducing standard bactericides and/or chemical inducers of systemic acquired resistance into transgenic plants containing forms of the MIM1 gene. -

The MIMI1 gene is the main component of the systemic acquired resistance (SAC) pathway in plants (Kuaiv ei a/., 19961. M

The MIM1 gene is associated with the activation of 5AK by chemical and biological inducers and, together with such inducers, is necessary for BAC and the expression of ZAC genes. The position of the MIMI gene was determined by i) molecular biological analysis of the genome of mutant plants known to have a mutant pit gene, which renders the host plants extremely sensitive to various pathogens and renders them unable to respond to pathogens and chemical inducers of BAC . The natural MIM1 gene from Agaridorziz was mapped and sequenced (SEQ ID No. Mo:1). The natural gene product MIMI1 (POSL. ID Mo:2) is included in the classical signal transduction pathway, which leads to the formation of both 5AK and resistance to the disease "gene-for-gene" in Agaridorvziz (Kuaiv « 2a, 1997). Recombinant overexpression of the natural form of MIM1 leads to the formation of immunomodulated c plants with a constitutive immunity (CIM) phenotype and, thus, creates resistance to diseases in transgenic plants. Increasing the levels of the active protein MIMI creates the same effect of resistance to ;" diseases, as well as chemical induction with the involvement of such chemical agents as VTN, IMA and ZA. See |joint US patent application, registration number 08/880,179), to which reference is made in this description.

In addition, it was found that the MIMI gene product is a structural homologue of factor I! kv, subclass oso, -and signal transduction in mammals |Kuaiv ei aiYi., 1997). Mutations of Ikv acting as super-repressors or dominant-negatives of the regulation scheme ME-k«V/Lkv have been described. Thus, certain modified forms of MIM' act as dominant-negative regulators of the signal transduction pathway of the ZAC. These altered forms of MIMI create - And the opposite phenotype in plants transformed by them as a mutant pisha; i.e., immunomodulated plants transformed with altered forms of MIM/1 are characterized by constitutive expression of BAC genes and phenotype

SEVEN. See (joint application PCT "METNOO5 ORE OBIMO TNE MIMI SEME TO SOMREK SBIZEAZE KEZIZTAMSE IM

CHT" RIAMTZ"I, the reference to which is given in this description.

Example 20: Transformation of plants with cosmid clones containing the natural MIM1 gene.

Cosmid 07 (deposited by ATCC on September 25, 1996 as ATCC 97736) was created from a clone that spans the region of the MIM'1 gene and thus includes the native MIM1 gene (SEQ ID No:1). Cosmid E1 was also created from a clone that spans the region of the MIMI1 gene and thus also includes the natural MIMI gene (SEQ ID No. Mo:1). i) Cosmids 07 and E1 were introduced into Adgobrasiegisht (Steitasiepe ASI-1 by conjugative transfer during ime) of a triparenteral cross with the helper strain HB101 (rKK2013) as described in |(US patent application

Mo 08/880,179). These cosmids are then used to transform a kanamycin-sensitive mutant line of sweat! with 60o Agaridorziz using vacuum infiltration (|Mipahypoz ei ai., 1994, Seiyi 78, 1089-1099). Seeds from the infiltrated plant were collected and allowed to germinate on OM agar plates containing kanamycin (5Omg/ml) as a selection agent. The rings that survived the selection were transferred to the ground about two weeks after being placed on the plates.

Plants transferred to soil were grown in a phytotron for approximately one week after transfer. 65 ZO0OMM of IMA was injected in the form of a finely dispersed aerosol until the plant was completely covered with it using a chromister. Two days later, the leaves were collected for RNA extraction and RK-1 expression analysis. The plants were then sprayed with Regopozrog ragaziiis (EtuUM isolate) and grown under conditions of high humidity in a growing chamber at 1929 day/172C night temperatures and 8h/1bh light/dark cycles. After eight to ten days after infection with the fungus, the plants were qualitatively and quantitatively evaluated for a positive or negative result regarding the growth of the fungus. Plants Moose and sweat! treated in the same way as controls for each experiment.

The total amount of RNA was extracted from the collected tissue using the extraction buffer

PeSiIphenol | Megmoega ei ai., 1989, Mys Asia Kez, 23621. RNA samples were processed on a formaldehyde-agarose gel and blotted on SepeZsgeep Ries (YuRopO) membranes. The blots were hybridized with a labeled ZR cDNA probe RE-1. 70 The resulting blots were exposed to a film with in order to determine which transformants are capable of inducing expression

RA-1 after IMA treatment.

To see if MIMI overexpresses any of the 07 and ET transformants due to insertion site (position) effects, primary transformants containing cosmids 07 or E1 were self-pollinated, and seeds were collected 12. Seeds from one E1 line and 95 lines 07 was sown on the soil and grown as described above. 75 When T2 plants had at least four normal leaves, one leaf was collected from each plant. RNA was extracted from this tissue and analyzed for the expression of RK-1 and MIM1. Plants were then inoculated with R. ragavziis (EtuUmMa) and analyzed for fungal growth 10 days after infection. Some of the transformants showed less than normal growth of the fungus, and in four of them, namely lines p7-2, 07-74, 07-89 and E1 -1, the growth of the fungus was not visually manifested at all. Plants in which the expression of MIMI1 and

RE-1 exceeded the normal and were characterized by resistance to the fungus, were evidence that overexpression

MIM1 creates resistance to diseases.

Example 21: Overexpression of MIM1 under the influence of its native promoter.

Plants constitutively expressing the MIM1 gene were created as a result of transformation of natural Mus plants with the genomic fragment VatNi-Nipany MmIM1 (SEQ ID No. Mo: 1 - bp. 1249-5655), containing 1.4 kbp. SU promoter sequence. This fragment was cloned in раОсСО01 and transformed into strain Adgobasiegiit o 53101 |РМРОО, Kopsz apa zZsenpei! (1986) Mine. Sept. Sepei 204:383-396). "MuUv" plants were infiltrated as previously described. The obtained seeds were collected and placed on ZM agar containing kanamycin (5 µg/ml). Surviving sprouts were transferred to soil and tested as described above for resistance to Rhegopozrog ragavziis, isolate

Etula. The selected plants were self-pollinated, and the next two consecutive generations were selected for the purpose of creating homozygous lines. Seeds from these several lines were sown in soil and 15-18 plants from each line were grown for three weeks and retested for resistance to Ethmzma without any prior treatment with a chemical inducer. Approximately 24 h, 48 h and five days after fungal treatment, the tissue of each line was collected, placed in a tank and frozen. Plants were left in the growth chamber for up to ten days after inoculation, after which they were subjected to quantitative analysis for Ethma resistance. at

RNA was prepared from all collected samples and subjected to analysis as described above |Oeiapeu ei ai, 1995)|. what-

The blot was hybridized with the Agaridorziz RK-1 gene probe |OKpez ei a), 1992). Five of the 13 analyzed transgenic lines were marked by an early induction of RK1 gene expression. For these lines, RK-1 mRNA was detected 24 or 48 h after fungal treatment. Fungal growth was also not visually evident in these five lines. The leaves were stained with lactophenol blue, as described in (Oieyispy, 1994), in order to check the absence of fungal hyphae in the leaves. In the other eight lines, the expression of the RK-1 gene was not induced after 48 h, and these plants had no resistance to Etm. A subgroup of resistant lines was also tested for increased resistance to the bacterial pathogen, "Rzeidotopaz zugipdae OSZO000", in order to evaluate the spectrum of proven resistance, as described in (OKpez ei ai. (1993)).

The experiments were carried out almost as described in (Gamzhmiop eyi a). (1996)). Bacterial growth was slower in those lines that were also constitutively resistant to Etm. From here it is possible to draw - And the conclusion that plants with overexpression of the MIM1 gene! under the influence of the native promoter have constitutive immunity against pathogens. o In order to study the overall characteristics of the SIM phenotype in these lines, uninfected plants are evaluated for both free and glucose-conjugated salicylic acid, and the leaves are stained with lactophenol blue, which allows assessing the presence of microscopic lesions. Resistant plants are sexually crossed with ZAK mutants such as maps and pa to establish epistatic relationships of the resistance phenotype with “with” other mutants and to assess how these dominant-negative MIMI mutants may affect salicylic acid-dependent feedback loop.

Example 22: Overexpression of MIM/1, which is stimulated by 355.

The full-length MIM1 cDNA (SEQ ID No: 6) was cloned into the Esok!I pSOM1761 site of the EMH ISotai ei ai. (1990) Riapi

My. Vioi 15, 373-381). From the resulting plasmid, a Hwai fragment containing an amplified Samu promoter was obtained

IF) 355, the MIM!1 cDNA in the correct orientation for transcription, as well as the terminator (the 3. This fragment was cloned into the double pSIV200 vector and transformed into SMZ3101. "Muz" plants were infiltrated in the manner described earlier. The resulting seeds were collected and placed on SM agar containing kanamycin (5 µg/ml)). Sprouts that survived were transferred to soil and tested in the manner described above. The selected plants were self-pollinated, and the next two consecutive generations were selected for the creation of homozygous lines. Nine out of 58 tested lines showed resistance under Etu/a treatment conditions without prior chemical treatment. Thus, overexpression of MIM1 cDNA also leads to the formation of disease-resistant plants.

Example 23: MIM1 is a homologue of IkVo. 65 Between the protein gene products of MIMI and Ikv, several sequences were aligned, with the help of which it was determined that the gene product of MIMI is a homologue of !/ kVo (Fig. 1). Sequence homology searches were carried out using VIABZT Ts|Ayesptsi ei ai., 9. Moi. Vioi 215, 403-410 (1990).

Alignment of multiple sequences was performed using Cyizia! M. INIDIPZ EI AII., SAVIO5Z 5,151-153 (1989)), which is part of the I azegdepe Viosotriipd Zoimzhage program package of OMAZTAK (Madison, Wis.).

The sequences used in this arrangement include MIM1 (SEQ ID Mo:2), I kKVo (SEQ ID

Mo:3, SepVapk Assayvsiop and: 1022734) in mice, IkVo (POSL. ID Mo:4, SepVapk assayviop MoMo 57674 and X63594;

Temagi ei ai., Mysivis Asids Kev. 20, 607 (1992)) in rats and IkVo (POSL. ID Mo:5, SepVapkK assayviop Mo721968; where

Magiip ei aI., EMVO 3. 12. 2773-2779 (1993); SepVapk assevviop Mo 517193, de Mapip eyi aY., Sepe 152, 253-255 (1995)) in pigs. The parameters used for the analysis (Siizia!) include "divergence penalty 10" 70 and "divergence length penalty 10". Evolutionary divergence distances were calculated using the РАМ250 weight table. (Oaupoi ei a. "A tode! oi emoishiopagu spapde ip rgoieip5. Maiygisez Tog aeyeesiipd aiviapi geiayopePpirv." in Ayaz oi Rgoivip Zedcepse ap Zygysiige, Moi. 5, Zirri. Z, M.O., Baupoiyi, ea (MaiopaiYi!

ViotedisaI Kezeagspy Roypaiop, Washington, DC), pp. 345-358 (1978). Residual similarity was calculated using a modified table (Oauyoy). |Zspugai» apa Oaupoiy, "A toaei!Yi oi 75 emoishiopagu spapde ip rgoivipa." in AIKMaz oi Rgoieip bedcepse apa igisishge, M.O. Byuapoi, hey (Mayiopai!

Viotedis! Proceedings of the National Academy of Sciences, Washington, DC) pp. 353-358 (1979); ShirzKom apa Vygdezz, Mysivys

Asidz Kez. 14, 6745-6763 (1986).

Homology searches indicate the similarity of MIM1 to the ankyrin domains of several proteins, including: ankyrin, ME-kv and IKV. The most complete homology is manifested to I kV and related molecules (Fig. 1). MIM1 contains 2 serines at amino acid positions 55 and 59; sequences at position 59 are in context (O/Exxxx5) and position (M-terminal), consistent with a role in phosphorylation-dependent, ubiquitin-mediated, induced cleavage. All IkVs have such M-terminal serines, and they are necessary for the inactivation of IkVs and the subsequent release of ME-kVs. MIMI has ankyrin domains (amino acids 262-290 and 323-371). It is suggested that ankyrin domains are involved in protein-protein interactions and are a ubiquitous feature of IkKV and ME-kV molecules. C-terminals of

KV are different. MIMI has a certain homology with the region enriched in OI (amino acids 491-499), whose Ge) has been found in the C-terminals of some IKVs.

Example 24: Creation of modified forms of MIM'1 - changes of serine residues 55 and 59 to alanine residues.

Phosphorylation of serine residues in IkVo from the human body is necessary for stimulus-activated cleavage of IkVo, which, thus, activates ME-KV. Mutagenesis of serine residues (532-536) from the human body

IkVo, with the formation of alanine residues, inhibits stimulus-induced phosphorylation, blocking in this way "- proteosome-mediated cleavage of IkVo (E. Vgiia-Mageep TgaepsKpeg ei ai., EMVO 4. 14: 2876-2883 і- (1995); Vgomup ei ai ., Zsiepse 267:1485-1488 (1996); Vgosktap ei aiY, MoIesshag apa SeiYi Vioyudu 15: 2809-2818 (1995); UUapo ei ai., 5siepse 274:784-787 (1996)). She

This altered form of I kVO functions as a dominant-negative form by retaining ME-kV in the Kk cytoplasm, thus blocking downstream signaling events. Based on the sequence comparison between MIMI and IkKV, it can be seen that serines 55 (555) and 59 (559) of MIMI are homologous to 532 and 536 in IikVo from the human organism. In order to create dominant-negative forms of MIM1, serines in positions of amino acids 55" and 59 are subjected to mutagenesis with the formation of alanine residues. This is done using any method known to specialists in this field, for example, using the OcikSpapde Zige Oigesied Myiadepeviv KI 020 s kit (5200518:51gagedepé). ts Using the full-length cDNA of MIMI (POSL. ID Mob), including 42 bp. of untranslated sequence 5 (TC) and 187 bp. OTK 3", the mutagenized construct is prepared according to the manufacturer's instructions, using the following primers in parentheses (POSL. ID Mo:b, position 192-226): 5-САА САС

STT SbA DOS SOT STI TOA SOS OSS OA TO-3 (RESOL. ID Mo:25) and 5-SAT SSO SO SOT SAA ASA SO STT - AND SOA DOS TO TO TO-3 (RESOL. ID Mo:26), where underlined nucleotide pairs correspond to mutations. The strategy consists in the following operations: KkDNA MIM'I!, cloned into the pO5E9UZb vector (|EEPeide ei ai., Rgos. Mai. Asad. Zsi. O5BA mni 88:1731-1735 (1991)) and Y i ineni iv, gib Poli : , are denatured, and primers containing changed pairs of nucleotides are hybridized. DNA polymerase I (Riish) straightens primers by non-chain rearrangement, resulting in the formation of cut 20 ring chains. The DNA is subjected to Orpi restriction enzyme restriction hydrolysis, which only cuts out the methylated sites (non-mutagenized template DNA). The rest of the circular 45DNA is transformed into strain "z" of E. soya XI 1-VIse. Plasmids from the resulting colonies are extracted and sequenced to verify the presence of mutated nucleotide pairs and to ensure that no other mutations have occurred.

The mutagenized MIM1 cDNA is subjected to enzymatic hydrolysis with Esoki restriction enzyme and cloned into pSOM 1761

Under the conditions of transcriptional regulation of the double promoter 355 of the cauliflower mosaic virus. The transformation cassette, including the 355 promoter, the MIMI1 cDNA and the terminator (those released from pSOM1761 by

IF) of partial restrictive enzymatic hydrolysis with the help of Hra! and ligate into the Hrvai ko site of dephosphorylated pSIV200. SEQ ID Nos. 7 and 8 correspond to the DNA coding sequence and the coding amino acid sequence, respectively, of this altered form of the MIM1 gene. 60 Example 25: Creation of modified forms of MIM1 - M-terminal deletion.

Deletion of amino acids 1-36 |Vhosktanp ei ai.; Zy!p ei a.) or 1-72 |Zip ei a.) IkVo; human body, which includes

K21, K22, 532 and 536, leads to the formation of a dominant-negative phenotype of I kVO in transfected cell cultures from the human body. An M-terminal deletion of approximately the first 125 amino acids of the MIM'I cDNA encoded product removes eight lysine residues that may serve as potential 65 extension sites and also removes possible phosphorylation sites at 555 and 559 (see Example 2). This altered gene construct is obtained by any means known to those skilled in the art. For example, using the method,

proposed (No ei ai., Sepe 77:51-59 (1989)). create a form of MIMI in which the DNA encoding approximately the first 125 amino acids is deleted. Below are the primers that create the 1612-bp ROK product. (PASS. ID Mo:b: from 418 to 2011): 5-dd aayisa-ATO SAT TSO TT OTO AST OT TTO-3 (PASS. ID Mo:27) and 5i-dda ay stA SAA ATS TOT ATA SSA TTO o -3 (SEQ. ID Mo:28), in which the synthetic initiation codon is underlined (ATO), and the linker sequence EsokK! shown in lowercase letters. For the amplification of fragments, a reaction mixture containing from 0.1 to 100 ng of template DNA, 10 mM Triglyceride, pH 8.3/50 mM KSI/2MM is used

MasI27/0.00196 gelatin/0.25 mM, each aMTP/0.2 mM of each primer, and polymerase (1 unit. "Pi) in a final volume of 5 Oml, using a device for PCR Regkip EItag Seiy5 9600. PCR conditions are as follows : 9492, Зхв: З5Х 70 (942С, ZOsec.: 522С, min: 7290, 2min): 7223, 10min. The RSC product is cloned directly into the pSK2.1 vector (creature, ip myo). The insert created by RSC into the ROK vector released by enzymatic hydrolysis (restrictase) using EsoK and ligated into the EsoK site of dephosphorylated pCOM1761, under the conditions of transcriptional regulation of the double promoter 355. The construct is sequenced to verify the presence of a synthetic initiating ATO and to ensure that no other mutations during RSC 75 The transformation cassette, including the 355 promoter, the modified MIMI1 cDNA, and the terminator (those released from pCOM1761 by partial restriction enzyme hydrolysis of Hwai and ligated into the site

Wow! rSIV200. POST ID MoMoe:9 and 10 correspond to the DNA coding sequence and the encoded amino acid sequence, respectively, of the modified form of the MIM1 gene, which has an M-terminal amino acid deletion.

Example 26: Creation of modified forms of MIM1 - C-terminal deletion.

It is assumed that the deletion of amino acids 261-317 of the I-kVo from the human body leads to an increase in its own resistance due to the blocking of constitutive phosphorylation of serine and threonine residues in the C-terminal.

The serine- and threonine-enriched region corresponds to amino acids 522-593 in the C-terminus of MIM1. The C-terminal coding region of the MIMI gene is modified by deleting the nucleotide sequence encoding amino acids 522-593. Using the method proposed by |No ei a. (1989)), the C-terminal coding region of "SM" and 3 TC of MIM1 cDNA (POSL. ID Mo:6: 1606-2011) are deleted with the help of PCR, creating a fragment of 1623 bp, using the following primers: 5'sddaayssATSTSTTTTAATTTOTOAATTT C-3" (SEQ ID Mo29) and 5'-ddaaystsSaAASASTT SATAATSTOSTSO-3 (SEQ ID Mo:30), in which the synthetic terminal codon is underlined (TOA on the complementary strand), and the EsoKiI linker sequences are indicated by lowercase letters register. The components of the RSK reaction are as described above, and the cycling parameters are " as follows: 942C, Xhv: 35x (942C, ZOsec: 522C, ZOsec: 7220, 2min); 722C, 10min). The ROK product is cloned directly into "- pSC2.1 vector (creator, ip migo). The insert created by RSC in the RSC vector is released by enzymatic hydrolysis using Esok and ligated into the Esok!I site of dephosphorylated pRSOM1761, which contains a double promoter 355. The construct is sequenced to verify the presence synthetic co intraframe terminating co dona and conviction that no other mutations occurred during RSC. The transformation cassette, including the promoter, modified cDNA MIMI and in. terminator (those released from pOOM1761 by partial restriction enzymatic hydrolysis of Hvai and ligated into the Chra! site of dephosphorylated pSIV200. SEQ ID No. 11 and 12 correspond to the DNA coding sequence and the encoded amino acid sequence, respectively, of the modified form of the MIMI gene, which has "

C-terminal amino acid deletion.

Example 27: Creation of altered forms of MIM1 - chimeric form with M-terminal/C-terminal deletion. From c The form of MIM1 with M-terminal and C-terminal deletion is created using the unique KrpI restriction site "" at position 819 (SEQ ID No. Mo:b). The form with M-terminal deletion (Example 25) is subjected to "enzymatic hydrolysis by restriction enzyme with using EsoKi/Krpi, and the 415-bp fragment corresponding to the modified M-terminal is recovered by gel electrophoresis. Similarly, the form with the C-terminal deletion (Example 26) is subjected to enzymatic hydrolysis with restriction enzyme with and using EsoKiI/Krpi, and the 790 bp fragment corresponding to the modified C-terminal is also recovered by gel electrophoresis. The fragments are ligated at 159C, subjected to enzymatic hydrolysis using EsoKiI, in order to remove EsoKiI concatemers, and cloned into the Esoki site

Sh- of dephosphorylated pCOM1761. The form with the M/C-terminal deletion of MIMI1 is controlled by the transcriptional regulation of the double promoter 355. Similarly, a chimeric form of MIMI is created, consisting of mutagenized sites of possible phosphorylation 555/559 (Example 24), fused with the C-terminal deletion

T" (Example 26). The structure is created in the manner described above. The constructs are sequenced to verify the correctness of the initiation and termination codons and to ensure that no other mutations occurred during cloning. The corresponding transformation cassettes, including the 355 promoter, the MIMI1 chimera, and the 22 Terminator (those released from pCOM1761 by partial restriction enzyme hydrolysis of Hwai and

GF) are ligated into the site Hra! dephosphorylated pSIV200. POST ID MoMo:13 and 14 correspond to the DNA coding sequence and the encoded amino acid sequence, respectively, of the altered form of the MIMI gene, which has both de M-terminal and C-terminal amino acid deletions.

Example 28: Creation of modified forms of MiIM1! - The ankyrin MIMI domains share homology with 60 ankyrin motifs in the amino acid range approximately 103-362. Using the method proposed by (Noi et al. (1989)), the DNA sequence encoding the putative domains of ankyrin (SEQ ID NO:/1: 3093-3951) is amplified by PCR (conditions: 942C, Exhv: C5x ( 942С, ZOsec: 6220, ZOsec: 7290, 2 min): 72920 10 min) from KDNK

MIM1 (SEQ ID Mo:6: 349-1128) using the following primers: 5'i-ddaaisadI ZZASTOSAASAASASOSSOS-3 (SEQ ID Mo:31) and 5-ddaaisTtSAASSTTTOSAAASTTOSTTSTOATO-5' (SEQ ID Mo:32). The resulting product is subjected to enzymatic hydrolysis with a restriction enzyme using EsoKiI and then spliced into the Esoki site of dephosphorylated pCOM1761 under the conditions of transcriptional regulation of the double promoter 355. The construct is sequenced to verify the presence of a synthetic initiation codon (ATO), an in-frame termination codon (TA) and to ensure that , that no other mutations occurred during RSC. The transformation cassette, including the 355 promoter, ankyrin domains, and the terminator (those released from roCOM1761 by partial restriction enzyme hydrolysis using XvaI and ligated into the

Wow! dephosphorylated pSIV200. POST ID MoMo: 15 and 16 correspond to the DNA coding sequence and the encoded amino acid sequence, respectively, of the MIM1 ankyrin domain.

Example 29: Creation of chimeric genes. 70 To increase the plausibility and probability of the corresponding spatial and temporal expression of the altered forms of MIMI1, the Nipap/VatnNi fragment with a length of 4407 bp. (PASS. ID Mo:1: p.n. 1249-5655) and/or a fragment

EsokM/Vatni with a length of 5655 p.n. (SEQ ID No. Mo:1: p.n. 1-5655), containing the MIM1 promoter and gene, is used to create the modified forms of MIM1, discussed above in Examples 24-28. Although the stages of creation may differ, the concept of their implementation is practically the same as that used for the /5 previous examples described in this text. Intense overexpression of altered forms can lead to undesirable consequences. Thus, the altered forms of the MIMI gene described in Examples 24-28 may be under the control of promoters other than the endogenous MIM1 promoter, including, but not limited to, the pos promoter or the small subunit of the Kibizso promoter. Similarly, altered forms of MIMI1 can be expressed under the control of the pathogen-sensitive RI-1 promoter (US Pat. No. 5,614,395). Such an expression creates

The possibility of intensive expression of altered forms of MIM1 only under the conditions of "pathogenic attack" or under other conditions, when the activation of ZAC occurs. In addition, resistance to the disease is detected in transformants expressing altered forms of MIM1 under the control of the PE-1 promoter, if they are treated with activator compounds

ZAK (ie, VTN or IMA) at concentrations that do not normally stimulate 5AK, thus activating a feedback loop (M/eutapnp ei ai., (1995) Reapi Sei! 7: 2013-2022). high school

Example 30: Transformation of altered forms of MIM'1 in Agabidors INaiapa The created constructs (Examples 24-29) are introduced into Adhorasiegitst (stetasiep by electroporation into strain CM3101. These constructs are used for i) transformation of Agaridors SoI-O and M/g-O ecotypes by of vacuum infiltration | 78, 1089-1099 (1994). or standard root transformation operation. The seeds of these plants are collected and allowed to germinate on agar plates with kanamycin (or another appropriate antibiotic), which acts as a disinfection agent. Only sprouts that are transformed are able to detoxify the breeding agent and survive. Sprouts that survive as a result of selection are transferred to the soil and tested for the CIM (constitutive immunity) phenotype. Plants are evaluated for visible phenotypic differences compared to natural plants. M

Example 31: Evaluation of the SIM phenotype in plants transformed with the natural MIMI1 gene or a modified form of the MIMI1 gene. me)

Collect one leaf from each primary transformant, isolate RNA

Kez. Ai., 1992). Conidia suspensions are prepared in concentrations of 5-10x107 spores/ml from two compatible strains of R. ragaziis, Etma and Moso (these fungal strains, respectively, cause diseases of natural plants UuUz-O and SoiI-O), and the transformants are sprayed with the appropriate isolate depending on the ecotype of the transformant. The inoculated plants are incubated under high humidity conditions for 7 days. The plants are evaluated for disease on the 7th day, and one leaf is harvested for RNA blot analysis using a probe, which allows quantitative characterization of fungal infection.

Transformants, which are characterized by the SIM phenotype, are taken in the 11th generation, and homozygous plants are determined.

Several tests are conducted with the transformants to detect resistance to diseases, the description of which is given later in the text. Infection with Moso and Etu/a fungi is repeated, and the leaves are stained with lactophenol blue in order to detect the presence of fungal hyphae, as described in (Oieigis ei ai!., (19943). Transformants o are infected with the bacterial pathogen Rzepidotopaz zuhipdae OSZO000, in order to evaluate the spectrum of evidence of resistance , as described in (OKpev et al. (1993)). Uninfected plants are evaluated for the presence of both free and glucose-conjugated ZA, and the leaves are stained with lactophenol blue, which allows the presence of microscopic lesions to be assessed. Resistant plants are sexually crossed with ZAK mutants, such as Mayo (pat.

Chl» USA Mo5,614,395) and pa, in order to identify epistatic relationships of the resistance phenotype with other mutants and to evaluate how these dominant-negative MIMI mutants can affect the BA-dependent feedback loop.

Example 32: Isolation of MIM1 homologues.

It is possible to obtain MIMI homologues by hybridizing them under moderate conditions either with the entire MIM1 gene from iF) Agaridorzis or, in the optimal version, with an oligonucleotide probe obtained from the MIM1 gene of Agabidorzis, which includes the adjacent part of its coding sequence with a length of at least about 10 nucleotides. Factors affecting the stability of hybrids determine the mode of intensity of hybridization. One of these factors is the melting temperature T p, which can be easily calculated using the formula given in (OMA RKOVEZ5, Seogde N. KeiPeg apa Mag M. Mapak, Mastiyap Rybriizpege CYA, 1993, Zesiop ope: MoiIesshag Nurbgiadigabyop Tesppoiodu; rad 8

Y. The optimal hybridization temperature is a temperature in the range of about 259 below the calculated melting temperature T y, preferably in the range of about 12-159С0 below the calculated melting temperature of Tut, and - in the case of oligonucleotides - in the range of about 5-10 below the melting temperature of 65 Tt.

Using MIMI cDNA (SEQ ID NO:b) as a probe, Agaridorz MIMI homologues are identified by screening a genomic or cDNA library from various cultures, such as (but not limited to) those listed below in Example 33. To standard techniques the implementation of this analysis includes hybridization screening of DNA libraries placed in dishes (or plaques or colonies; see, for example, |(Zatgook ei aI., MoiIesshag Siopipo, edv., Soid Zrgipd Nagrog I aroghaiog Rgezv. (1989))) and amplification by by RSK using oligonucleotide primers | see for example, Ippiz ey aI., RSK Rgoiosoyiv, A Scide yuyu Meiypoav, apa Arriisayopz edvz., Asadetis Rgezz (1990)Y. Identified homologues using genetic engineering methods are inserted into the expression vectors described in this text and transformed into the cultures listed above. Transformants are evaluated for increased resistance to the disease using 7/o Appropriate pathogens of tested cultivated plants.

Homologs of MIMI1 in the genomes of cucumber, tomato, tobacco, corn, wheat, and barley were detected by DNA blot analysis. Genomic DNA was isolated from cucumbers, tomatoes, tobacco, corn, wheat, and barley, subjected to restriction enzymatic hydrolysis with Watni, Nipai, xHvrai, or ZaiII enzymes, separated on 0.8965-th agarose gels, and transferred to a nylon membrane by capillary blotting. After cross 7/5 Stitching (UV) with attachment. The DNA membrane was hybridized in slightly rigid conditions M19685A; 520 mm

МаРОо,, рНН7.2; 795th lauryl sulfate, sodium salt; mM EDTC; 250 mM sodium chloride) at 5592 for 18-24 years with radiolabeled R. cDNA of MIMI Agarbidorziv (Nayapa. After hybridization, the blots were washed in mildly stringent conditions (6X55С for 15 min. (X3) ХХ5С for 15 min. (X1) at 55923; 1X55С is 0 , 15 M of Ma, 15 mM of citrate of Ma (pH7.0)) and exposed to X-ray film in order to visualize the bands corresponding to MIM1.

In addition, to identify homologues, expressed sequence tags (ESTs) are used, which are detected by their similarity to the MIM1 gene. For example, several rice expressed sequence tags (ET) were identified by similarity to the MIM1 gene. Multiple sequence alignments were created using Syizvia! M | Niddiph, Oeztopa b. apa Ratshi M. 5Nagr (1989), Ravi apa zepzyyme tiyirie zedoepse Ge aiidptepiv op a tisgosotry(eg, SAVIOS 5:151-153) az rap oyi (Pe OMK" (1228 Zotsii Ragk 5igeeii, Madison, State (5)

(Wisconsin, 53715), a package of programs for the I azegdepe Wiosotriyypud bzoiymage for Masipiosp (1994)). Some areas of the protein

MIM'1 are homologous in amino acid sequence to 4 different protein cDNA products from rice. Homology was detected using MIM1 sequences in the study of SepVapk VI AZT. Comparison of areas of homology in

MIM1 and rice cDNA products are shown in Figure 2 (See also SEQ ID Mo2 and SEQ ID MoMo: 17-24). From 36 to 4895 identical amino acid sequences from 4 products - obtained from rice - were found in the MIMI protein fragments. These rice E5BTs are particularly useful for the isolation of MIMI homologues from other monocots. -

Homologs are also obtained using RSC. In this method, comparisons are made between known homologues (for example, from rice and Agabridorziz). Areas with a high degree of similarity or identity of amino acids and DNA are further used to create PCR primers. Areas rich in - amino acid residues M and MU are optimally followed by areas rich in amino acid residues РЕ, У,

C, H, O, K and E, because these amino acids are encoded by a limited number of codons. After the identification of the corresponding region, primers for this region are created with different substitutions in the C to codon position. This variety of substitutions in the third position can be limited depending on the target species. For example, since corn is rich in CC, such primers are created in which, if possible, C is used in the ZMU with position C or C. The RSC reaction is carried out from cDNA or genomic DNA under different standard conditions. When the "" band becomes clearly distinguishable, it is cloned and/or sequenced to determine whether it is " a homolog of MIM'1.

Example 33: Expression of the MIM1 form in cultivated plants.

Those constructs that produce the SIM phenotype in SoI-O or Muvz-O are transformed into cultured plants for evaluation. - In another variant, the modified native MIMI1 genes, which are isolated from cultivated plants according to the previous example, are returned to the corresponding cultivated plants. Although the MIM1 gene can be inserted into the cell of any plant from the variety of classes listed above, it is particularly useful in the cells of cultivated plants such as rice, wheat, barley, rye, and corn, potatoes, carrots, sweet potatoes, sugar beets, legumes, peas, chicory, lettuce, cabbage, cauliflower -l 70 cabbage, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, zucchini, pumpkin, zucchini, cucumber, apple, pear, quince, melons, plums, cherries, peaches, smooth peaches, apricots, strawberries and strawberries, grapes, raspberries, black currants, pineapples, avocados, papayas, mangoes, bananas, soybeans, tobacco, tomatoes, sorghum and sugar cane. Transformants are evaluated for increased resistance to diseases. In the optimal version of the invention, the expression of the MIMI1 gene reaches a level that is at least twice the level of expression of the native MIM1 gene in natural plants and, in the best version, exceeds the natural level of expression by ten times.

Example 34: Synergistic disease resistance created by introducing a standard bactericide into a transgenic plant overexpressing MIM1.

The plant lines that are used in this example (bE and 7C) are created as a result of the transformation of 60 natural plants Agabridorziz (paiyapa (ecotype Muz) with the genomic fragment of Vatni-Nipaiya MIMI (SEQ. ID Mo:1 - p.n. 1249-5655 ), as described above in Example 21. The fungicides metalaxyl, fosetyl and copper hydroxide, prepared with a concentration of 2595, 8095 and 70905 of the active component (a.i.), respectively, together with a carrier in the form of a moistened powder, were introduced in the form of a finely dispersed aerosol in leaves of three-week-old transgenic "Muv" plants constitutively expressing the MIM1 gene. For control, only the moistened powder was injected. After three days, the plants were inoculated with a suspension of conidia of the Etma isolate from Regopovzrog ragaviisa (1-2x10 5 spores/ml), as described (Oeiapeu ei a. (1995)).After inoculation, the plants were covered to maintain high humidity and placed in a Percival chamber (Regsima!I) for growing at 17 C with a light/dark cycle of 14 h/10 h |OKpez ei a! ., 1993). The tissue was collected 8 days after inoculation.

The progression of the fungal infection was followed for 12 days by studying it under an analytical microscope

With a microscope, quantitatively evaluating the development of conidiopores in this way (Oeiapeu, eiiaI. (1994); Oiekisn, ei ai. (1994)). To study the growth of the fungus in the leaf tissue, individual leaves were stained with lactophenol trypan blue. Fungal growth was quantified using a fungal rRNA probe obtained by RSC according to (MUpiie eii a). (1990; RSK Rgoyuosoiv: A dtside yu Meiypodz apa Arriisayop, 315-322)| using primers M51 and M52 and Etuma DNA from R. ragaziis as matrices. RNA was isolated from frozen tissue by phenol/chloroform extraction after lithium chloride precipitation.

RMAB, 84: 7542-7546). Samples (7.5 μg) were separated by electrophoresis through formaldehyde-agarose gels and subjected to blotting analysis on nylon membranes (Nubopa-M--- Ategzpat) as described in |A!egrbei ei ai!. (1987).

Hybridization and washing procedures were performed according to Spigsy apa siireg (1984, РМА5, 81:1991-1995)).

Relative transcript abundances were determined using the Itadeg RIozrpog device (MoiIesshag Bupatisv, 7/5 Sunnyvale, CA) according to the manufacturer's instructions. Sample loading was optimized by probing scraped filter blots of constitutively expressed p-tubulin cDNA from Agaridorziz. Infection of untreated plants corresponded to 095 inhibition of fungal growth.

The use of metalaxyl, fosetyl or copper hydroxide in plant lines with MIM 1 overexpression created a greater than the total, i.e. synergistic, effect of resistance to diseases. This effect was defined as the synergy factor (SE), which is the ratio of the investigated (0) effect to the predicted (E) effect. The following results were obtained: o component I: overexpression of MIM'1 (line 6E) component II: metalaxyl He)

Test Mo Synergy factor O/E

THEY ARE DOGS NOAH PAUL m 161 with his wife! 8101 - with zboms! I'm sorry! 60010893 т ne z ж- "component I: overexpression of MIM'1 (line 6E) shsh component II: fosetyl c Test Mo Synergy factor O/E g"

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MIe-natural M/v5 o component I: overexpression of MIM'1 (line 7) and component II: fosetyl

Test Mo Synergy factor O/E including 1911 lm m a frmivoty 7.11 persuade 211 b i o mmuvoty 86172113 5 |MIM1) 0.5 g/l 56 16 3.5 -A3-

component I: overexpression of MIM'1 (line 6E) component II: copper hydroxide

Test Mo Synergy factor O/E i t 6 mu a miayuti 061 z |mivat 6111 i myst o 17777111 vommugot|) 6600100600556 vommioyatya 4003164 m

From component I: overexpression of MIM'1 (line 7) component II: copper hydroxide Ge)

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As can be seen from the above tables, synergistic disease resistance effects were detected in plants with MIMI1 overexpression by using metalaxyl, fosetyl and copper hydroxide. For example, in ts of an untreated plant with MIM' (line b6E), a 1090th inhibition of the growth of the fungus was observed in comparison with an untreated natural plant; this confirms that the constitutive expression of BAK genes for such a MIM1 overexpressor is correlated with resistance to diseases. However, as shown above in Table 37, due to the introduction of fosetil at a concentration of 5.Og/l (that is, at a concentration that is usually insufficient to ensure effectiveness) in an immunomodulated ("on ZAC") plant with overexpression of MIM1! the investigated level - the inhibition of the growth of the fungus was increased to 9395. The synergy factor of 5.5, calculated on the basis of these data, clearly demonstrates the synergistic effect achieved by the introduction of the bactericide into the immunomodulated ("BAK included") plant. In another example, in an untreated plant with MIM1 (line 7C), 14965-e inhibition of fungal growth was observed compared to an untreated natural plant, and this is confirmation that -ko 270 constitutive expression of BAK genes for such an overexpressor of MIMI1 is correlated with disease resistance .

However, as can be seen from Table. 40, due to the introduction of copper hydroxide at a concentration of 2.Og/l (i.e., at a concentration that is usually insufficient to ensure effectiveness) in immunomodulated ("Included ZAC") plants with MIMI1 overexpression, the observed level of fungal growth inhibition was increased to 7795. The synergy factor of 5.5, calculated on the basis of these data, also indicates a synergistic effect, which is achieved by introducing a bactericide into an immunomodulated ("included ZAC") plant.

Gee! Thus, it is the combined use of immunomodulated plants with MIM1 overexpression! with bactericides at low, usually ineffective concentrations to achieve disease resistance, where it provides advantages that are obvious to the agronomist. By taking advantage of the synergies disclosed herein, toxic or otherwise undesirable concentrations of bactericides can be avoided. 60 In addition, as a result of reducing the amount of bactericides needed to ensure a given level of plant protection, a significant economic effect can be achieved.

Example 35: Synergistic resistance to the disease created by introducing the chemical inducer BAK into transgenic plants with MIM1 overexpression

Transgenic plants containing a fragment of MIMI genomic DNA under the control of its own promoter 65 (Example 21) were also analyzed for the reaction to different concentrations of VTN depending on the line of natural

Uh8. Seeds from each line were sown and germinated as previously described. About three weeks after -dA-

after planting, leaf samples were collected from each line (day 0 - control), and the remaining plants after that were treated with NO, 10 μM VTN or 100 μM VTN. Total samples were collected on the 1st, 3rd and 5th day after treatment.

After collecting the samples on the 3rd day, a subgroup of plants from each line was separated separately and treated with the Etois isolate

From Regopozrog ragaziysa, as described above. RNA was prepared from the collected tissue, and Northern blot analysis was performed using the RK-1 gene probe. Plants were quantified for resistance to the fungus 8 days after infection.

The results of Northern blot analysis for Muz and four MIM overexpressing lines (ZA, 58, 6E and 7C) are shown in Fig.3. The expression of the RK-1 gene in the natural line of Mus was clearly manifested after treatment with VTN at a low 7% concentration, i.e. 10μM (to achieve effectiveness, it is usually necessary to use VTN at a concentration of 100-300mμM). "MuUv" plants as a result of this treatment also remained sensitive to the fungal pathogen R. ragaziysa (Eplmua). However, in all lines with overexpression of MIM1, a significantly more intense reaction to the expression of the RE-1 gene was observed after treatment with VTN at a low concentration. In addition, all lines with overexpression of MIM1 treated with tOMK VTN were marked with complete or almost complete resistance to R. ragaziis. Staining /5 leaves with lactophenol blue in order to identify the presence of fungal hyphae. (1994), confirmed the absence of fungal growth in lines with MIM1 overexpression. The expression of the RK-1 gene in leaf tissue after treatment with 100 μM VTN was also significantly more intense and faster in lines with overexpression

MIM1 compared to natural lines. Thus, immunomodulated plants are able to respond much faster and to much lower doses of VTN than natural plants, as evidenced by the expression of the RK-1 gene and resistance to R. ragavziis. These data confirm that synergistic resistance to the disease is achieved by introducing a chemical inducer of systemic acquired resistance, such as VTN, into an immunomodulated ("enabled ZAC") plant, such as a plant with overexpression of MIM'1.

Thus, it is the combined use of immunomodulated plants with MIMI overexpression together with chemical inducers of BAC, such as VTN, in low, usually ineffective concentrations, with the aim of achieving resistance to diseases, that provides advantages that are obvious to specialists. Using the advantages of synergies disclosed in this description, it is possible to avoid usually toxic or for other reasons i) undesirable concentrations of chemical inducers of ZAC. In addition, a significant economic effect can be achieved by reducing the number of ZAC inducers required to ensure a given level of plant protection.

LIST OF SEQUENCES «E zo (1) GENERAL INFORMATION: () APPLICANT: - (A) NAME: Momagiiv DO M (B) STREET: Schwartzwaldallee 215 (C) CITY: Basel me) (E) COUNTRY: Switzerland ych- (EE) POSTAL CODE (21IR): 4002 (F) PHONE: 41 61 69 11 11 (3) TELEFAX: i 41 61 696 79 76 (I) TELEX: 962 991 « (M) CONVENIENT FORM OF READING ON A COMPUTER: in s ( A) MEDIA TYPE: floppy disk

And (B) COMPUTER: compatible with IVM RS and? (B) OPERATING SYSTEM: РО-БО5/М5-0О05 (Д) SOFTWARE: Raiepiip Keyease 41.0, Megviop 41.30 (i) TITLE OF THE INVENTION: METHOD OF PROTECTING PLANTS -I (ii) NUMBER OF SEQUENCES: 32 (2) INFORMATION FOR FOLLOWERS ID Mo:1: o () CHARACTERISTICS OF THE SEQUENCES: -I (A) LENGTH: 5655 pairs of nucleotides (B) TYPE: nucleic acid - (C) CHAIN BRANCHING: single "(D) TOPOLOGY: linear (its) TYPE OF MOLECULES: DNA (genomic) (ii) HYPOTHETICAL MODEL: NONE 5B (m) ANTI-SENSE: NONE (their) CHARACTERISTICS: (F, (A) KEYWORD: exon ka (B) LOCATION: 2787..3347 (M) OTHER INFORMATION: /product- "MIM1 exon 1" 60 CHARACTERISTICS: (A) KEYWORD: exon (6) LOCATION: 3427..4162 (0) OTHER INFORMATION: /product- "MIM1 exon 2" ( them) CHARACTERISTICS: 65 (A) KEYWORD: exon (B) LOCATION: 4271..4474

(M) OTHER INFORMATION: /product- "3rd exon MIMI1" (their) CHARACTERISTICS: (A) KEYWORD: exon (B) LOCATION: 4586..4866 (D) OTHER INFORMATION: /product- "4- th exon of MIM1" (their) CHARACTERISTICS: (A) KEYWORD: СО5 (B) LOCATION: union. (2787..3347, 3427..4162, 4271..4474, 4586..4866) 70 (xi) DESCRIPTION OF THE SEQUENCE: END. Mo ID: 1:

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Suv siu yuv A1ia Suz Meb Puv Pec Prep Azr Agu Suv Buv biz I1e I1e 215 720 225

STO AAb OTST AAT STA SAT ATo STT AST STT SDA AS OTSA TO SSO SAHA 3594

Whoaie Poovz Beh Up Whoaie Ayer Meb Whoa! beck Bay Beach Was Beck Pay Rco Sich 230 235 240

SdAO STT OSTT AAA SAS ATA ATT SAT ASA SOT VAA SAS StTt sot TTOo SAS 3647 siv Hey Mai pub sich Ii I1e ABr Aho AkoO pub pizza rep S3)u Pec siy s » 245 250 255 o

STA SST AVA STA DA AAA SAT OTO TS AAT OTA SAT AL OSA StTT SAS З6550 zo Uai rRkgo Puz Maiii Gu Guk Nish Ua! beat A5gp oUa1! Niv tsu Aza Gem ABr h 260 265 270 275 - ' in.

TOSS bAT SAT ATT bas TTA STO AAsS OTTO STT OTTO AAd SA SAT SAS dos 13738 Geo)

Beg Azr Ar I1ie sii Pec ua1i Tuv Without Without Pes Buv sich Avr Niye TNE M 280 285 290

JSC STA JSC SAT oso tot St o sttT SAT OTTO OST o sttT OSA TATO TOS DAT 3785 «

Ap oMem ABr o ABr o A1ia SuB dia iesh niv Re Aia Ma1i Aia Tut Suv ABrp - s 295 300 105 ;»

STO AAS ACC bSA ASA SAT OSTT OTTA AAA STT SAT STT SSS SAT STO AAS 3834

Uaiii Puv Tk Aza TE o Azropech Pem Buv Mech Ar Pets Aza Ar oUai Ap ikh 310 315 320 (95) -I SAT ASO AAT SSO OR OA TAT dSO STO STI SAT ST OST Sa to seb 3882 -oUu 70 Ni Ag ABpoRgo Aga s1u Here Tg Uai Tez Niv uai Aia A1a Mes AgCh

I" 325 330 335

AAS OSA SSA SALA OTTO ATA STA TStTOSTA TT baAA AAAA OST SSA dotT ssA 3930 -

F) ko 60 65

I was there Rkgo beep oven I1e ep I was without ray beach I was biu A1ia Beh Ai1a 340 345 359 3255

TSA SALA SSA AST OTTO AD OST ASA ASS OSA STO ATO ATS ATS OSA AAA SAA 3978

Beh bitz Aia Tu res bij b1u Ago tig o Aza Ped Mebye Y1i6e Aia tsub bip 360 355 370

SSS AST ATS OSOo STT OSAA TOT AAT AAT ATS SSO AS SAA TOS AAO SAT ya26 dia Tyh Mes dia uai Sat Suk A5poA5p 116 Rgo si bip Sub uv Nivb 375 UVO Zc5

TST STO AAA SOS SOA STA TOT OTA SAA ATA STA SAb SAA bAA SAS AAA 4074 bek Pec Buv biu Ahu Pech Suv Mai b1p Iie rep bi sip beach ABr GuB 390 395 400

ССА САА САА АТТ ССТ dBA PAT стр ССС СОС ТСст ТТ СА hundred осо сс 4122

Ak sich sip T1e Rgo Aga AvroUa1i Rgo Rgo bek Re Aza Uai Aza A18 s 405 410 435 (5)

SAT SAA TT AAS ATO So Sta STO SAT StTT SAA AAT ASA b 4162

A5zr persons Pemy Buv Mee TCt Ped Without Avr volume bij Avp o Age Y ago 425 430 - and -

OTATSTATSA AOTSTTATTTT STTAATSTT TOAATTAAAT TTATSTOSTS TSTATTASOOA 4272 s

AASTOASTOA ASTAATOATA ASTATISTTT STOTTSSTSSSA STOTTTAS TT OSA st 4278 te chUuai Aa ce 435 « shi s SST SAD Set STT tTtT o sSSA AS SAD OSA SA; OST OSA ATO SAS ATS SOS 4326 "z Aia bip Aga yep Rpe Rgo Tit 010 A1a bip dia Aia Meb Si ize Aza i 440 445 450 - -i SAD ATO AAS OSOA ASA TT SAS TTS ATA hundred ASTT AOS Sto sAb sstT SA 4374

Ha beach Mee puz biu Yes Sub beach Rpe I16e Ua1! quiet back rap fight Rgo Avr -1 1455 460 465 shu 20

Im SOT STO AST OST ASO AAb o ASA ASA tTSA SSO OST STA AAb ATA SSA sst 4422

Ahu pen tTpg osiu Tpg Puz AgYa Tv bek Rgo biu Ma) rub I1e Aia Rgo ato 475 48o

GF) TT ASA ATS STA SAA OAS SAT SAA AST ASA STA AAAA OOSO SstTtT OST ALA 4479 ko Rpe Agu Iie Pech sich bets Nivz bip be Agud Mech Buv Aza Pe beg Guz 60 65

485 430 495

АСС b STATSSATTS TSASSSSASTT SATSSSASTS STTTATSASAAA ADAASALAAS 4524 tТпх 5

TAAATSATST TTAAASATOS TTTTOTTAST TOSRTOTSTOA SSTTOTTTTT TTTATSATSA 4584 s te SAD ST bob AAA SsSA TT tto SSO SOS tot Tso OSA sto ste 4629 uai b3iz pe bs1u Gu Ago Rpe Rre Rho dku Sub bBeg A1ia chai Ppech t 505 510 515

SAS SAS ATT ATO AAS OTOT SAS SAS OTTO AST OSAV STO OST TOS SbA SAA 4677 yo bip Ti Mee Ap Sub S)1its Ayer Pbep Tu bip rez Az1a Suv siu 1 570 525 530

САС САС AST OST САС DAH SSA STA SAA AAS AAO SAA SO TAS ATO SAA 4725 in Ar Ar trt Aia Si Buye Ag Peo beep pub Buye beep Aga Tug Meb Styy s 535 540 545 i)

ATA SAA SAS ASA STA AD OADO SSS TTT AST SAS SAS DAT OTTO SAA TTA 4773 - zo tI1e Sip biz Tk o Pech Bu5 bue Aza Re beg sSits Ar Avp o ben 013 Bey 550 585 550 - i-

OSA AAT TSo TSS STO ASA SAT TS AST TST TOS ASA TO AAA TSA dos 4821 so siu Ayep Beg Zek Gen Tit Avr Bet Tu beh beg Tkh beg Buv bek Tvg cha 565 570 575

OST SSA LO ASo tet AASOSST AAA Sto TST SAT SOT SStT SO tod av « siu b1u Tsuv Ah Beg Avp Ag Pub cem bek Ni dk Ago Ag /x shi s 580 585 590 z

САСТСТТОСС ТСТТАСТСТА АТТТРТОСТо ТАССАТАТАА ТТСТСТТТТО АТОАТОАСТО 4926 і ТААСТСТТТА ТСТСТАТСОТ ТООССТСАТА ТАСТРТСОСТ СТТОСТТІТО САТОСТСТОТ 4986 (95) - АТТАТТОСТО САОСТОТОСТ ТСААЛСАЛАТ СТТСТААСАА ТТТСААССАА ТОСТАТАСАЄ 5046 - 50 ге АТТТСТААТА ТАТАТІТАТО ТАСАТСААСА АТААСССАТО АТССТСТТАС АСАСТТОСТА 5106

SAATSAAAST STSALATAAT STSAVATTST TSATSTSTTO SATATTTTSS ASSAAASAdSS 5166

F. AVAASAATAT TSAASTROSS TSAASTTSTO SSAASATTSA TOTTATATOT ATSTTOSTAA 5226 ko bo 65

TTSTTSSTTT AASSTTTTOT AASTSSDATT ASASASSAAAS TTASTTTTSAS STOSTABASAT 52856

AASASAASAS TOASTOSOSO TOTAASOSTOS ATTSTOSTAV TSASSTSSSAT TOSATSSAAS 5346

ATTTOSTOSAATtT SASASAASTT AASAATOSTT TOSASSATTT STOOSTOSAT ASATOSAAAAS BOB

TTISTTSSATT SAAASTTOSSS ASATOTOSAS STOSSTTSOS TOTSASTSAT ABASSAASAS 5456

ASTOSAVASST TTSASAAAATT OSSSSTSAdDAT STISTOKTTS TATOSTSATO ASTOSATATS 5526

TSSSASSAST SOSTSATOASS SASASSSSASS TSATTTTOAO OSAATTOSOS TAASSATTTS 5586

СОСАОСТТСТО АСТССТИСТТ TITOATSTOS TITATOTASS AATSAAATTS TTSSTISTOA 56456

STISTOSAT 5655 (2) INFORMATION FOR POST. ID. Mo:2: () CHARACTERISTICS OF THE SEQUENCE: ( sch

A) LENGTH: 594 amino acids (B) TYPE: amino acid i) (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQUENCE. ID. Mo:2: "Mex Avr Tii TpPI I1e Ar Siu Rne A1a Avr bei Tuk bij I1e seg 5eg "- 1 5 10 15 eat o

I Bek Rpe Mai Aia TNK Avr d5dp Trg Ar o bek beh I1e Ua1i Tug Pe

Avia Aia bi bid Mai rei tigobiu Rgo ABr o wai bek Aia tsets siy Gen " "0 45 - s y tsez Bek AvpobBeh Rpe Si" bet Uai Rpe Azr Bet Rho Ar ABr Rpe Tug y? 50 55 50 t -I zek ABr Aia Guz Petz Mai petsyp beg Avor biu Aga Sich Mai bek Rre niv syu 65 70 75 vo -I - 50

Thursday"

F) name) because b5

Atch Sub Whoa! Pei bBect A1ia Ak bet beg RPpe Ripe Buv bek Aia Pem dia 85 80 95 dia dia was pub c) gub Avzrobeh Abp o ABp oТh Aza Aza Uai Tsu5 Beh 100 105 110 to Sic rem tsu ZZo) i11e Aza Buv Avr oTuh siy Uai b01u Rbe AvBr Beth Uai 115 1720 125 75 Teas tpk o ua1i Ppep dia Tug UuUa1! Tukht Beg Bek Ag Uai Ag Rko Rgo Rgo 130 135 140 ru siu Ua1i Beh biy Sue A1a dor sich Abzp o Suz Suv Niv Ma1i dia Suz 145 150 155 160

Aha Rgo dia Uai ABr o RBe Mee Bez bi ai Be Tut Bei Aza rRpe Ti1ie 165 170 175 s

Gee)

Rpe Buz i1e Rkgo sitz pem I1ie TI Gay Tug bip Akd NiB Pe Pe Avr 180 185 190 « chua1i Mai ABr uv Ua) Mai I1eee biz A5r o trtI Be UMai Iie rech Buv Gay -- 195 200 205 - so

A1ia Azp 116 Sub siu Was Aia Sub Mee Guv Mech Pec Ar Ag Suv Was ich- 210 215 225 bin YiI1e i11e Mai Was Bek A5p Mai Ar Mee Ua! Bek Bez C) Buv beig « 0 225 230 235 240 Z s with em Rhgo siy bib Ppez uUai pub bis i16e I1ie ABr Ag9d Aku Guv bij Pec 245 250 255 - . biu rech bi Uai Rgo Buv Ua1i Iluv Iluv Nib5 Uai beg Ap Uai niv Gu s 260 255 270 -I - Asia this Avzr Bek Avr ABr I11e bio ce mai Puz ytets pbem tech Buv 1

GT» 275 280 285 rya Ar Ni Tat ABp o rey Akr o Avr Diaz Sub A1a tseu Niv Rpe Aza Uai dia 290 295 300

F) kobo 65 -5D-

tuk Suv Azp Mmai Puv Tig o ALia Tit AvroBech Petz Tsu5 Bech ABr o Pem Aza 305 310 315 320

Ayer mai Avp Ni Ahu AvpoRgo Ago s1u Tug Tk oUai Pes Ni Uai A1a 325 330 335

Aia Mes Ag Buv siy Rgo sip Pe I131e Ben bet Pei Gez bii Buv b1iu 340 345 350

Aia seg Aiiiiv beg stand Aza Tikh o Pei biz biu Ako Tr o Aia Pie) Mee Ii y 355 360 355 dia uz zip dia Tph Mes Aza uai s) Suv Abyep o ABp I1e Rgo sim bii 379 375 380

Suv Buv Niv Bet Gay uv S1iu Ag this Suv uai 51 tie tech sii bip 385 390 395 400 sch o b195 A5v Sub Dgch bi bip H1e Rgo Ag AzroUai Rgo Rgo beh Rpe A1ia 405 410 415 " uai dia Aia Azr bij pey Ppuv Meb Trg oPpeip Petz Ar Pbech S1M Ap Ah «- 420 425 430 | ich- (ze) uai dia Ppezy Aia sip Asya Pec Rpe Rgo Tig bii dia siv dia Aza Meb m y 435 440 445

Sich I1e Aza si Mebs puye Siu Tpu Suv bin RBe i11e wai TNK bek this " 20 450 455 460 2 s z" si Rgo Azr Ag GPem tTVh biu Tak Gu Ak tTpgobeg Rgo Siu Mai Gu 455 470 475 480 i I1ie Aia Rgo Rpe Agu I1e tsech bi Si Ni b1ip beg dgo Pech Was dia (95) 485 490 495 -I - 70 rec Bek Was So Uaii si Pe biu uv Agu Rpe Rpe Rko AkYA Sub bek

T» 200 505 516 dia Mai ces Avr beep I1e Mes A5p Suv bis Abvr this Tvh beep Tec dia 515 z20 525

F) ko Suz b1u bis Ar Ar tTBh dia bi uz Ak pe) sip uz Tsuv bip Ako 60 65

530 535 5ad

Tuh Mee biz Ii11e Sip bid TBhorez puv tsuv Aza Rpe zekg siy A5r Avp 545 250 555 5BO

Ivch biz Hey biu Azp obeg beh em TI Ar o beg Tpg bek bek TpPg bek to 555 570 575 pu Beh Tak siu b1u Tsuye Agu beg Avp Agu uz Bec bek Niv Ago Ako 58 585 5980

Yes (2) INFORMATION FOR POST. ID. Mo:3: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 314 amino acids (B) TYPE: amino acid (B) CHAIN BRANCHING: not important (D) TOPOLOGY: not important (y) TYPE OF MOLECULE: protein sch (хи) DESCRIPTION OF THE SEQUENCE: ID Mo:3: o

Mes Rpe pip Rko Aia Siu Ni Siu bip Avr Tgr Aa Mes siy biu rRgo 1 5 10 15 « zo -

Aha Avrob1iu Pec Buv There was a plague Ago Pez Ua)! Ar Abzr o Ago Nibv Abor Bek 20 25 zo - so biu tsez Avr bBet Mes Mru5 Avr bis biz Tuk bij bib Mes Uai yiuv bij - 35 40 45

Bez Aga b1ch Iz1e Akd Pem bip Rkgo bip s3iy0 Aia Rkgo Pe) A1ia Aza b1x " 50 55 vo in s ;" Rko TIR Puv siv biv pes TVi 01h Abvr o siu Adbzr bek Riv Beh Niz Tez -I (95) -I shi 20

Thursday"

F) name) bo 65

65 70 75 80

Aza I1e I11e Niv bij bib Buv Rgo Ppep TVgoMee bij Uai T1e Ssiu bip 85 80 35 wai uv siu dvr without dia Rre Pec Avp o Re pip Ap ABao Gem bir bib 100 105 110 tru oRgo BPech Ni Bec dia Udi I1a Trt Aep bip Rgo b1Uu I16e Aza siy 115 120 125

Aia pe pcesh was A1a Siu Suv Abr Rgo si Pyemi Aku Avr Rpe Ak9 s1u 130 135 149

Depot Tu Rto Pemy Niv Pes Aia Suz b01h sip b1u Su Pem Aia bei Uai 145 150 155 150 s

Aia UMai Pcez Tik biv Tvg Sub TBgoBgo bip Niv Pen Nib Bek Uai Bey o 165 170 175 bip dia TVg Azpotug ABp ob)iu Nib TVg Sub Pec Nik Pem A1a bek Trg « 3o 180 185 190 - -

Niv siu Tut Pem Aza I1e MUai siy Niv yeech Mai Tk Bez" siu z1a Ar so 195 200 205 o o - chai Avp Aza pip biv Rgo Suv Avp biu Ag Tag Aza pech niv Gech dia 210 215 220 « o, s uai Avr Be bip AzpoRko AvroPei Uaii beh rem Gem Pec tsu Suv S1uU z» 225 230 235 240

Aia ABr Mai Ap o Ako Uai Tu Tuk lunch biu Tukh Bek Rko Tug bir yeu ikh 245 250 255 (95) -i TI Tgr b1iu Aku Rgo bek Tg Aku i11e beep beep beep Pei biu beep rez - 70 260 265 270

Thursday"

TptI Bechts 010 Ap o remy bij Mes this Rgo bij bek bi A5r sitz bij beg 275 280 285. o Tut A5r TpPh b310 Beg Si Re tТг o bis ABrobich Pei Rkgo Tuk Avr Avr o 299 295 zo bo Suv Mai Rpe s1u siu Sbip Aga Bem TK pech 305 310 (2) INFORMATION FOR POST. ID. Mo:4: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 314 amino acids (B) TYPE: amino acid

(B) CHAIN BRANCHING: does not matter (D) TOPOLOGY: does not matter (i) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQUENCE. Mo ID: 4:

Mee Rpe bip Rgo Aia b1u Niv sSiu bip Avr Tgr Aia Meb si siu rRgo 1 5 10 15

Ah Ar o biu em tsu Buv Mi Ag Petz Uai ABr o Azr Aga niv ABr Beg 20 25 zo sS1u rem Avr bek Mes Buv ABr sich Ar oTug bich bip Me Ua1i1i Buv s) 35 40 45 iem At siyaa Iie Agu Teo bij Rgo bip 10 Aia Rgo Bay dia Aia Jan 50 55 vo

Rgo Tr Bub5 beep beep Pes TNK s1u Ar sSiu A5r bek RBe Be Niv Bey 65 70 75 vo s o

Aza t1e T1e Niv biz siy Tsuzv TB ore Tr oMei bi mai i11e biu bip 85 90 95 " zo Uai tsTsuv s1u Azr Pecs Aia Rpe Gets dep Re S)p Avp o ABp o Bei bip Sip "-- 100 105 -110 m.

Tih Rgo rem Ni Pes Aza Uai Tie Tit dep sip Rgo b1u I1e dia siy o 3 115. 120 125 - " in s ;" -I (95) -I shu 20

Thursday"

F) ko 60 65 dia rey pe Buv A1ia siu Sub Avr Rgo bis Pec Acad Avr RPe Atud biu 130 135 150

ABp otvVg orRgo pe Ni be A1a Suz bii Sip biu Suv Pec Aza bBet Uvi 145 150 155 160 dia Uai rem tah o bip Tig osuv TB orRgo bip Nibye Gay Niv bek Mai Bey 155 1750 175 sSip dia Tako AbzpoTuh dep o siu Nib Tag o Sat Bez Niye Pec Aia beth i1e to 180 185 190

Niv biu Tug rap siu 716 Uai y0its Nie Pen Ua! TPppPgoHbets O1u Aza Ar 195 200 205

Uuai Avp o dia sSip bij Rko Suv Avp o piu Agu Tpt o Alia Sei Nibv em A1ia 210 215 220 sch o chai Avr bez) sip Avp o Rko ABr o Geh Uai bek Petz Pen Pes Buv Suv s1Uu 225 230 235 740 «

Aia ABr Uai ABp Ago Uai TPpt Tug osSip biu Tug b5ek Rgo Tut bip Gay "- 245 250 255 "a : so

TI Ttir o spiu Agu Rgo bei Tit Agu 71ie sbip bip bir Pe" Siu bip Gay 260. 265 279 y- tik opPets 010 A5p opech bip Tak Bei Rgo bi beg bi Avrobich si beg " du 275 280 285 z s "z Tukh Avr TI biz Beh bi) Rpe Tk biz Ar obi Be) Rhto Tuig Avr Ar 290 285 300 and Suv Uai Rne biu biu sip Ag4d Pez Tk This (95) 305 310

Ш- (2) INFORMATION FOR POST. ID. Mo:5: -o 70 () SEQUENCE CHARACTERISTICS: (A) LENGTH: 314 amino acids

T" (B) TYPE: amino acid (B) CHAIN BRANCHING: not important (G)

TOPOLOGY: does not matter 59 (i) TYPE OF MOLECULE: protein (hi) SEQUENCE DESCRIPTION: SEQ. ID Mo:5:

F) name) because b5

Meb Rre bSip Rgo Aza bij Rho biu biu a1mh Tgr o dia Mes biz p1iu Rgo 1 5 10 15

AgChch o AzroAia em was Guv Si Agu Bez Gay Avr Avr Ago Ni Ar Bbeg 20 25 30 siu pez Avr o bBei Me Kub A5r biz bi Tug biz bip Mee Uai Gpuye S1C 35 40 45 res Aga bis Iie Ak Pec bi Rgo soiy 56105 Aza ro Ago sbiu Aza si shchi 50 55 bo

Rgo TgroBu5 Sip bip ope Tip obi Azr o biu ABr Beig Rpe ie No Pec

AND

65 70 75 in

A1a I1ee I16e Niv si) si: pub Dia Pe) TPph Mes siy Uai Uai Aha a1py 85 90 95 sch o chai Bruz a1iu Avr obem A1ia Re Pei Abzp Re Ssip Av) Ap Pes siv bip 100 105 110 «

TVg Rkgo ep Niv Pem Aza Uai y1ie TVI Ap obip Rgo biz y16 Asia 01 "-- 115 120 125 chn so z5 Aia pes pes bis Aza biu Su5 ABr Rgo bib Tez Agu Avr Re Aga b1u m 130 135 150

Azp oti Rgo Bec Ni Bez dia Suv Si bip siu Suz Bez Aia beh Uai1 « 20 145 150 155 160 z s i "» sSiu Ua1i Pei Trgobip Rgo Aku biu Tpu o bip Niv em Niv beg 71e Pech 165 170 175 -I (95 ) -I shi 20

Thursday"

F) ime) bo 65 siy Aza TagoAvp otukh Avp o b)iu Niv Tk oSuv Bets Ni rech Aza beg I1e 180 185 180

No si1u Tug Without biu i1e Ua! siy Pe Pe Ua1 bBeh Pe O01Uu A1a Avr 195 200 205 uai Ab5p Aza bip Si Rgo Sub Abpos1iu Ag TVk o A1ia rei Ni5 Prep Ai1a 210 215 22 uai A5r Pec bib o Ap o Rgo Ar oGei Uaiii 5ect Bei Pec Bei Buv Sub 01u 9 225 230 235 240

Aia ABr oMaii ABpP oActo Uai Tph Tukhk bip bSiu Tuk bek Rko Tuk bip o Gay 245 250 255

TrI Tkr p1i1Uu Ahu VBgro bBetg TPg Aga I1e beep bid beep bez siu beep Bez 260 265 270 sch o

TI Pcez bets Abzp oBech bip Mee pecs Rhho biy bek bi. Avr battle 061 Bbeg 275 280 285 «

Tuk Avr Tit biz Beig biy Re Tu bib Avr o bish pev Rgo Tuk Avr o ABr «- 290 295 300 ichso

Suv wai Pe siu biu biv Agd Bey Tygoiets 305 310 th- (2) INFORMATION FOR POST. ID. Mo:6: () SEQUENCE CHARACTERISTICS: « (A) LENGTH: 2011 nucleotide pairs with | . (B) TYPE: nucleic acid c (C) CHAIN BRANCHING: single-stranded: c" (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: cDNA (m) SOURCE OF ORIGIN: - 15 (A) ORGANISM: Agabidorviv Ipaiapa (their) CHARACTERISTICS : (95) (A) TITLE/KEY: Misc. mark -1 (B) LOCATION: 1..2011 (M) OTHER INFORMATION / note - "MIM1 cDNA Sequence" - 2 (its) SIGNS:

I" (A) NAME/KEY: SO (B) LOCATION: 43..1824 (U) OTHER INFORMATION :/product- "MIM1 protein" rya (xi) SEQUENCE DESCRIPTION: SEQ. ID Mo:6:

F) name) because b5

SATSTSTRTA ATTTSTOAAT TTSAATTSAT SOSAASSTOT TO ATO SAS SS AS 54

Mes Ar Tikh Tpg

ATT SAT SOA TO OSS SAT ST TAT SAA ATS AOS AOS AST AST TS STO 102

Iiee Abzr biu Rpe Aza Avr beh Tut biz I1e bei bek TI bBet Rpe Uai 70 Z 10 15 20

OST ASS SAT AAS ASS O SAS TOS TStT ATT OTT TAT SR OSS OSS AV SAAd 159 dia tph AvBr o AbZp oTpg der bet beh T1e Uvi Tukh Pes Aza dia 5195 b1p 25 30 35

STA ste ASS OBA SST SAT OTA TStT OST Sto SAA OTTO STO TOSS AS dOaSss 198 uai ce Tpg obiu Rgo ABr UaZi bei A1ia Pe) bip Pe Gay Beh Up bekg 40 45 50

TTS SAA TSO OTO TT OAS TS SSO OAT SAT TI TAS AOS OS OST up to 745

Rpe Si Bek Uai Re Ar bek Rko Ar Ar Rpe Tukhk beg Ar dia was se 55 60 65 Go) stt tt sto tTos SAS sas so SAA sSTT TStT tt SAS SOS toSs st TtIS 254 thing Ua! tsem bBet Avr b1u Agu sii Ma) beg RBe Ni dgo Suv Uai Pec « 79 75 vo - u

TSsA SO ASA AOS OTST TT TTO AAb AS OST TTA SSS OSS OST AAS up to 342 so

Beg Aiia Ag Bet Bek Rpe Rre Dub bek Aza Pech dia Aiia lLia Muv Gu m - 85 of 5 100

SAS ADA SAS OTOS DAS AAS DSS OSS OSS sto DA STO OS STTOAddS SA 390 « si Sub Azr o BbbBei Azp o ABp oTVu Aia Aza mai There was em bim Bez There was bij -o 105 110 115 s with -I (95) -I - 50 "g" (F. ko) bo b5 a55 460 465

STO AST OST ASb AAS DSA DSA TSA SSO OST STA AAS ATA SA SsSsT TS 1494

Hech tng biu Taho uv At TvI begt oBro biu Uai Buv tZe Ata Rgo Re yo 475 480

APA ATO STA SAA SAS SAT SAA AST ASA STA DAA SSO STT TST o AAA ASo 1542 dgCh t1ye bez bii bi Ni bip beg Ago Be Buv Asia Bbets bei gub Tig 485 490 195 500

STO SAA Sto 0 AAA SOd TTS tt ss Sas tot to SA STO STO SOS 1590 wai b10 Bbem siu tsu Akd Rpe Rpe RIo Ak Sub Beh A1ia Uai Gech Avr 5О5 510 515

SAS ATT ATO AAS OTOT SAS SAS OTTO AST SAA STO OST OTOS OSA SAA SAS 1638 bip t1e Meb ABp Suv bij Ar o Geu Tgobip May Aza Suz S1iu Si Avr 220 525 530 Ge o

SAS AST OST SAS AAV OSA STA SAA AL AAS SALA dos TAS ATO SAA ATA 1686

Aer o tTpI A1iia sib Was dhu Pe spid gub Was pip Ak Tuk Meb bij It11e 535 540 545 " "-

SAA SAS ASA STA AASOADO SSS TTT AST SAS SAS AAT OTTO SAd'iTTA bSA 1734 che b1ip biz TVVI rey Buv Puv A1ia RBe bek sits ABr Ashp o Bey bib Ge) s1iu so 550 555 550 ' m.

JSC OTTO TSOS STO ASA SAT ORTSO AST OTST TOS ASA TSO AVA TSA DSS ost 1782

AzpoBech beg rem TP o Avr Bech Tig bBei bek Tig bek Ptsuve Betgt Tik sSiu « 55 570 575 zvo shi s z» SBA AAb Or TST AAS SOT AAA STO TST SAT sot sot Sov TO 1824 " siu tsu Ag bet Azp Aga o Bub this Bech No Aga Aga - 5 85 530 -and here Sustastas Tsttstasta Attttostototostata Tastststststttttz Atoatoastoo 1884 - and 20 Taastastatita Toastastostatotstosttaststtttttttttttttttt.

ATTTTOTTA 2011

F) (2) INFORMATION FOR POST. ID. Mo:7: o () SEQUENCE CHARACTERISTICS: (A) LENGTH: 2011 nucleotide pairs 6o (B) TYPE: nucleic acid (B) CHAIN BRANCHING: single-stranded (D) TOPOLOGY: linear (u) TYPE OF MOLECULE: cDNA (u) CHARACTERISTICS : b5 (A) NAME/BILE: СО5

(B) LOCATION: 43..1824 (G) OTHER INFORMATION: /product- "altered form of MIM1" / note - "Serine residues at positions of amino acids 55 and 59 in the natural MIM1 gene product are changed

TO alanine residues." (i) SIGNS: (A) NAME/KEY: different sign. (B) LOCATION: 205..217 (G) OTHER INFORMATION: / note - "nucleotides 205 and 217 changed from T to C compared to the natural 70 sequence." (xi) SEQUENCE DESCRIPTION: SEQ ID Mo:7:

SATSTSTTTTA ATTTOTOAAT TTSAAATTSAT SSOSAAASSTOT TO ATO SAS SS des 5a

MeE Avr o TntI Tpg 1

ATT SAT PERSON TT OSS SAT TST TAT ODA ATS ABS AOS o AST AStT tt ste 192

T11e Azr 01u RBe Aza Ar beg Tug bs)1sh Iie bBei Bek tPt bek Re uai 2 10 15 20

OST ASS SAT AAS ASS SAS TOS TST ATT STT TAT STO SSS OSS SA SAd 150 dia Tvh AbroABp Tu Avr obek obBet і11e Uai Here it is Aza Aza bits siv sch 25 30 35 (8)

STA STO ASS OSA SST SAT STA TST OST Sto SA) OTTO SRS TSS AAS dos 195 chai es tu piu RKO Azr Uai bZei dia tey pip grec Pec bek A5p beg "I 40. 45 50 "- cha so i - shi s ;" -I (95) -I - 50 s»

F) name) 60 b5

Pec o puv biu Ag Pezp Suv UMai Si I1ie Besh bich bip sich Avr o PCuB At 390 395 400

SAA SAd DTtT o SST ASA SAT o stt o sst SSS tet TTtT OSA sto bSo SS BAT 1302 Jan bip o IZZe Rgo Akgu AvroMa1i Rgo Rgo beg Rne Aia Uai Aiia Aia Ar 405 410 415 420

SAA OTTO AL ATO SO Sta STO bAT STE SAA AAT ASA TT SOSA STT OST 1359 bij rez Buz Mee Tk opem rem Azr o Pec bis Ap Ago Ma1i Aza vo Aza 425 430 435

SAA sSOaT StTtT TT SSA AS PAH OSA SALA OST OSA ATO PAS ATS OSS SAA 1398 sSip AgYA Bey Re Rgo Trg ob) Aia bip A1ia Aia Mes Si I1e v1ia 21 440 445 450

ATO AS OSA ASA TOT OA TTS ATA OTO AST AOS STO to SST SAS Set 1446

Mes puz biu Trh SuB bii Re I1ie Ua! Tu Beg Pep biy Rgo Avr Aga s aBj 46o 455 ge)

STO AST o сOT AS DA ASA ASA TSA SSO Ost SOTA AAb AtTA OSA SST TS 1494 thing Tpg o biu TK oЮu5 Atu Tk obet Rgo spiu Uai pub T1e Aia Rkgo Re « 470 475 480 «- -

APA ATS STA SAA SAS SAT SALA AST ASA STA AAAA SSO STtT TST DAA dos 1542 s

Aka Iie Pec si biz niv b1ip bBeg Ag pe Puv Aia bPec bek pu TP M 35 . 1485 450 495 500

STO SAA Sto bos A SSsA TT TT SSO SOS tot To OSA sto ST САС 15990 « 20 uai siy em b01u Pu5 Actu RbBe Rpe Rgo Ag Suv bBeg Aza Ua) Tec d5r z s 505 510 515 z

SAS ATT ATO AAS OTST OA SAS TO AST OSAA STO OST OTOS OSA sVA SAS 1638 bip ch11e Mee ABp Suv Siysh ABr Pec Tv obip Beh Aia Suv sa1u 61 Avr sh- 520 525 530 (95) -I SAS AST OST SAb AAA Sad STA SAA AAS AAS OSAA dos TAS ATO PAA AtTA 1586 -oUu 70 ABr Tpg A1ia biz duv Aga Pes beep Puye Puv sip Agud Tuk mes si 116

T" 515 340 545

SAA SAS ASA STA LA Aa OSS TTT AST SAS SAS DAT OTTO SAASTTA SSA 1734 99 bip bib TV opech Buv Guv ATa Re Bek S1SH Ar o ABp o bem bis res b1U

F) 550 555 550 ko) 60 65

AAT o TtTs tToS STO ASA SAT tob dAST TST TSS ASA TSO AAA TSA AS bat 1782

Avp o cem bZei cem Tr oAvr obeg o Trh obeh bek Tgobek Gub5 Bek Tyh b1U 565 570 375 580

SOA AAb o ASo TST AAS SST AAA STO TT SAT SsStT sot sob tod 18724 biu tsuv Aga bet Ab5p o Aga Buv Pets Beg No Aga Aku AkYa " 585 590

САСТСТТОС ТСТТАСТОТА ATTTTTROSTO TASSATATAA TISTOTTTTTS ATOATSАСТО 1884 t TAASTSTETA TOTSTATOSST TOSSOSTSATA TASTITSSSST STTSOTTTTO SATOSTOTOoT 1944

ATTATTOSTO SASOTSTOST TSADASAAAAT STTOTAASAAA TTTOAASSAA TOSTATASAS 2004

ATTTSTA 21 (2) INFORMATION FOR POST. ID. Mo: 8: () SEQUENCE CHARACTERISTICS: ch (A) LENGTH: 594 amino acids (B) TYPE: amino acid i) (D) TOPOLOGY: linear (i) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQUENCE. ID. Mo:8: « zo Mesie Avr Tiph TpE Iiie Ar biu Rre Aza Avr beih Tug 014 Iie Beh bBeg - 1 5 10 15 m

TrI Bek Rpe Ua1i Aia Tu Ar Ayep Tys Ar Beg bek yiye Mai Tut HCem at 20 25 zo -

A1a AtTa biz bip wai Pec tvg obiu Rgo Ar Ua1i bek A1a rech sSip Bec « 35 40 45 - s i z ets Bek Avp o5eh Rde biz Aza Uai Rpe ABr Aza Rko ABr Avr RBe Tukh " 5O 5 vo - -1 Bet Ar Kia Guz Pez Uaiii Pem bet Ar biu AkYaya bii wai bek rRnNe No o 65 70 75 vo - At tuh Mes bis i11e bip siy TBg opez was Buv A1ia Rpe bek 0310 Avr Avp t 545 550 555 560

ХТ» rem sim Pec Siu AyaepoBrech Bek Me TP oABr obeti TPg bek bek Trg obeg 565 570 575 o z Muz bek Tipg obiu Siu Buv Agd bek Abvp Ah Puv rey Beg Niv Aga Aha z8o 5a5 580 bo

Acc.

(B) TYPE: nucleic acid (B) CHAIN BRANCHING: single-stranded (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: cDNA (i) CHARACTERS: (A) NAME/BILE: СО5 (B) LOCATION: 1..1410 70 (M) OTHER INFORMATION: /product- "Changed from MIM1" /note- "M-terminal deletion compared with natural

MIMI1 sequence. M (xi) DESCRIPTION OF THE SEQUENCE: END. ID Mo:9:

ATO pAT TSo otTtTosotTo AST OST TTOo ST OTAT STI TAS AS AOS o ASA Sto av 75 Mes Avr Bek Uvi Mai TpPu Mai Me Az1a Tukh Mai Tuk beh bet Aka Uvi 1 5 10 15

ASA SSO SSO SST AVA OSA STI TST OOAA TOS OSA SAS SAS AAT TOoS TOS 96

Azhch Vgo Rko Rgo Puv biu Mai Bek bish Subv A1ia ABr S ABYSSuv Suz 20 25 30

SAS sto ost tOS sob Ssoeo oso Sto SAT TKS ATO TO SAS OTTO ST TAT 144 сч (8) « «- исо m. « shi s z -I (95) -I - 50 s»

F) name) 60 b5

No Mai A1ia Sub Aga Rgo Aza Ua) A5r Rre Mee pech sii Ua1i pe here 35 40 45

TT OST o tТtTS ATO TT DA ATS SST SAA TGA ATT AST STO TAT SAS AS 192 rei A1ia РPE I1e РPE Puv I16e rRgo 210 Pes І116e TPgoyets Tug bip Ago 50 55 5О

SAS TTA TO SAS OSTT STA SAS ALA STI STT AtTA bab SPAS ASA TT sTtT 240

Niv rap pech Avr oUuai Uai Avr o Buv Mai Mai t11e bij ABr TBh Gay Uvi 65 7o 75 vo

ATA STO AAS STT OST AAT o ATA TT SOT AddA OST tat ATO ADO STA To 28V

Iie this poo Without A1a AB I1e Sub siu Puz A1a Sub Mee Gu5 Gen Gay 85 Zo 35

SAT ABA tot AAA SAS ATT ATT STO AAb TST AAT OTA SAT ATO TT ASsT 136

ABr Aga Suz Was bi) I16 I1e Uai Was back AvBp uai AbBr Mee Ua1i bBeg 100 105 110 s o

STT SAA AASOTSA To SSO SA SAS STI STT ALLA bAb ATA ATT SAT ASd 384 yeech bib Buv bBet Bey Rgo bis biz bets Uai Bub5 bis i1e I16e Ab5r Ag « zo 115 120 125 -

SOT AAA SAB STI Sst TI SAS STA SST DAdd STA AAb o AAA SAT ST OTO 437 -

Ako Buv biu Bei biu Teo biz Ua) Rko Guz mai Bubz uv Ni Uvi BbBeg (ze) 130 135 140 im-

AAT OTA SAT AL OSA STT SAS TSo SAT SAT ATT SAS TTA ST AAb To 480

Avp o Mai NiB Tsuv Aia Petz Abzr beg dBr Azr i131e bits Petz Uai Muz Ben « 045 150 155 160 Z s :z» STT OTTO AAA SAS SAT SAS ASS AAT STA SAT SAT o bSo tot OST ST SAT 528

Bez Bey was M) Avr oNiv Tpt Azp orey AdB5r o Avr o Aza Suz A1a Sem Niv 165 170 175 -I o TTS OST TT OSA TATO TOS AAT Sto AAb o ASS bSA ASA SAT OSTT TTA ADA 576 -i Re Aia Ua1i A1ia Tukh Suv Ayep Mai pub TVg Aza TVgoAbr ope Geo was - 70 180 185 130

Thursday"

STT bAT STT BSS bAT STO AAS SAT ASO DAT SSO Or SbA TATM.ASOo oto 524 zv Tez ABr Pe A1ia Ar Maiii Avp Ni Aga dep Rgo AkYa biu Tut TnNg Ua) 195 200 205

F) name) bo 65

STI SAT TT OST OSO ATO SSO AAb SAS SSA SAV OTTO ATA STA TST STA 612

Petz Niv uai Aia A1ia Mee Ago pub Jan Rgo Sip Petz I11e Petz Beh Petz 210 215 2720

TS SAA AAdA SOT OSA AT OSA TA SOAA OSA AST OTTO SAVOSOT ASA Se t20

Gez bii Buz S1u Aia beg Aza bek pich Aia Tikh Pe bin biu Ak9 Tpg 225 230 235 220

SSA STO ATO ATS OSA AAA SAA SSS AST ATO SSS o StTtT SOVA TST LAT DAT 7658 dia Pec Mei i11e Aia PuB Sip Aza TB oMebse Asia Ma1i siy Suv Avp o AB t5 245 250 255

ATO SSS SAA TOS AAS SAT TST STO ALA SOS SSA STA TT OTA OA 816 2 11e Rhgo sich bip o Sub5 tsTsubv Niz bek this Buv biu AkChch Bez Suv Ua) sio 2590 255 279

ATA STA САС o САА Az САС AAA ССА САА СДА АТТ ССТ АСА SAT OsTT СсТ вва сч ch11e Bey bi sip bib AvBroBuv Aga biz b)ip ch11e Rgo Ako ABr Uvi rkgo o 275 280 285

SSS TSTtT tTtTtT OSA hundred oso OSS SAT SAA TTO Ado ATO SO STO STO SAT 912 "I

Rgo bek Re ATia Uai Aza Aia Ar sim Bec Pub5 Mes TI Pec Pech Avr «- 290 295 300 m

STT SAA DAT ASA STT OSA STT OST SAA SsT o StTT tTTT SSA DSO SAA ssSA zva o 39 rem beach ABp AKkd Uai Aia Pec diva bip At Bez Rpe Rgo Tr biz Aza - 305 310 315 320 «

SAA OST OSA AT SAS ATS SSS SAv ATO AD SSA ASA TOT Oda TO AtTA 1908 7 s bip Aia Aia Me 015 I1e Asia Si Meb uz biu Trg o Suv 116 ts 325 330 335 i

STO AST AS STO SAS SST SAS SOT STO AST SOT ASb AAO ASA SA TsSA 1956 -I Uuai Tig o bBeg rep siy Rgo Avr o Ahgo this tigoSiu Tik Buv Ago TTN Zeg so 340 345 350 -I tsu 5 SS ost STA AAb ATA OSA SST OTTS AbA ATS STA SAA САС SAT САА dot 1104 l Rgo biu Mai uv I1e Aia Rgo Ribe Agu IzZe resh biz beach Nib5 bip b5eg 355 z6o 365

ABA STA ALA SSO STT TStT AAlL ASS OTO OAA ST bob AvA SO Me TT 1152

GF) Aga ren Puv Aia Pez becht Buv TI Uai bij rap siu pub Ako Ve re

GI 370 375 zvo bo b5 sss SOS tot tTSo OSA STO STO SAS SAS ATT ATO AAS OTOT SAS SAS To 1200

Wgo Aku Suvz beg Aia uai rech Ab5r o bip i1ie Mes Ap Sub bi ABr yets

Z85 390 395 400

AST SAD Sto OST TOS SA PAA SAS SAS AST OST SAS AAA SSA STA SAA 1248

Tiu beep without A1ia Su5 biu C1) der db5roTvyg Asia biz Buv Agd Without beep 405 4150 415

AASOAAAS SAA ASS O TAS ATO SALA ATA SA SAS ASA STA AAo AAs soSs TT 1296

Guv Muv bip AgYa Tuk Mes b) Iziye bip bi TBgoBech Guv5 yuv Aza RBe t 420 425 430

AST OSA SAS AAT OTTO SAA TTA OOA AAT OTTO TOS STO ASA SAT OTO AST 1344 bek bij Azr Azp pev biz Pe biu Azp oBets beg rech Tit Ar beg TNg 4235 449 445

TST TOS ASA TObb AAA TSA SS SOT OA AAZ AOS TST AAS SOT AAA ST 11392 sch

Bek Beg TiPI bek PCuv bek Tyh siu si1u Ptsuv Agu bBet Avp Ako MuB Beh o 450 455 or

TOStT SAT SOT SOT sob tOA SASTSTTOSS TSTTAOSTOTA ATTITTTOSTO 1440 «

Beh Ni Ag Aga AkYa 7 | "- 455,470 hours

TASSATATAA TISTSTTTTS ATSATOASTO TAASTSTITA TOTSTATOST TOSOSSTSATA 1509 and -

TASTTTTSOST STTSSTTTTOa SATOSTOTST ATTATTOSTO SASOTOTOST TSAAASADAT 1550 « ю STTIOTAASAAA TTTOAASSAA TOSTATASAS ATTTSTA 1597 with p (2) INFORMATION FOR POST. ID Mo:10: "» () CHARACTERISTICS OF THE SEQUENCE: " (A) LENGTH: 470 amino acids (B) TYPE: amino acid (G) TOPOLOGY: linear - (i) TYPE OF MOLECULE: protein oo (xi) DESCRIPTION OF THE SEQUENCE: SEQUENCE. ID Mo:10: -1 beg No Ago Aha Aga "7 - 0 dB5 470 iz" (2) INFORMATION FOR SELF. ID Mo:11: () CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1608 pairs of nucleotides (B) TYPE : nucleic acid o (B) CHAIN BRANCHING: single-stranded (G) TOPOLOGY: linear ime) (i) TYPE OF MOLECULE: cDNA (i) MARKS: 60 (A) NAME/BILE: СО5 (B) LOCATION: 43..1608 ( D) OTHER INFORMATION: /product- "Changed from MIM1" /note- "C-terminal deletion compared to natural

MIM1." (xi) DESCRIPTION OF THE SEQUENCE: SECOND ID Mo:11: I b5

SATSTSTITA ATTTOTSAAAtT TTSAATTSAT SOSAASSTOT TO ATO SAS AS dos 54 months Abr tah Tapg

ATR BAT Both TTS OSS SAT TST o TAT SAA ATS DOS AOS AST OT ORI OTO 102

T12 ABr b1Uu Rpe Aia Ar bBeg Tuk bih Ii begt Bet TPg bBeti RBe Uai 5 10 15 20

OST ASS bAT AAS ASS OBAS TOSS TSTO ATT STtT TAT STO OSS ССС САА САХ 150

A1a Tk AvBr Abp TV Ar o bet bei I1ie Uai Tug Pec Aia Aza biz b1p to 25 30 35

STA STO ASS bbA SStT AT STA TST OST Sto SAdA OTTO STO TSS AS AOS 1938 Uai Bem tiph biu Rgo Ar ouaii beg A1ia rem bip rez rey beg Abzp beg 40 45 50

TTS SAA TOS STO TTT SAS TSS SSO SAT SAT TTS TAS AS PAS OST Ado 245 sch

Rpe siy bek Uai RCesAb5r Beg Rro Avr o ABr o RBe Tut bek Avr Aia Buv o 55 Bo 65 "I "- u (ze) - " o, s ;" -I (95) -I - 50 "g" (F) ko bo b5 stt o stt sto tToS SAS SOS sob SAA o stTt tot TT SAS oSos tTosS stTT TI 254 ivch Uai rem behg Avr siu Ahga biz Uua1i Zeig Re Niye Ag Sub chai Pec 7 75 in

TSA SS AOA AOS OTST TIS TT AL dos Ost TTA SSS OSS OST AAb AS 142 bek Aza Aga beht Beh Re Ripe Puv5 bet ATa Pec dia A1a Aza Puz Bu to 85 30 95 109

САС AAA САС TOS AАС АС АС Oso СС ОТО ALLO STO САС STR AAOo САС 390 siv pub Ar bek AzpoAbp o Tak Aza Aia Ma) Muz Bez bij Pe Buv 1 105 110 115

ATT OSS AAS SAT TAS ODA STO Ost TS SAT tob st sota ASstT stTT TI 4138

Iie Aza Buye Ar o tTui siy chai bSiu Rpe Azr bBehg Uai1i Ua) Tit Uai Gech 120 125 1360

OST TAT STT TAS AS AOS ASA STO AbA SSO SSO SST AAA SbA ORE OST 486 sch

Aza tut Uai Tut beg back Aga Uaii AkYa Rko Rgo Rgo Was siu Ma1i beg o 135 140 145

SAA TOS bSA SAS SAS AT oS toS SAS ost ost otoSs sos sso ssSsa sto 534 "I bio Sub Aza Avr biy AbpoSuv Su Niv Ua1 A1a Su Aga Rgo dia Uav1 "- r. 150 155 160 m (ze)

SAT TS ATO TTO SA OT ST TAT OTTO OST TO PBX TS AS PBX SST 582

Zo Ar Rpe Mes Pec bi Ua) Pech tTuhk Pech Aza Rpe 116 Rpe BuB I1e RgOo - 165 170 175 180 «

SAA TTA ATT AST STO TAT SAb AS SAS TTA tTtTO SAS OSTT TA BAS AvA 530 z s sich rey I1e tah Tez Tukh b1p Ag Nib rey pe AvroUa)! Mai Apr Buv y 185 190 195 i» 15 STT OTT ATA SAS SAS ASA TTO OTTO ATA STO Ado STTOOST AT ATA tt 57V -i chai mai I1ie beach der TT Hep Uai I1e pem Buv GPechp Dida Ap te Sub s 200 205 210 -I pz OST AAA OST TOST ATO AdO STd TTO SAT ASA TSOT DAA SAS AT ATT STO 7258 siu Bu5 Aia Suv Mee Puz MtsTsets cem Azr Ag Suz pub 010 116 Iie Uai » t 215 220 225 --

AAS OTST AAT STA SAT ATO STT AST OStTtT o SDA AAS OTSA TO SSO SAD SAS 7174

F! Puv Beh A5p oMaii Ayer Mes Ma) beg Bey piz Buv behg Pec Rgo bis sic ko bo b5

230 235 242 2 sStTtT OTT AAA SAS ATA ATT SAT ASA SST ALLA SAS sStTtT sot Ta SAS STA 822 rey wai pu siy I1e 712 Avr Agod Agya was b1ch Pe biu rech b1o Mai 245 259 255 260 to SST AAA STA AAS OAAA SAT STO TS DAT OTA SAT AAb OSA SsTT SAS To 870

Rgo Buv Mai Buv PCuv Niv Uai Beh Ab5p Mai Ni puz Aza geo ABr b5ethg 265 270 275

SAT bAT ATT SAS TTA STO Adb OTTO STI OTTO AAA SAS SAT SAS AS AAT 518

Ar o Avr ot11e Sich Ppech Uuai Buv Pec ray Bem Buz 51 Absr Ni Tit Up 280 285 290

STA SAT OT SSO tot OST STT SAT TT OST sStTtT OSA TAT TOS AAT STO 956 pech Azr o ABr o Aza Sub Aza Bemch No Rpe dia Uai Aza tug Suv Avp Uai1 295 300 305 s and Ge)

AO ACC OSA ASA SAT ST TTA AAA ST SAT STT OSS SAT OT AAS SAT 1014

Pukh TIK o Aia Tg o Ayar Bey rets PCub Bez Avr o Pets Aza Ar Ua! Ap No 310 315 320 "And "-

АСО AT SСО АС sОА TAT о АСОо STO STT SAT STT OST Co ATO CO As 1052 h-

Aktu dsp RKO Akou YaaTu Tut TI Zai cem Ni5 Mai A1ia Aia Me dko Buz s 325 330 335 za i -

CA ССА САА OTTO ATA STA TStT STA TTO САА AAVOSOT OSA AST OSA TSA 1110

Si Rgo bip Bechy Ii Bem Yaehk Pes Pes biz Buv Ssiu Aza bek dia Beg « 20 345 350 355 z s » SAA OSA AST OTTO SAA OST AbA ASS BSA STO ATO ATS OSA DAA SALA OSS 1158 syn Aia tru ope Si biu Ato TBgoA1ia pe Me Iiiie Aza iuv biv Aza 360 365 z7o -I (95) AST ATS Ob OTTO SAA TOT AAT AAT ATS SSO AS SAA TOS AV SAT OST 1206 -i TtTpk o Mee Aia Ud1i bbits Su A5p Azp I1e Rgo bij bip Suz Puv Niv beg pz 375 zvo 385

Thursday"

STO AAA SOS SOA STA TOT STA SAD ATA STA SAB SAA SAD pAS AllA SOSA 1254 ийеч ццувБ Siu At yem Suv Uai bij I1e Pez si sbip bid ABr Tu vho 99 390 395 400

F) ko SAA SAA ATT SST AsA SAT SoTtT SST SSS TST TT OSA hundred SSS SSS SAT 1302 bo b5

Jan bip 116 Rgo Agu dbr o wai Rgo Rgo Zeh Rre Aia Ma1i dia A1ia Ar 405 410 415 420

SAd OTTO AAO ATO ASO STO STO SAT StTT OAA AAT ASA STI OSA STT OST 1350 siy cem bukh Mes TtpI Bem rem Avrobets biz Abp Aga Mai A1a Bbets Div 425 430 435

SAD SST STT TTT o SSA AS SAA OSA SALA OST OSA ATO SA ATS SS SAA 1398 beep Agu this Rpe Rgo Tk siy Aia sip Aia Aia Mes biz i11e A1a siy 440 445 450

ATO AKA OSA ASA TOT SAS TTS ARA OTO AST AOS hundred oda SST ZAS SOT 1546 month was Z1u Tu oSuz bid Re I1ie Uai TNK bek yechp S1its Rko ABr Ago 455 460 465

STO AST OST ASO AAb ASA ASA TSA SOS OST STA AAS OATA OSA SsSsT TS 1494

Geo TnNI biu TBgoBuz Ak TBu beh Rgo biu ma! was I1e A1a Rgo Rie 470 475 avo cm o

ASA ATS STA SAA by SAT SA) AST ASA STA AAAA Wasp STE ST AdA OSS 1542

Ak і116ee Pech biy biz Nib sib beg AgYa Be Ptsu5 Aza Geny bek Buv TaG « zo 485 480 495 500 «-

ОТО BAА СТО Сб AAA СОА TS tt ССО СОС tot To ССА sto ste САС 1550 -

Uai bi Pesp biu uz Aqua Re RiPe Rgo Ag Suz bek Aza Uai rech ABr She 505 5190 515 -

SAS ATT ATO AAS OTTO TA 1508

Sip T1ie Mee Ap o Suv yah h 520 Z - (2) INFORMATION FOR POST. ID. Mo:12: a () CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 522 amino acids (B) TYPE: amino acid -I (G) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein o (xi) DESCRIPTION OF THE SEQUENCE: SEQUENCE. ID Mo:12: - pizza Vgo Abr Aga Pec TPg o biu Tbgopuye Aguu TBt o beg Rgo b1u Uai Buv - 00 465 470 475 480

Thursday"

Iie Aza Rko Rre Ago Iie Pec siyu Si niv bip o beg Ahuru Bez uv Aza 485 490 4z5 o Hey Bek Gu Tato Uai bib Bez b1u Puv AgCh RBe RBe Rgo Ak Suv Zeg dk 500 505 510 bo dia Mai1i cem Ar bip Ti May Up oSuB xx 515 520 (2) INFORMATION FOR MESSAGES ID. Mo:13: 65 () SEQUENCE CHARACTERISTICS: (A) LENGTH: 1194 nucleotide pairs

(B) TYPE: nucleic acid (B) CHAIN BRANCHING: single-stranded (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: cDNA (i) CHARACTERS: (A) NAME/BILE: СО5 (B) LOCATION: 1..1194 ( D) OTHER INFORMATION: /product- "Modified from MIM1" /note-"M-terminal/C-terminal chimera." 70 (xi) DESCRIPTION OF THE SEQUENCE: END. ID Mo:13:

ATO SAT TSO STT STO ASTOOTT OTTO OST TAT OSTT TAS AOS AOS ASA sto 48

Mes Ar Beh Ma! Mai Tyh Mai Pez Aia Tukg Mai Tuh Beh Beh Akad Mai 1 5 at 15

АСА ССС ССО ССТ AAAA OSA STR TST САА TOS OSA САС САС AAT ToS TOs 96

AkYa Rgo Rgo Rgo Tsuv bi1u Ma1 bek bi Suv Aza Azr sin Ap o Subz Suv zb 20

SAS hundred SStT o TOosS sob ss oso hundred SAT TKS ATO TO SAS o sStTT Sto TAT 144 niz Uai Aza Suz AhuU Rko Aza Mai A5r Rpe Mes ce bi Uai rep Tukh s 25 ge) « «- che so v. " o) c ;" -I (95) -I - 50 s»

F) name) 60 b5

35 40 45 s tt sst o Tto ATS TTS AAS ATO SST SAA TTA AT AST STO TAT SAb lob 132 ietsp Aza Re Ize Rpe Buv I1e Rgo bi Pes 11e tk otsei Tukhg bip At9

BO 55 6o

T0 САС ОтТТТА TTO САС ОСТТ STA САС ОААА СТТ ST? ATA SAS SAS ASA Tto sTT 240

Niv me Petz Ayer o Mai Ua1i Abr o Puye Ma! Uai Yi1e biz Azr Tak Tech Uvi 55 70 15 at 15

ATA STO AAb StTT OST AAT ATA TOT SOST AAA OST TOT ATO AAS STA TI 285 tI1e Bec Buv Bems Aia Abp oi1e Su5 si1u uz Aiia Sub Me Pyuv5 Pe Gecz 85 90 95 20

SAT DSA TOT AAA SAS ATT ATT ATT Sto AL TST DAT STA SAT ATO TT AstT 336

Avr Aho Sub was 010 t11e Izie Chai Puv Bek ABp oMai Avr Mei Mai Bek 100 105 110 sch 25 o

STT SAA AAO TSA TTO SSO sAA CASS STT STT AddA SO ATA ATT SAT DOA 384 rech bid pu5 Bek Pec Rkgo biz bi Bec Uai Bub bij i1e I1Tee Ar Ago 115 120 125 « 30 -

SST AAA SAS ST osot TO BAZ STA SST AVA STA AAb AAAA SAT ost OTSo 437 m

Aga pu b10 Pen siu rem si Uua1i Rgo Buv uai BuB Buv Nib uai beh 135 135 140 o 35 m.

AAT TA SAT AAS SsSA STT SAS TSO SAT SAT ATT SA TTA STO vdo to 480

ABpoUai Ni Puv Aia Bez Azr o BbBetg Abr Ar t1eeee beach Pe Udi Buv Bey « 145 150 155 160 40 - s z» STI TT AAA SAb SAT SAS ASS AAT STA SAT SAT Oso tot OST STAT SAT 528 " oven without tsub5 sii Avr NivB TrI Ayeporets Abzr Ar Aza Su Aia Gech Ni 15 165 i170 115 -I syu TT OST OTT OSA TAT TOS AAT STO AAb OASS OSA ASA SAT STE TTA DAV, 576 - Rpe Asia Mai Aza Tuk Suv Azp oUMai Buz Tort Aiia Tit AvBro without rez Guz 180 185 1990 shu 20th» STT OAT STT OSS SAT STO AAS SAT OR DAT SSO ASOO OSA TAT des sto 624 ed AvroPez Aia AzroUai Ap o Ni Agu Abzp oRgo Aga a1u TukZTrk o Uai 195 200 205 o

GF STT SAT stTtT OST o 0sSo ATO SOS AS SAS SSA SAD OTTO ATA STA TST STA et2 60 65 rep No Uai dia A1ia Meb Ago Buv Si Rgo bip Gay T1ie Pech Beh Pets 7210 215 220

TTO SAA AAA SOT OSA AST SA TSA SAA OSA AST OTTO BSAA ST ASA SS 720 rez bij guv sSiu dia bet Aia beh biz Aza TBgopech bi siu Ag tak 225 230 235 240

SSA STO ATO ATS OSA ALLA SALA SSS AST ATO SSO StTT SAA TST AAT AAT 768

Aza rep Mes t11e Aia tsuv bip A1ia TI mes dia Uaii bij Sub Up dep 245 250 255

ATS ССС САС САА ОТОС AdОо SAT TST ST AAA СОС ССА STA TOT STA САА ИБ

I1e Rho beach bsip Suv Buv Niv bet Pei Tsuv piu Ak Pe Su Uai bij 260 265 270

ATA STA SAS SAD bSAA SAS AAA SBA SAA SAD ATT SST ASA OATOSTT SStT 864 11 rech sich bip biz Azr Puv Ato biz bip t11e Rho Agu AvBr ouai Rgo re 275 280 285 o

Ssoes tet TT OSA hundred oso OSS SAT SA; TTO AAb Dt AS hundred hundred SAT 312

Rgo bek Re A1a mai A1a A1ia Ab5r persons Becp duv Mes Tph Gech Pem ABr « 280 295 300 -

STT SAA AAT ASA STT OSA STT OST SAD SST OSTT TTT SSA ASO SAD OSA 960 -

Petz biz Avp Aga Ua)! Aza Pets Aia sip Alya rey Rpe Rgo Tpu b1h Adi i. 305 310 315 320 -

SAA OST OSA ATO SAS ATS OSS sAA ATO AAbOSOdD ASA TST SAS TT AtTA 1008 bip Aza Aza mes bib Iiiiie Aza Sp)3o Meb buye biu Tk o Suv bi) Rpe 116 h 70 325 330 335 n- s;» sta AST AOS ST SAS SST SAS SST STO AST SOT ASO ANA DSA ASA TSA 1056

Uuai TOPI bek Ppei bij Rgo Avr At Bei Tvgobiu Tikh Buv Aga Trk o behg shchi 340 345 350 (95)

SeoZ OST OTA AAb ATA OSA SST TT APA ATS STA SAA SAS SAT SAA dOot 1104 7 Rgo siu Ua1i! tsuz T1e Aia Rgo Rie Atu i1e Pech bib biz Niv bip 5eg - 355 360 365

I" "

ASA STA AAA SSS STTOTST ADA SS OST SAA STO SOS AAA SSATTIS TIE 1152

Aha Bech pu Aia pey Beh Guv Tig o Ua! b) Without biu Bub5 Ag RNe Re o 170 375 380 ko sso SOS tot to sСsSA STO STO SAS SAS ATT ATO AASOTOoT To 1194

Rgo AkgCh oSuv Beg A1ia Mai Bez ABr bip t11e Meb AvpoSuv " bo 35 390 395 (2) INFORMATION FOR SEQUENCE ID Mo:14: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 398 amino acids 65 (B) TYPE: amino acid ( D) TOPOLOGY: linear

(i) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ. ID Mo:14:

Mes ABr bei Ua1i Uai Tu Uai rem Aia Tukh Uaii Tuk bek Bek Aku Ua1 9 1 5 10 15 vkCh Rgo Rgo Rko Pub5 p1u Ua)! Bek bitz Suz Aia A5r bij Ap Suz Suv 20 25 30

No Chai Aiia SuB Agu Rgto A1ia Uaii ABr Rpe Mes Bez pizza Mai rem Tug 35 40 45 cem Aza RBe I1e Rre PCub5 I1e rko s0310 Sword I1e TBg Pecs Tug bip Aga 50 55 6o

Ni Ge Bech ABr o Mai Uai Azr uv Uai Uai i11e 010 Avr o Tg oiez Mai 65 7o 15 in t11e Ge Puv this Aza Azp oI1e Sub b)u Buv Aia Suv Mee Buv Pem rech s 85 90. 35 o)

Avr o Ago Sub uv bij I1e ITe Ma1i1 Tsuz beg ABp Ua) Avr o Mee Ua) beg 100 105 510 Z «- tsech bis Buv Bek Bemsh Rgo bij biz Pecs Uai Bbuz bis I1e I16 A5r Agd i- 115 129 125 (ze) h- h-

Aha tsuz biz Bei siu yesh biz Uai Rgo Buv UMa1i Tspuv Ptsuv Niye Uuai beg 130 135 150 «

With c ;" -I (95) -I shu 20

Thursday"

F) ko 60 65

AzpoMai Niye pub AzZa GPez Avr becht Abr Avzr 116 bij Pesh Uai Puv peh 145 150 155 160

Petc cem tsu sich ABr No Tk oAbzpoBec AvroABr Aza Suv Aia pe No 155 170 175

Ripe lia uai Aia Tuk Sub ABp o Uaii Was Tpu Aia Those Avr Without them Was 180 185 190 yeez ABroBrets A1a Avr oMa1i ABpoNibv Ag Ap o Rgo Ago o biu Tukh Tik Uvi to 195 200 205 rez Niv Ua1i dia dia Mes Agu Bu 014 Rgo beep bet 116 ben bet ben 210 215 220

Pech biz Puv s1iu A1ia beg A1ia bet sii Aa TBt Without pech a1u Aha TBGg 225 230 235 240 sch

Gee)

A1a rep Mes I1e Aia Pu5 bip ATa TrtI Mee A1a Uai Sits Suv Avp Avp 245 250 255 «

Iie Rgo bii beep Suv Tsub5 Niv bei yeech MDub5 b1u At rep Suv Ma1i s1) «- 250 255 270 M (ze) t11e be si beep b10 Avr oPuv Aga bi beep 116 Rgo Aga Avr o Uaiii Rgo 275 280 285 i-

Rko b5eht Rpe dia ua1і1 Aia A1ia Ar sii Pes yub5 Mesye TNh Pec Pem ABr « du 230 295 300 sh-v s "z iz biz Abp odho Uai Aza pes A1ia bip Ahu bez Rpe Rgo tTPpu biv dia 305 310 315 320 - bip Aza Asia Mes bis Ii Aia bish Mes Tsuv biu Tig Suv sich Rce Iie (95) 325 330 335 -I -oUu 70 Ua1i Tyh Beh Hech biy Rgo AbBr Ak Ge) Tg o biu Tpg Buv Ahko Twk beg

GT" 34 345 350

Rgo siu Ua1i Buv I1e A1ia Rkgo Rbe Aga I1e re sim bii NietO1p 5eg 59 355 zvo 365 (F) ime) Ah Petcsuv A1a Pes beh Puv Tyh UMai1i Si beo biu Shuv Ak RNe Rre 60 470 3785 380

Rgo Agu Sub Yaog Aza Ma! Pecs AvroSip 11e MeeE ABp Sat x 385 390 395 b5 (2) INFORMATION FOR POST. ID. Mo:15:

() SEQUENCE CHARACTERISTICS: (A) LENGTH: 786 pairs of nucleotides (B) TYPE: nucleic acid (B) CHAIN BRANCHING: single-stranded (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: cDNA (i) CHARACTERS: (A) NAME/ BILE: СО5 70 (B) LOCATION: 1..786 (GI) OTHER INFORMATION: /product- "Changed from MIM1" /note-"Ankyrin domains of MIM1." (xi) DESCRIPTION OF THE SEQUENCE: END. ID Mo:15:

ATO SAS OTSS AAS AAS ASS o sss ssSS Ogo DA ST SAS STT AVS BAS ATT AV /u5 Mee Ar Bek Avp Avp TrI Mia Aza Ma) Puv yets biz Gay PuB sich I1e 1 5 19 15

SSS AAS SAT TAS SAA ST SsT o TtTo SAT TOb stTt sto AST st tt ost ze dia tsub Avr o Tut biz Mai siu RBe Ayer bek Uai Uai Tpgr oUMai cem A1a 20 25 zo

TAT STT TAS AOS AS ASA STO ASA SSS SSO SST AAA OSA STT TST OA 144 s

Tut Mmai Tukh Beh bek Ag Mai Akgod Rkgo Rgo Rkgo tsuz sS1u Ua1i beh siy o 35 40 45

TSS SA SAS SAS JSC o TOoS TOS SAS hundred ost tTOoSs sob SSO So ST SAT 192 «

Suv Aia Avr siy Ap Suvz Suz Niv Mai A1a Suv Ago Rgo dia Ua1 ABr - zo 55 50 - cha so

TTS ATO TTOo SAS o sTT Sto TAT TT OST TTS ATS OTTO AAb ATS SST SAA 240 Kk vpe Mes Pez bi Mai Pekhz Tut tsem Aza Rpe Tie RBe Buv T1ee Rgo sits "shi s;" -I (95) -I - 50 s»

F) name) 60 b5

65 70 75 in

TTA ATT AST STO TAT SAS DOP SAS TTA TTO SAS OOTT OTA SAS AVA STT 288 rep tIie Tpgotsech Tug b1ip Aku No Pem rem AvroUai Ua) Azr o Buv Uai 85 90 85 710 STT ATA SAP SAS ASA TT OTTO ATA STO AAs STT OST AAT ATA tot OT 136 cha) ti1e Sits ABr o tTygoiech Uai Y1eeee cem Puv Pem Aza Ap 116 Suv s1iu 100 105 110 12 AAA OST OTOT ATO AAS STA TTO SAT ASA TOT AAAA SAS ATT ATT STO AAb 384

Puv Aia Sub Meb Buv Pec Pec Avr o Ago SuvB Buv sich I1ie T1e Uai Guz 115 120 125

TST AAT STA SAT ATO STT o ASTT o STT SAA AL TsSA TTO ССС сАА ОДо СТТ 4172

ShZet AzpoUai AzroMes Maii Beh Pec (1 Was Zec Pec Rgo seven b)m Gay 130 135 140 s

STT AAA SAS ATA AT OSAT ASA SST AAA SAS OST SOT TO SAS STA SST 480 (8) wai Puz bi t11e 112 Ar Aga Aga Buv SR Ppech siu Pes bis Uai Rho 145 150 155 1650 " "-

ADA STA AAb AAA SAT SRS TSS AdtT STA SAT AAS OSA STI BAS TS SAT 528 mtsuv uai pub Puv Niv Uuai beh Avp Ma1i NibB Buv Aza Pec Avr 5eg Ar - 165 170 175 so i -

SAT ATT SAS TTA STO AAb OTTO STT OTTO AAA SAS SAT SAS ASS AAT STA 575

Avr o t1eee bib press Uai Tsuz rez Ppem Pec uv biz Abr Ni TBgodvp Gay 180 185 190 " o, s SAT SAT oso sRBOOtT OST StTT SAT TT ssStT stTtT SA TAT o TOS DAT STO DAO 524 :z" Ar Avr o Aia SuB Aia Petz Niv Re Aza ua1 Aia Tug Sub A5p Uai Buv 195 200 205 -I

ASS OSA ASA SAT STI TTA AAA STT SAT STT SSS SAT STO AAS SAT dos 672 o Tykh A1a Tphu Azr obech yey pub Pecs Azr o Bem Aia dor Uuai Avp Ni Aga -i 210 715 220 - 50

T» AAT SSO Or SSA TAT ACC hundred STT SAT sSTT ost Sat ATO SSO Av to 729

Avp o Rgo Agu Siu Tukh Trh UMai Ptsep Niye Uai Aiia dia Mebye Ag pu Jan 225 230 235 240 o SSA SAA OTTO ATA STA TST STA TTO SAA AAA SST OSA AST SSA TSA SAA 768 name) bo b5

РрРго бип цей и1е рем бекг Пем Пец бий руб биу Ая бехт Ая бех Sim 2745 259 255

ССА АСТ ОТТО САА ОСТ To t8e dia Trg o pey biz b1u 260 (2) INFORMATION FOR POST. ID. Mo:16: () CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 262 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ. ID Mo: 16:

Mei ABr o Beg Ap o Avp oTyg o Aza A1a wai There was a thing 10 pei There was a Sim IІ1Е 1 5 10 15

A1a tsuv Ar Tukh bi Uaiii b1iu Re Azr bek mai Uuai Tpg Uai Pem dia 20 25 30 s 29 Tukh uai Tukh bekt bek dgd Ua! Ag Rgo Rgo Rgo Iz biu Mai check Ss1ts (o) y « zo Suz Aia Ar biy AvpoSuv Suye Ni Ma1i Aia Suv Agud Rgo A1ia uai Avr bo tb i-

Rpe Mebs pe si Mai ce Tukh Pep Aza RBe Ii116 Rpe iuv I1e rRgo biuts i 35 65 70 75 vo M

Tez I1ie Tk rez Tug Sip Akud Niv tez pech Ar o Uai Mai Avr was Udi 85 en 35 h 40 shi s Uai Iie bij Avr Tiig Pec uai 116 rep puz Bez dia Ab5p oi1ee Su« siu :» 100 105 110 -1 35 Pub5 A1a Sub Me Guz pech Bez Avr o Ago Suv Puv sii I1e i1e uai Buv 115 120 125 (95) -I - 50

Thursday" 55

F) name) because b5

Bek ABp Mai1 ABr Mes Uaiii Beh Bez sin Bek Bez rRgo beach 51 Be 130 135 140 chai Puv Sich 116 i1e A5r AkYa Ak Beb bis Bez s01u Bez bis Uai rgo 145 150 155 160 tsuv Mai uv Gu Ni Uai bek Abvp oua1i Ni Ppuv AtTa Be Ar Beh Azr 165 170 175 75 Avr o i1e bij pe Uai puv rem Bez Pec Il/ib5 Sich Azr o Ni Taho dvp Bey 180 185 190

A5r ABr Aza SuB A1a Pes Ni Rpe A1a Uai dia Tut Sub5 Avp oUa1i Gu 20 195 200 205

TBI Asia Ttg o Abropei tsets pu Pec Avr o Gay A1ia ABr uai Ap Niv Ag 218 215 220 s 25 o

Ap oRgo Ak siu Tug Tpu Uai pech Niv Mai Aza A1ia Meb Agu tsu Si 225 230 235 240 « 30 Rgo bip Mei I1iee Bey behg Pec Pech sit Buv Sbiu dia beg Aza bek SsSI1ts -- 245 250 755 i - so dia TOh ie siy b shchi 35 1U and - 250 (2) INFORMATION FOR POST. ID. Mo:17: () CHARACTERISTICS OF THE SEQUENCE: "(A) LENGTH: 41 amino acids with 40 s, (B) TYPE: amino acid s (C) CHAIN BRANCHING: does not matter :z" (D) TOPOLOGY: does not matter (i ) TYPE OF MOLECULE: peptide (hi) SEQUENCE DESCRIPTION: SEQ. ID Mo:17: -i tI1ie Ago Agu Meye dGYA Ago A1a Greek Avr Aia A1a Ar t1e bi Hey Uai (95) 1 5 10 15 -I shu 20 tsub ce Me UMai Mes biu si biu Bsyush AzroBez Avr o Avr o Aza Pe Aza with

Uuai Niv TUuk Aia uai sSip Nib Sub Avzp 15 "9 (F, (2) INFORMATION FOR SEQUENCE ID. Mo:18: ka () SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 amino acids in (B) TYPE: amino acid (B ) CHAIN BRANCHING: not important (G) TOPOLOGY: not important (y) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID No. Mo:18: b5

Rkgo TVt o siu Puv TBh Aza Pe Nib Pec A1a Aza bi; Mee Uai bek Rgo 1 5 10 15

ABr Mes Ua) Beh chai rep Bretz Avr Niv Ni Aza Avr Haa Ap RPpe Ag 20 25 30 70 TBVI Haa Abr s1u wai Tng 35 (2) INFORMATION FOR POST. ID. Mo:19: () CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 41 amino acids (B) TYPE: amino acid (C) CHAIN BRANCHING: not important (D) TOPOLOGY: not important (i) TYPE OF MOLECULE: peptide - (a description of the sequence : POST ID Me:19:

I1e Ahku Aga Mes Ag Ag Aiia Bey ABr Aza Aza ABr t1ie sich Hey Uai sch 1 B 10 15 o

Bu pem Mes Ua1ii Mes s1u bi siu Pes Abzropetsy Ar Avr A1a Gay dia 270 75 30 «g «-

Uai Niv Tuk Aia Ua1i bip Ni5z Suv Ap k (2) INFORMATION FOR POST. ID. Mo:20: so 35 () SEQUENCE CHARACTERISTICS: - (A) LENGTH: 27 amino acids (B) TYPE: amino acid (C) CHAIN BRANCHING: not important « (G) TOPOLOGY: not important 40 (i) TYPE OF MOLECULE: peptide - c (xi) SEQUENCE DESCRIPTION: SEQ. ID Mo:20: z" Aka Aho Rko Avr obeh Buv Tit dia Pem No Bez Aza A1ia Siish Mee wai 1 5 19 15 - Bek Rgo Avr Mes Uai Bbet Ua) this ray ABr bip (95) 20 25

Sh- 24. (2) INFORMATION FOR POST. ID. Mo:21: -o 70 () CHARACTERISTICS OF THE SEQUENCE:

GT" (A) LENGTH: 41 amino acids (B) TYPE: amino acid (B) CHAIN BRANCHING: not important (D) TOPOLOGY: not important (y) TYPE OF MOLECULE: peptide

GF) (xi) DESCRIPTION OF THE SEQUENCE: END. ID Mo:21: name) 60 b5

Izye Agd Agu Mes vVgu Ak Aza Pem ABr oAia Mia ver ch11e bij Pec Mai1i 1 5 19 15

Tuz rap Mes uai Mmeye Siu bii biu Bei Avr Pem Ar ABr Aia hechsh dia 20 25 30 wai niv Tug Aia Uua1! Ssip NiB Suv Avp 35 chn) (2) INFORMATION FOR POST. ID. Mo:22: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 amino acids (B) TYPE: amino acid (B) CHAIN BRANCHING: not relevant (D) TOPOLOGY: not relevant (y) TYPE OF MOLECULE: peptide (xi) DESCRIPTION SEQUENCES: SECOND. ID Mo:22:

Ako Agu Rgo ABr obBet PtsuB Tpt Aia Bay NivB Gay A1a dia b1z Mes Uai 1 5 10 15 s

Zek Rkgo ABr Meb UMaii bek Ua! Pei Mech Avr beep at 20 25 (2) INFORMATION FOR POST. ID. Mo:23: « zo () CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 41 amino acids -- (B) TYPE: amino acid m (C) CHAIN BRANCHING: not important (D) TOPOLOGY: not important co (y) TYPE OF MOLECULE : peptide m (xi) SEQUENCE DESCRIPTION: SEQ. ID Mo:23: Ah Ago Mes dgo Agu Aia Ge Avr Aza Asia AbBr o t11e 010 Pen Uai 1 Z 10 15 " - s uv Gez Mes uai1 Mee siu bichts siUu Be ABr Mei der Avr A1a Pech dia 20 25 30 ;" wai niv tTukg Aia Mai bip Niv Suv Avp

Ш- 35 40 o (2) INFORMATION FOR POST. ID. Mo:24: -I () CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 amino acids - (B) TYPE: amino acid "y" (C) CHAIN BRANCHING: not important (D) TOPOLOGY: not important (y) TYPE OF MOLECULE : peptide 5B (xi) SEQUENCE DESCRIPTION: SEQ ID Mo:24: o Rho Tk biu pub TI A1ia rep Niz Pec Aia A1ia bi) Mes Uuai beh rRgo z 1 5 10 15 vo ABr Meiese Udi (2) INFORMATION FOR SEQ. Mo ID: 25: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 nucleotide pairs (B) TYPE: nucleic acid bo (C) CHAIN BRANCHING: single-stranded

(7) TOPOLOGY: linear (its) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: /desc. - "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ. ID. Mo:25:

СААСАССТТС САдОосСссИСТ TTOАСОСОСС ОБАТО 35 (2) INFORMATION FOR POST. ID. Mo:26: () SEQUENCE CHARACTERISTICS: 70 (A) LENGTH: 35 nucleotide pairs (B) TYPE: nucleic acid (B) CHAIN BRANCHING: single-stranded (G) TOPOLOGY: linear (its) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: /desk. - "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ. ID Mo:26:

SATSSSOSOS STSAAASASO SSTTSSAAOS tTotto 35 (2) INFORMATION FOR POST. ID. Mo:27: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 pairs of nucleotides (B) TYPE: nucleic acid (B) CHAIN BRANCHING: single-stranded (G) TOPOLOGY: linear (its) TYPE OF MOLECULE: other nucleic acid ch 29 (A) ) DESCRIPTION: /desk. - "oligonucleotide" Ge) (xi) SEQUENCE DESCRIPTION: SEQ. ID Mo:27:

ССААТСААТ ССАТТСОСТ ОТОАСТОТТТ To 12 (2) INFORMATION FOR POST. ID. Mo:28: "I () CHARACTERISTICS OF THE SEQUENCE: - (A) LENGTH: 28 pairs of nucleotides (B) TYPE: nucleic acid - (C) CHAIN BRANCHING: single-stranded (D) TOPOLOGY: linear so (her) TYPE OF MOLECULE: other nucleic acid acid i- (A) DESCRIPTION: /desc. - "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ. ID Mo:28:

SSAATTSTAS AAATSTOTAT ASSATTOOo | 28 « du (2) INFORMATION FOR POST. ID. Mo:29: z () SEQUENCE CHARACTERISTICS: c (A) LENGTH: 31 pairs of nucleotides: z" (B) TYPE: nucleic acid (B) CHAIN BRANCHING: single-stranded (G) TOPOLOGY: linear - 15 (its) TYPE OF MOLECULES: other nucleic acid (A) DESCRIPTION: /desc. - "oligonucleotide" (95) (xi) SEQUENCE DESCRIPTION: SEQ. ID Mo:29: -i SSOSAATTSOSA TOTSTITAAT TTOTODATTT С з -к 70 (2) INFORMATION FOR POST. ID. Mo:30: () CHARACTERISTICS OF THE SEQUENCE:

T" (A) LENGTH: 29 pairs of nucleotides (B) TYPE: nucleic acid (B) CHAIN BRANCHING: single-stranded (D) TOPOLOGY: linear

HF) (her) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: /desc. - "oligonucleotide" o (xi) SEQUENCE DESCRIPTION: SEQ. ID. Mo:30:

SSAATTSTSA ASLattSATA DTSTOSTSOYA 29 60 (2) INFORMATION FOR POST. ID. Mo:31: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 nucleotide pairs (B) TYPE: nucleic acid (C) CHAIN BRANCHING: single-stranded 65 (G) TOPOLOGY: linear (its) TYPE OF MOLECULE: other nucleic acid

(A) DESCRIPTION: /desk. - "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ. ID Mo:31:

SHAATTSAAT SVASTOSAAS DASASSOoSSO C 3 9 (2) INFORMATION FOR POST. ID. Mo:32: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 nucleotide pairs (B) TYPE: nucleic acid (B) CHAIN BRANCHING: single-stranded (G) TOPOLOGY: linear (its) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION : /desk - "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ. ID Mo:32:

Claims (29)

The formula of the invention 1. A method of protecting a plant from damage by pathogens by synergistically increasing disease resistance, which includes the following stages: a) obtaining an immunomodulated plant that has the first level of disease resistance, and the immunomodulated plant is: () a mutant plant that carries a constitutive immunity gene ( sit), (i) a mutant plant that has a gene that causes a lesion that resembles a disease, (ii) a plant that was obtained by recombinant expression in a plant of a gene with systemic acquired resistance (SAC) and b) treatment of an immunomodulated plant with at least one a microbicide that creates a second level of disease resistance, wherein treating the immunomodulated plant with the microbicide creates a synergistically enhanced third level of disease resistance that exceeds the sum of the first and second levels of disease resistance. " 2. The method according to claim 1, in which the indicated sit-mutant plant is selected from the population of plants in accordance with the following "- stages: a) evaluation of the expression of ZAC genes in uninfected plants that are phenotypically normal, which means the absence of the phenotype in the indicated uninfected plants , which resembles the damage caused by the disease, and b) carry out the selection of uninfected plants that constitutively express ZAC genes in the absence of viral, bacterial or fungal infection. in. C. The method according to claim 1, in which a mutant plant having a lesion that resembles a disease is selected from a population of plants according to the following stages: a) evaluation of BAC gene expression is carried out in uninfected plants that have a phenotype that resembles a lesion "disease" and b) carry out the selection of uninfected plants that constitutively express the genes of ZAK in the absence of viral, bacterial or fungal infection. "» 4. The method according to claim 1, in which the BAK gene is a functional form of the MIM1 gene, which encodes the MIM/1 protein, "involved in the signal transduction pathway that leads to the creation of systemic acquired resistance in plants. 5. The method according to claim 4, in which the MIM1 protein contains the amino acid sequence presented in SEQ. Mo ID: 2. 6. The method according to claim 4, in which the MIM1 gene contains the coding sequence presented in SEQ. Mo ID: 1. - 7. The method according to claim 1, in which the BAK gene encodes a modified form of the MIMI protein, which acts as a dominant-negative regulator of the ZAK signal transduction pathway. 8. The method according to claim 7, in which the modified form of the MIMI protein has alanine residues instead of serine residues in - positions 55 and 59 of the amino acid sequence presented in SEQ. Mo ID: 2. -oUu 70 9. The method according to claim 8, in which the modified form of the MIM1 protein contains the amino acid sequence presented in the SEQ. Mo ID: 8. driving" 10. The method according to claim 8, in which the modified form of the MIM1 protein is encoded by the nucleotide sequence presented in SEQ. Mo ID: 7. 11. The method according to claim 7, in which the modified form of the MIMI protein has an M-terminal shortening of the amino acid 2o sequence, which approximately corresponds to the position of amino acids 1-125 in SEQ. IDMOo:2. at 12. The method according to claim 11, in which the modified form of the MIM1 protein contains the amino acid sequence presented in the SEQ. Mo ID: 10. yu 13. The method according to claim 171, in which the shape of the MIMI protein is changed! encoded by the nucleotide sequence presented in SEQ. Mo ID: 9. 60 14. The method according to claim 7, in which the shape of the MIMI protein is changed! has a C-terminal truncation of the amino acid sequence, which approximately corresponds to the position of amino acids 522-593 of SEQ. Mo ID: 2. 15. The method according to claim 14, in which the modified form of the MIM1 protein contains the amino acid sequence presented in SECOND. Mo ID: 12. 16. The method according to claim 14, in which the shape of the MIMI protein is changed! encoded by the nucleotide sequence 65 presented in SEQ. Mo ID: 11. 17. The method according to claim 7, in which the modified form of the MIMI protein has an M-terminal shortening of the amino acid sequence, which approximately corresponds to the position of amino acids 1-125 in SEQ. ID Mo:2, and the C-terminal truncation of the amino acid sequence, which approximately corresponds to the position of amino acids 522-593 in SEQ. Mo ID: 2. 18. The method according to claim 17, in which the modified form of the MIM1 protein contains the amino acid sequence presented in the SEQ. Mo ID: 14. 19. The method according to claim 17, in which the shape of the MIMI protein is changed! encoded by the nucleotide sequence represented by SEQ. Mo ID: 13. 20. The method according to claim 7, in which the altered form of the MIM1 protein consists mainly of ankyrin motifs that approximately correspond to amino acids in positions 103-362 of SEQ ID NO: Mo ID: 2. 70 21. The method according to claim 20, in which the modified form of the MIM1 protein contains the amino acid sequence presented in SEQ. Mo ID: 16. 22. The method according to claim 20, in which the modified form of the MIM' protein is encoded by the nucleotide sequence presented in SEQ. Mo ID: 15. 23. The method according to any one of items 4, 8, 11, 14, 17 and 20, in which the MIMI1 protein is encoded by a nucleotide sequence that hybridizes with the nucleotide sequence presented in SEQ. ID Momo 1, 7, 9, 11, 13 or 15 under the following conditions: hybridization in 19685A, 520 mM MarPo, pH 7.2; 795 lauryl sulfate, sodium salt, mM EDTC, 250 mM sodium chloride at 5593 for 18-24 hours, and washing in a 6-fold solution of chloride and sodium citrate for 15 minutes. (3-fold washing), in a 3-fold solution of sodium chloride and citrate for 15 min. (1x wash) at 55260. 24. The method according to any of items 1 - 23, in which the microbicide is a fungicide selected from the group that includes: 4-(3-(4-chlorophenyl)-3-(3,4-dimethoxyphenyl)acryloyl |morpholine ("dimethomorph"), 5-methyl-1,2,4-triazoloi|3,4-51(11,3|benzothiazole ("tricyclazole"), Ha 3-allyloxy-1,2-benzothiazole-1, 1-dioxide ("probonazole"), A-(2-(4-chlorophenyl)ethyl|-A-(1,1-dimethylethyl)-1H-1,2,4-triazole-1-ethanol ("tebuconazole") , i) 1-((3-(2-chlorophenyl)-2-(4-fluorophenyl)oxiran-2-yl|methyl|-1H-1,2,4-triazole ("epoxyconazole")), A-(4 -chlorophenyl)-A-(1-cyclopropylethyl)-1H-1,2,4-triazole-1-ethanol ("cyproconazole"), 5-(4-chlorobenzyl)-2,2-dimethyl-1-(1H- 1,2,4-triazol-1-ylmethyl)-cyclopentanol ("metconazole"), "I 2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl)- propyl-1,1,2,2-tetrafluoroethyl ether ("tetraconazole"), methyl-(E)-2-12-(6-(2-cyanophenoxy)pyrimidin-4-yloxy|phenyl)-3-methoxyacrylate (" ISI A 5504", "azoxystrobin"), -- methyl-(E)-2-methoxyimino-2-| 2-(2-phenoxyphenyl )-(E)-2-methoxymino-M-methylacetamide, o (2-(2,5-dimethylphenoxymethyl)-phenyl|-(E)-2-methoxymino-M-methylacetamide, h- (1kK, 35/15, 3K)-2,2-dichloro-M-((K)-1-(4-chlorophenyl)ethyl|-1-ethyl-3-methylcyclopropanecarboxamide ("KT 3616"), ethylene-bis-dithiocarbamate-polymer complex of manganese and zinc ("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-ylmethyl)-1H-1,2,4-triazole. ("difenoconazole"), 1-(2-(-2,4-dichlorophenyl)pentyl|-1H-1,2,4-triazole ("penconazole"), - with cis-4-IZ-(4-tert- butylphenyl)-2-methylpropyl)|-2,6-dimethylmorpholine ("fenpropimorph"), c 1-(3-(4-tert-butylphenyl)-2-methylpropyl|-piperidine ("phenpropidine"), "» 4- cyclopropyl-b-methyl-M-phenyl-2-pyrimidinamine ("cyprodinil"), methyl ester (K5)-M-(2,6-dimethylphenyl)-M-(methoxyacetyl)-alanine ("metalaxyl"), methyl ester (K)-M-(2,6-dimethylphenyl)-M-(methoxyacetyl)-alanine ("K-metalaxyl"), -I 1,2,5,6-tetrahydro-4H-pyrrole|3,2,1 -|)quinolin-4-one ("pyrochilone") and acid ethyl ester of phosphonic acid ("fosetyl"). Mn 25. The method according to claim 24, in which the indicated fungicide is metalaxyl. -AND 26. The method according to any of items 1-24, in which the specified microbicide is a benzothiadiazole derivative, an isonicotinic acid derivative, or a salicylic acid derivative. - 27. The method according to any of items 1-26, in which the immunomodulated plant is treated with two microbicides at the same time. 28. The method according to item 27, in which one of the microbicides is a fungicide selected from the group that includes: 4-(3-("4-chlorophenyl)-3-(3,4-dimethoxyphenyl)acryloyl|morpholine ("dimethomorph" ), 5-methyl-1,2,4-triazolo|3,4-51(11,3|benzothiazole ("tricyclazole"), F,Z-allyloxy-1,2-benzothiazole-1,1-dioxide (" probonazole"), co A-(2-(4-chlorophenyl)ethyl|-A-(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 ("epoxyconazole"), 60 A (4-chlorophenyl)-A-( 1-cyclopropylethyl)-1H-1,2,4-triazole-1-ethanol ("cyproconazole"), 5-(4-chlorobenzyl)-2,2-dimethyl-1-(1H-1,2,4-triazole -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)-2-12-I6-(-2-cyanophenoxy)pyrimidin-4-yloxy|phenyl)-3-methoxyacrylate, (ICSI A 5504), "azoxystrobin" ), 65 methyl-(E)-2-methoxyimino-2-| 2-(2-phenoxyphenyl)-(E)-2-methoxyimino-M-methylacetamide, (2-(2,5-dimethylphenoxymethyl)-phenyl|-(E)-2-methoxyimino-M-methylacetamide, (1kK,35 /15,3K)-2,2-dichloro-M-((K)-1-(4-chlorophenyl)ethyl|-1-ethyl-3 methylcyclopropanecarboxamide ("KT 3616"), ethylene bis-dithiocarbamate polymer complex manganese and zinc ("mancozeb"), 1-(2-(-2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-ylmethyl|-1H-1,2,4-triazole Spropiconazole") , 1-2-(2-chloro-4-(4-chlorophenoxy)phenyl|-4-methyl-1,3-dioxolan-2-ylmethyl)-1H-1,2,4-triazole. ("difenoconazole") , 70 1-(2-(-2,4-dichlorophenyl)pentyl|-1H-1,2,4-triazole ("penconazole"), cis-4-3-(4-tert-butylphenyl)-2-methylpropyl )|-2,6-dimethylmorpholine ("fenpropimorph"), 1-(3-(4-tert-butylphenyl)-2-methylpropyl|-piperidine ("phenpropidine"), 4-cyclopropyl b-methyl-M-phenyl- 2-pyrimidinamine ("cyprodinil"), methyl ester (K5)-M-(2,6-dimethylphenyl)-M-(methoxyacetyl)-alanine ("metalaxyl"), methyl ester (K)-M-(2,6 -dimethylphenyl)-M-(methoxyacetyl)-alanine ("K-metalaxyl"), 1,2,5,6-tetrahydro-4H-pyrrolo|3,2,1-|)quinolin-4-o n ("pyrochilon") and acidic ethyl ester of phosphonic acid ("fosetyl"), and another microbicide is a derivative of benzothiadiazole or a derivative of isonicotinic acid or a derivative of salicylic acid. 29. The method according to claim 28, in which the fungicide is metalaxyl, and the other microbicide is a benzothiadiazole derivative. s (8) " "- cha so i - - s ;" -I (95) -I - 50 s» F) ime) 60 b5