JPS59501495A - Broad-spectrum plant protection from disease sources - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Broad-spectrum plant protection from pathogens field of invention This invention relates to the protection of members of the plant kingdom from pathogens, and more particularly to the protection of members of the plant kingdom from pathogens. from the phylogenetic source or its ancestral components to avoid or halt the progression of virus infection. The ubiquity of plant viruses related to the use of interferon (IFN) molecules has increased raises the overall issue of economics. in either hemisphere of this earth , food crops and ornamental plants can escape from the ravages of plant viruses. do not have. For example, virus-induced plant diseases are fatal to coconut palms in the tropics and It attacks wheat, barley, and other grains in northern Canada. Currently, I still feel Primitive method of "picking" to remove infected plants before they spread to uninfected plants There are no therapeutic studies or methods to help. Microscopy of infectious viral organisms physical nature, and inability to detect infection with the naked eye before picking is too late It will be clear that a fundamentally different method is required.

What is necessary is (1) to improve the plant so that its inherent resistance to viruses is strengthened. Being able to change the germplasm and ( 2) Against the complete spread of various plant viruses on already infected plants. is broadly protective, thereby preventing further damage to the crop, and Substances that stop the continued spread of infectious agents or safe substances can be added externally. Is Rukoto. This invention demonstrates that the beneficial physiological effects of IFH are demonstrated in members of the plant kingdom. The above requirements are met by providing an effective method.

Concerning placing exogenous human interferon (I (u IFN)) in plants Research has been conducted.

0rchansky et al., Proc, Natl, Acad, Sci, USA (7 9) +2278, (1982). Similar studies have shown that so-called intracellular mediated Concerning the exogenous placement of a substance (2'-5' oligoadenylate synthetase) into plants oDevash et al., 5science 216, 1415 (19 See 82).

Summary of the invention This invention addresses the need for a type of natural protein called interferon (IFN). Amino acid sequences or chemical fragments thereof can be transmitted to plant viruses once the plant has been infected. terminating the proliferation and cell damage of various chronic or latent plant viruses. It is possible to prevent spread to normal, uninfected plants, i.e. infection in the first stage. It is derived from the inventor's determination that it can be done.

The above essential amino acid sequences are referred to in the specification and claims as "anc. estral) j array. The “ancestral” sequence has been That is, changes can occur over at least a million years, perhaps tens or hundreds of millions of years. This is one of the most conserved sequences. To prevent this invention, "ancestral sequence" or The "ancestral fragment" is itself naturally occurring and produces an antiviral response in the plant. Any amino acid sequence that has the ability to

The inventors believe that the ancestral sequence contains, at least in part, the entire molecule or large molecule containing the ancestral sequence. It has been found that the fragments are endowed with IFN properties.

This invention can be implemented in many different ways and can be based on any of the following: .

(a) at the genetic level, i.e. the ancestral sequence or sequences It encodes the biosynthesis of contained molecules (e.g. total Hu IFN) in plant cells. Introduction of genetic information to program.

(b) Mechanical level, i.e. external physical addition of IFN or its fragments , i.e. already infected plant tissues or plants at imminent risk of virus infection. Local addition to tissues.

In addition, double-stranded RNA (ds RNA) can provide the same results (if the target plant cell is programmable) (containing genetic elements), and various interactions between plant cells and viruses. It also enhances treatment results.

One of the most important decisions leading to this invention was that the entire A smaller portion than the IFN molecule is sufficient. In other words, and details Accordingly, the present inventors believe that a certain amino acid sequence in the IFN molecule is a complete IFN molecule. determined to produce an antiviral response substantially equivalent to that produced by . The inventor further states that the sequence is ancestral, i.e. , that they have evolved over time, and that their sequences have evolved in plants and animals. found that it is common to molecules in many organisms, including both.

Very simply, this specification means that the invention extends beyond the scope of all IFN molecules. It means that it can be carried out. In other words, the (ancestral) amino acid sequence described later Any molecule containing can be applied topically to produce an antiviral response. . In addition, any gene containing the ancestral sequence or a molecule containing the ancestral sequence Any plant that can be "triggered" to produce the sequence or molecule. Sunawa However, some plants cannot be genetically modified to produce all IFN molecules. This means that plants are genetically programmed to produce their own IFN. IF N-like reactions can be induced. The term "r　IFN uniform" is used in the specification and It shall be understood to have this meaning throughout the claims. Of course, plants are IFN. genetically modified to contain the gene encoding (its entire molecule) In some cases, the plant can also be artificially induced to produce total IFN. .

Furthermore, the inventors have discovered that the plants themselves produce substances containing the ancestral sequence, i.e. It has been confirmed that plants directly provide interferon' (PIIFN). .

That is, plants are engineered to contain a functional gene that expresses plant interferon. It is also within the scope of this invention to be genetically modified.

The present invention further provides for the production of IFN in plants by causing a uniform IFN response in plants. Provides to halt or prevent virus infection. How to achieve this is One of three different routes can be taken.

(1) One or more of the IFN genes so that the plant actually produces its own IFN. Genetically modifying a plant to contain multiple copies. transferred gene may function from the beginning or may be initiated by the use of an inducing agent. genetically Degeneration is particularly prevalent in plants that have no or very few IFN genes. It is for use.

(2) Local application of molecules containing ancestral sequences to plants.

(3) Produce molecules containing ancestral sequences in plants that have not been genetically modified triggering. This method allows for the “onset” or is activation.

Typically applicable for protection against viruses (i.e. IFN-like response) Exotic plant treatment agents useful in this invention as a source of , various whole IFNs, ancestral (i.e., essential) molecular fragments, IFN-inducing agents (limited dsRNA), and exposing the cells to the ancestral IFN bioactive fragment. In plant cells, molecular changes related to virus resistance that occur specifically due to Contains specific oligonucleotide (vehicle) substances that can cause the same reaction.

Topical applications or treatments can affect the biological activity of drugs in a wide variety of ways. You can do this. Such treatment methods include soaking the plants or seeds, spraying, or crop dusting.

``IFNJ includes (a) human alpha, beta, or gamma interferon. (b) animal (e.g. human) interferon; in a non-human organism to which a gene or a fragment thereof (resulting in a hybrid gene) has been added. “synthetic” animal interferons produced; and (c) these interferons. chemical derivatives of erons, e.g. modified polypeptide chains or modified glycosyl Contains zill units.

This invention, of course, has important and wide applications, the most direct and economical of which. Of great importance is the protection of major crops produced nationally and even globally. Ru. Furthermore, by strengthening the natural defense mechanisms of plants, this invention commonly occurs when farmers attempt to change a plant's natural geographical growing area. Environmentally stress plants without succumbing to possible viral infections enable. Many tropical plants cannot grow in non-tropical areas . This is because the accompanying "stress" increases susceptibility to pathogens, including viruses. For example, the mother plant is generally relatively weak in the tropics. Although it does not contain the virus, it would be difficult to grow it in California. Everyone is infected with a deadly virus. Therefore, this invention can be used directly Not only - not obvious even to trained observers in agro-economic biochemistry Even so - it will provide additional agricultural benefits as it becomes increasingly implemented.

Additionally, there are viruses whose life cycles are associated with the utilization of intracellular products of plant cells. Other pathogens are also inhibited by IFH as taught by this invention.

“5elective Inhibitorg of Viral Funct iona jW+A, Carter; Chemical Rubber C Company Press; Cleveland, Ohio; 1973. child This includes various non-viral species that are particularly sensitive to IFN effects at high IFNi levels. Contains a comprehensive review of viral pathogens, which is incorporated herein by reference. Incorporate into.

Brief description of the drawing Figures 1a and lb show human-IFN-β1 (HuIFNI) and human-IFN. -r (Hu IFN-) and the structural identity of IFN over a long evolutionary process (i.e., most, if not all, IFNs have The determination of having common structural elements is a new structure of the three-dimensional model shown in Figure 1. It can be proved by the composition. This model establishes a close three-dimensional resemblance The region containing the biologically active site, i.e. the target cell (of clear mammalian or plant origin) Clarifying the commonality of the parts of the IFN molecule that confer the biological activity of the complete molecule on cells) It clearly shows.

Figure 1 shows a two-dimensional three-dimensional model using the data shown in Table 1 below. It is something.

0 γ5-61.29 0,88 0.99βq-121,350,770,75 γ2. -3o1.38 1,01 0.86β32-35 1.14 0.92 1.00γ45-a61.21 0.91 0.79165-681.33 0,98 0.84β71-7+s 1.44 0,84 0.74186-8 91.39 0,83 0.697 β11o-413,1,120,800, 91γ132. , 1,08 0.86 0.81β, 36. , 1.18 0 ,85　1.01β141-161　0.96　4 each of 1.16 amino acids and calculate the probability of the existence of certain matching structures within the complete IFN molecule. Ta. For example, if you choose the existence of reverse turns, u and Fasman (Annual Reviews of Biochemi Using the formula of I calculated it. Using the same formula, an average range of 10 to 20 contiguous amino acids α-helix and β-delete sheet (pleated sheet) The existence of was inferred. P, is related to the possibility of a lipper's turn, and P is an α-helix. with respect to the possibility of formation, and P with respect to the possibility of formation of β-bleto sheets.

The numbers in the left column indicate the relative positions of the amino acid residues starting from the amine terminus of the IFN molecule. show.

The overall and far-reaching inference of the structural homology of IFNs has never been clarified in the past. has not been published or published. This is because some already known and the creation of isolated facts and additional insights derived from the inventor's novel research. This is because it is related to physical integration. Comparative nucleotide sequence data, comparative protein sequence data data, mutation frequency data, matching data, and Markov's chain analysis (all with different This research using IFN in the form of An ancestor of IFN that can provide antiviral activity in all life forms of Alternatively, the determination of a primitive array is derived. In addition to this anti-American arrangement, The amino acid composition is determined. To emphasize further, these ancestral sequences present in large molecules The columns do not separate naturally and are themselves biologically active. and gives plants virus resistance, that is, resistance to virus multiplication. can be given. The inventor believed that millions of years ago it had already mutated and no longer exists in nature. Several amino acids were mutated in the ancestral DingFN molecule, which is obvious to not exist in It was established that the tertiary structure in the sequence is conserved even when Today's plants, including humans, In organisms and animals, only traces exist in the form of amino acid sequences essential for biological activity. Ru.

Preferred molecular moieties (i.e., biologically active amino acid sequences) of IFN include a series of possible uses. can be easily isolated by techniques such as sser andori, G11lepie, Analytical Bioch emystry, Vol. 129 + p. 357, 1983), or genes The product is expressed in protein form on nitrocellulose paper (Bresser et al. ProceedingsNational Academy 5science , USA, in press 1983: Bresser et al., DNA, in press 1983) column can be determined. and I having medical value as described in this specification. Many possible variations in the solid phase synthesis of peptides to obtain active fragments of FN can be carried out. i.e., beneficially influences plant cell function, and Any route (solid-phase conjugation) of the above amino acid sequences to prevent virus infection. synthesis, or synthetic production by recombinant DNA techniques in bacteria, yeast or animal cells) is also within the scope of this invention.

The essential amino acid sequence is typically about 10 to 15 times longer than the normal molecular length of IFN, Approximately 25-45 amino acids in length instead of the usual approximately 162 constituent amino acids It is an acid. Determination of these essential amino acid (takuputide) fragments was based on the inventor's inference. Many types of IFs have been made possible and conserved during the evolution of vertebrates. A few dozen of the theoretically possible arrangements of several regions of H Facilitated by analysis. The absolute positions of these regions vary depending on the IFN molecule. Na For example, some IFNs (e.g. This is because it is shortened by ~15%. What is important about active fragments is that the intact fragment It is the three-dimensional activity of the fragment rather than its relative position from the child's amine end.

In addition to favorable physicochemical properties, IFN fragments or sequences also have a cost-effectiveness point of view. It is also useful because of its small size, which accounts for a small portion of the total production cost of the IFN molecule. It can be produced in large quantities in minutes and can be produced using various manufacturing methods, e.g. It can be produced by recombinant DNA techniques and solid phase synthesis methods described above.

Of course, this invention applies to any natural or synthetic DNA techniques) can also be performed using full-length IFN molecules. However, in this case These interferons contain the necessary biologically active regions and are biologically active. Hiding sites from the plant cell surface or relativizing biologically active regions through structural distortion Any molecular modification (usually sugar) that renders it partially or completely inactive to plant cells or carbohydrate moieties). Generally cardiac or peripheral orico The presence of a small side chain in the zanokalide may be due to its direct or structural mechanism may interfere with expression of the activity (e.g., W, Carter, LifeS sciences, Vol. 25 + pp. 717-728 + 1979; and Pharmacology and Therapeutics, Vol. 8 r359-355, 1980).

■Active interferon region Genes encoding interferons have been cloned (e.g. De rynk et al., Nature, Vol2851542-547, 1980 ). These genes have been sequenced (Goeddel et al., Nature rV QI 290. pages 20-26 + 1980; Derynk No.

Nature, Vol. 285+, pp. 542-547, 1980), 5 Nine primary structures of various interferons were determined. combination=-tar Using the program, the inventors were able to investigate several biological properties of the interferon system. Determined the required area (G, 1llespie and Carter, Han dbook of Experimental Pharmacology of Interferon, in press).

The ancestral amino acid sequence of interferon that produces an antiviral response is sequence 25- 40 and 115-141. Published gene sequences (Derynk et al. and Goeddel et al., supra), these results were found for each of human-IFH. It is possible to determine the existence of at least two ancestral sequences of Ashi, and the above and Similar analyzes can be applied to IFN in other animals.

Interferon domains determined to be essential for binding to specific cellular receptors The range contains amino/acids 115-141 (G11lespie and Carte r.

1982). The location of this area may be partially determined or Buunit (Lai.

Journal Biol, Chem, Vol 252, 7249-72 56, 1977), i.e., competes with interferon for binding to receptors. facilitated by homology with substances that

Ancestor fragments different from those described above exist in nature, and this invention is disclosed herein. It is not limited to the ancestral sequences shown. The concept of this invention is that the ancestral array or the ancestral sequence itself. Additional Identification of fragments is also within the skill of the art.

■Preparation of interferon fragments with one selected region How many interferon fragments are there? It can be prepared by the following method. By way of example, one suitable method is described below. . Other methods, such as chemical modification of natural interferon or cloned (Synthetic) Chemical modification of complete interferon, interferon derivatives, etc. This is readily apparent and is within the scope of this invention.

The cloned interferon gene has a specific restriction endonuclease can be used to cut at specific locations. Obtained interferon gene The fragment encodes a fragment of interferon polypeptide, and this is translated into RNA. & limerase promoter, IJ g seam binding site, start codon and other requirements can be fused with an expression vector containing a specific signal. This recombinant DN A is the introduction of interferon fragments into suitable host cells for synthesis. Can be done.

For example, in order to express a HuIFN gene fragment in a host, the following method may be used. can be done. ligated to polyA dT and cloned in pBR322 The human interferon-β gene 51 was incubated at 37°C for 2 hours for 20 m M Tris, pH 7,4, 10mM MgCl2.50yy+M(NI(4) 2so4 and 50 units of End in 100rnq/- bovine serum allamin.

The interferon gene is converted to base 2 by incubation with R-PI. The single strand of 03-206 was cleaved. DNA in calcium acetate 0.3 M and precipitated from ethanol.

The DNA was dissolved in 100-50rPtyi potassium phosphate, pH 7,4°6, 7mM. of MgC42, 1rnk/1 of mercaptoethanol.

Class 35 deoxyadenosine and deoxyfuthynones and Estherichia coli D 25 units of the Flenow fragment of NA polymerase. DNA with potassium acetate 0.37-1M in um and precipitated from ethanol. precipitation 100-30mM acetate) IJum, pH 4,6-50mM Na, Cl , 1 trM ZnSO4P, 5 units of glycerin and 25 units of 81 nucleic acid. and incubated for 1 hour at 37°C. This makes it smooth The complete interferon gene fragment up to nucleotide 204, with the end The first one was located at the codon that coats leucine at position 45. Sodium acetate um was added to 0.3M, and the DNA was precipitated from ethanol. .

Separately, ligated to poly dA-dT and cloned in pBR322 Human interferon-B gene 51 was incubated at 10 rnM for 2 hours at 37°C. Tris, pH 7, 9, 6nM MCI, 10mM MgC72, 1mM Nothiothreitol and 50 units in 1.00 m9/ml of boar serum alramin It was incubated with a kit of Endo RMbo II. DNA as ethanol and treated with S1 nuclease as described above.

288 base pairs containing nucleotides 401-688 of the interferon gene The blunt-ended fragment was purified by electrophoresis on 3-colour agarose. of this fragment A 25 ml aliquot was treated with Pst1, treated with DNA polymerase and It was introduced together with SLS1 nuclease-treated DNA 59-.

The DNA thus obtained was an interferon with amino acids 46 to 111 deleted. Interfuses with deletion of nucleotides 205 to 400 encoding polybezotide It contained the eron gene.

Recombinant interferon fragments can be isolated from bacterial or mammalian cells using conventional techniques. can be inserted into a number of suitable vectors for expression in.

Interferon or interferon fragment c) Purification by a standardized method Can be done. However, this invention does not apply to less than 1 inch of interface unless harmful substances are present. It can be carried out using Feron. For example, containing interferon Proteins in solution are oxidized in the absence of ethylene or polyethylene glycol. Mbu/l/-(Cibachrom blue) Binds to Sepharose . The column is treated with various solutions that remove most proteins other than interferon. Wash and remove interferon from ethylene glycol or polyethylene glycol. or other suitable eluent (Carter et al.).

Pharmacology and Therapeutics, Vol. 8, pp. 359-377, 1980). Routine use of binding and elution reservoirs, if desired. The interferon-containing solution containing anti-interferon antibodies was prepared using the following conditions: Purification of the Cibachrome Blue fraction or other material by passing it through a column base that can be further done.

The above recombinant DNA may contain some or all of the same enzymes under different conditions or in a different order. or using modifications of the enzymes or interferon genes listed above. can be manufactured. Other suitable recombinant DNA may be produced using other enzymes and other methods. It can be formed by Finally, in order to obtain interferon fragments, recombinant D Using NA is only one way to generate interferon fragments. This is an example.

Other means, such as solid phase synthesis, intact materials, or genes prepared in bacteria and yeast Similarly, proteolytic cleavage of interferon cloned in be able to.

The present inventors have discovered that plants that produce high levels of both plant and human interferons are It was determined that a wide range of strains would be resistant to the pathogen.

Accordingly, methods of creating such plants are provided. Created by the following method The plants were genetically engineered to carry novel A, human and plant interferon genes into recombinant DNA. Cloning; B. Inserting this recombinant DNA into protoplasts and introducing plant and human interfaces. Isolate cells that have high levels of long-term synthesis; and c. producing a mature plant from the protoplast;

For example, the tobacco plant Nicotiana (N1 cotiana) was used. Below is an example Described as .

A. Cloning of human and plant interferon As shown in previous 2, human interferon The eron gene has been cloned (eg, Deryck et al., @).

Several commonly used methods are also used to clone plant interferon genes. All you need is a suitable glove to find the recombinant DNA. It is. One probe that can be used for this purpose is the human interface Highly conserved portions of Ron or, but not limited to, nucleotides 345-423 (G11lespie and Carter 1982 + Handbook of Experimental Pharmacol ogy of Interferon, in press). There are other suitable gloves as well. There will be. Importantly, this invention clones the plant interferon gene. The residual effects of the final plant do not result from the selection of the method used to plant the plant. This results from historical newness and increased desirability.

Analytical methods described for HuIFN in the Kono specification have been used in various animal or plant species. Comparable ancestral sequences in a given IFN can be readily determined. recorded in this statement. The method for isolating, inserting, and expressing the loaded gene fragment is described in HuIIF. Comparable, and if so identical, to the method described for the ancestral sequence method would be used. Protoplasts are formed and DNA becomes a regular (Bourgin et al. + 1979 + Phys. iol, Plant.

45: 288-292: Caboche, M., 1980.

Planta 149: 7-18; Cocking et al., 1981°Na ture 293: 265-270) o Difficulties include recombination and therefore these It does not become a stable part of the physical genetic material. Agrobafter (Agrob) Acter) Ti DNA plasmid (Davey et al., 1980, P I.

Sci, Lett, 18: 307-313; Wullems et al.

1979 r InAdv, Protoplast Res, Proc, Sth.

Symp:407-424) is an exception to this, but how many DNA plasmids does this have? It has certain disadvantageous properties. Among these grasmids, T1 plasmid DNA is too much, the DNA inserted into this plasmid will not express the proper protein, and Cells infected with this plasmid do not grow properly. Agropactor's R i DNA cilasmids allow proper growth of plants, but other undesirable have quality.

Use the following plasmid to insert foreign DNA into the Nicotiana chromosome be able to. It can be modified to contain one undem copy. child The plasmid has a unique Eco R1 site, and this plasmid has a plant and/or The human interferon gene is inserted. Instead of this method, thin p BR322 can be used.

Many copies of the plasmid containing the plant interferon gene were transferred into microorganisms. Nzekunyong, calcium phosphate precipitate, infection by Agropacter, protoplus It can be introduced into Nicotiana protoplasts by fusion, etc. transformation The produced protoplasts can multiply as plant cells and the chromosomal DNA In-chie molecular hybridization of pBR322 probe to A can be released. These cells induced by poly■:C -Globally pure inplants for increased levels of feron mRNA production. Insitino hybridization using the biointerferon gene 7. can be tested. These cells produce elevated levels of plant mRNA. cells are associated with antiviral growth factors for increased synthesis of plant interferons. It can be tested using conventional biological tests.

Nicotiana cells that produce elevated levels of plant interferon are proton 0 last can be reconverted and, as mentioned above, this time the human-interface Transformation can be performed with the above-mentioned vector containing the feron gene. shape Transformed protoplasts can multiply as plant cells and rise Levels of plant interferon + human interferon mRNA ’% and and Yin/Qi molecular hybridization and antiviral protein, respectively. can be tested by biological assays for growth factors. These transformations The converted proton-0 last can then be cultured into mature tobacco plants.

More specifically, this method can be performed as follows.

Protoplasts were prepared by Chupeau et al. (C, R, Acad.

Sci, Ser, D (/’Li) 278:1565-1568゜1974) can be separated from ==+ Chiana spp. Remove the skin from the sterilized leaves. and containing sea-croses, 02% Macero zyme) To medium containing R10,01 cellulase otsuzu (10, 3mM NH4No3.9.4mVI KNO3, 1.5mVi Cact2 ・7H2oX075F? IM MgSO4 7H20,062 class KH2Po4 ．． 0.1 m’A of FeSO4”7H20% o, 1 +ruM of Na2ED TA 116 class H2BO3, 06rrM MnSO4 H2O-, 35tn Ni ZnSO4’7H20% 0.2rrM) CuSO4”5H20,0, 22mM ALC13, 0, 13mM NICt2・6H2o18μM Nonico Chinic acid, 2 μM calcium pantothenate, 0.047 biotin, 161 μM of naphthalene acetic acid, 44 μM of 6-bennoadenine, 58 m+M of 7 yuk Loin, 440? can be incubated in rIM (mannitol) .

Released protoplasts can be washed by low speed centrifugation in To medium. . As long as it is possible to isolate living single cells without a substantial portion of the cell wall, Other methods of rotoplast separation can be substituted.

Transformation of Nicotiana spp protoplasts carrying the interferon gene Together with the vector DNA or with bacteria containing such NA, a large number of can be incubated using any standard transformation method ( Davey et al. 1980, PI Sci, Lett, 18: 307- 313; Willems et al. 19 79, In Adv, Protopl ast Res, Proc, 5th.

symp, : 407-424). Such vector DNA can be used as described above. E containing eel plant repeat sequences.

It can also be a plasmid or can be multiplied in microorganisms, and It may be any other vector DNA that can be integrated into the toplast chromosome. .

The transformed 70 rotoplasts were cultured in To medium in the dark at 25°C for 4 days. and then placed under fluorescent light (2500 lux, 16 hours a day). can. After 30-60% of the protoplasts have divided once, they are centrifuged. and AC medium (10??IM KNO3, 3mM CaCt2”2 1 (20゜3yy+M MgSO4・7H7H2O11rr KH2Po4.0 ．． 1 rrM FeSO4'7H, 010, 1 nM Na2EDTA, 4 9mM H2BO3,1,8FFIM MnSO4 H2O1104rnyi ZnSO4・7H2o1004 class CoC42・6H2O1036μM Cu So4・5H2o10, 4.1 μM NaMoO4・2H20,0,66tt M Atct3.0.40mM NiC42'6H20, 0.18μM KI, Vitamins as in To medium, 053 μM naphthane v vinegar et 4.4 μM 6-pennoradenine, 58yyiM sucrose, 440rrM can be washed once in mannitol, class 1 glutamine).

Cells can be plated in AG medium on dishes (Caboche , Planta 149: 7-18 +1980). Next, use this dish as standard Incubate in a closed transparent box at 25° C. under light conditions.

Under this condition, transformed protoplasts form colonies.

Individual colonies were picked, made into single protoplast suspensions, and placed in AC medium. (fl) Can be inoculated onto caprates. Replica plate, vector Regarding expression of interferon gene or recombinant interferon gene, (1) Molecular hybridization presence of specific DNA sequences due to the presence of specific DNA sequences (Robins et al., 1981.J.

Mo1ec, Appl, Gen, 1: 191-203, 2), ( 2) Presence of specific RNA transcription products by molecular hybridization (Bra hic and Haase, 1978.

Proc, Nat, Acad, Sci, USA 75: 612 5-6129), and/or (3) excessive immunological or biological measurement. Presence of interferon protein (5ela11982, Interferon Sci, Memo.

Memo No. Iall 34, January) can be evaluated by measuring . Other methods of measuring excess interferon production are also suitable.

Colonies showing the presence and expression of new interferon genes were collected in AG medium. can be cultured for m2 months and plated on solid R4 medium for germination. (Bourgin et al. + 1979r Physiol.

Plant, 45: 288-292). Transfer the sprouts to rooting medium B. can form seedlings (Bourgin et al. + 1979, p. hysiol, Plant, 45:288-392). Rooted seedlings It can be transplanted into pots and grown in a greenhouse. transformed plants Other methods of transferring single cells into mature plants can also be used.

Young plants were tested for their interferon production and for tobacco mosaic virus ( 5ela, 1981.

Adv, Vir, Res, 26:201-234) or other exogenous agents. Tolerance can be tested using standard evaluation methods. interferon Highly productive, disease-resistant strains are selected and sexually propagated by any conventional and effective means. It can be tightened.

The above method is an implementation of this genetically novel plant generating method with favorable properties. This is written to explain the possibility of implementation. Needless to say, this rapid change In an increasingly evolving field, many variations of this method are outside the scope of this invention. It is hoped that this will be possible.

■Applicable to 8 locations As already mentioned, the therapeutic agent of the invention can be used in a conventional manner (e.g. in a greenhouse). This ranges from spraying (spouting) to more complex methods (e.g. spraying onto hundreds or thousands of crops). It can be applied topically by a number of methods, including mist application).

A preferred method of carrying out this invention involves the use of biological agents at the time treatment is required. and mechanical factors. For example, permanent protection of seed crops is Alterations in Noyamplasma due to increased number of P. (plant and/or animal IFN genes) It would be preferable to do this by In contrast, plants have already covered large areas. Dusting of crops may be the preferred mode when on occupied agricultural land.

For example, specific topographical conditions (such as molecular stability, wind shear effects, etc.) ) and biochemical conditions (young leaf cells vs. mature leaf cells) or uptake efficiency due to old and weak leaf cell structures) must be taken into account. this In this case, preferred embodiments consist of lower molecular weight components. These are generally larger Provides excellent thermal and physical stability and enhanced cellular uptake/receptor binding It is et al. Oligonucleotides and IFN fragment peptides directed to ancestral IFN is a relatively heat-resistant and swirl-resistant active ingredient resulting from the basic invention.

For topical administration, at least one IFN or other substance containing the ancestral sequence. (or triggering agent) in a suitable agriculturally acceptable carrier, such as water.

That is, it is applied as a solution. Concentration is approximately o per ml of solution, o o o O1~1 The range is 00,000 IRU (International Comparison Unit). IFN to dust method Dry interferon if you want to disperse it as a dry powder for better application. from about 111 pm to about 1 part 710-1 A weight concentration of 0.16 parts is effective. A suitable carrier is a crop gestation method. Known about the law.

Interferon can be mixed with other plant treatment agents such as fungicides, leather removers, oils, etc. can be used in combination. In this case, all additional drugs are provided that it does not affect the biological activity of the column.

The final concentration of IFN or other ancestral material is determined by the crop to be protected, the virus to be protected, and It will depend on the conditions of the farmland. The final concentration also determines the molecular moiety that is non-ancestral ( In other words, it will also differ depending on the part of the molecule). For example, the amino acid sequence 11!5 In the synthetic rFN fragment consisting essentially of -141, the non-ancestral region is approximately O. Probably. In contrast, in all IFN molecules, the non-ancestral molecular portion is approximately 06 be. That is, about 60% of all IFN (e.g. Hu IFN) molecules are non-ancestral. It is.

Typical applications of IFH to crops include, for example, 1 ppb (pI)b; HuIF For N, 1 m is approximately 10 IRU). Surface application (d) (needing about 100 gallons per knee car), no more than 1 m9 per knee car All of the HuIFNs listed below will be required. Crop dusting methods are typically surface-applied 115 to 1/10 the volume and a comparable amount of interferon. , or the efficiency of topical application.

However, in some cases it may add additional stability to IF, N or its ancestral fragments. to withstand harshness of application and prolonged exposure to environmental factors. It is desirable to do so. Let's describe this next.

Interferons, like other proteins, are relatively unstable chemicals. water The solubility and biological activity in solution is highly specific for various linearly arranged amino acids. It depends on the three-dimensional arrangement (see Figure 1).

Specifically, it is an interface that functions to bind to cells and induce complex biological reactions. The regions of the feron molecule must be properly exposed and aligned. death However, many other non-functional orientations are also possible and can be freely formed. . These different conditions are facilitated by heat, certain foreign chemicals, and long-term storage. lengthened.

One solution to prevent the formation of different inactive states is Form molecules with a limited number of different states, such as the interferon fragments mentioned above. It is to accomplish. Another solution developed by the inventor is the interface The formation of different states can be limited by immobilizing Ron on a carrier molecule or structure. That is.

Active enzymes are often found as part of complex structures attached to cell membranes, nucleic acids, proteins, etc. found, and enzymes are usually more stable in the complexed state.

To evaluate the feasibility of such a method, the inventors investigated Several column bases containing various ligands thought to be Figure, Carter et al. + 1980, Pharmac, Ther.

8:359-377). Characterize cycharum by binding albumin Ta. 100ml containing 11,500 units and 0.99ml of protein in 1ml ml of non-dialyzed interferon preparation was added to 60 m It was applied to the column at a flow rate of me/cm2/h. Albumin column, 0.15 Using 0.02 M sodium phosphate (pH 7.4) containing M NaC7, I compared the balance. The eluate from the column was divided by a flow splitter in a ratio of 1:9.

The 10th part of the eluate was treated with Q, 12M phosphoric acid, 0.15M phosphoric acid and 0.15M phosphoric acid. NaCl was collected in 1 rnl of a candy solution of bovine serum albumin containing 1. used to measure interferon activity. Measure the protein concentration in the 90φ portion of the eluate I used it to. Both portions contain approximately 98% of the applied protein; contained less than 1% of the applied interferon activity.

50 (v/v)% ethylene glycol and 50% 004M phosphate (pH The column was further eluted with 7,4) and 0.30 M NaCl. stomach The original protein has very little residual interferon activity (86%) (less than 2%). ) was recovered. Some of these experiments have recently been published (Carte r W, A, rMethods in Enzymology 78: 5 76-582 y1981).

The above experiment shows that interferon can bind to serum albumin, but Most other cellular proteins were shown to be unable to bind. Other immobilized proteins, e.g. For example, experiments using cytochrome C are common in that interferon binds to proteins. This shows that it has the following properties. The present inventor has discovered that the strong hydrophobicity of interferon is hypothesized to forcibly interact with the normally hidden hydrophobic pocket of the protein. And the above experiment suggested that this assumption is correct.

Regardless of the mechanism, the above experiments demonstrate that the formation of a complex with a protein is an interface. It was shown that feron can be stabilized.

The method developed by the present invention for this purpose is as follows. human interf from any biological source - human cells, recombinant microorganisms, etc. - by any conventional means. can be separated by After purification, human serum albumin or other protein The white matter carrier can be added in excess, typically 3 m/-. Phosphate buffer solution can be dialyzed against salt solution or other suitable buffer and lyophilized. Ru. In a typical example, 1 million units of purified interferon are combined with 316 It was lyophilized with 1 m of sodium phosphate.

In this rapidly changing field, many variations of this method can be used. . Carrier proteins other than albumin or cytochrome C can be used. other Carrier molecules or structures such as lipids, small hydrophobic ligands, membrane fragments, or solid particles can also be used. I can be there. Other means of bonding are also suitable, such as ionic or covalent bonds. . 1st generation salt or buffer, or other concentrations of salt or buffer (salt or buffer (including cases where it is not used) can also be used. However, clearly this The method of Stabilizes IFN against structural changes.

Topical administration of HuIFN to plants generally prevents the virus from spreading from one plant to another. effective in preventing the spread of To prove this, The young leaves of the potato plant are entirely infected with potato virus N or potato cigar virus. I forced it. A disk was punched out from this leaf and human interferon-α The cells were cultured at 25° C. for 7 to 10 days in a nutrient medium containing or without. child After incubation, the leaves were ground in 1% Br1j35 and Particulate matter was removed by heart separation. Viral proteins are measured using light The amount was quantified by immunoassay (ELISA) using the chemical concentration. High optical density The value corresponds to an increased amount of virus. The results are shown in Table 2 below.

Table 2-) (UlFN inhibits the spread of pareizovirus) Potato oil S N 7 0.519 0.342 0.184 Potato virus N 10 1.331 0.702 0.838 As the data shows, the inhibition of the virus is interferon was found to be stable, i.e., inhibition of the virus over the range tested Although it does not increase linearly with increasing concentration of Demonstrates an anti-virus effect. Additionally, topical administration dramatically slows down the virus; The effectiveness decreases somewhat over time, and this means that the Indicates that repeated application is required. Importantly, the inventors have Periodic application of interferon to plants infected with In addition to preventing the spread of the virus from these plants to uninfected plants, It was determined that this would prevent the transfer of the virus. The present inventor has proposed the above-mentioned Hu A series of photos were taken of the leaves during the IFN experiment, and when the virus life cycle was stopped. At the same time, I observed that the leaves were growing and thickening.

Very low purity IFN is sufficient for increasing crop yield by topical application ( (i.e., no expensive purification methods are required), making repeated applications very economical. It is possible. For example, based on this experiment, the inventor has determined that approximately 100 square feet of We found that you can protect your home for about $10 a month at current prices. Ta. IFN may induce contaminating organisms (bacteria, etc.) or plant diseases, or Meets the criteria of not having an intact virus that can cause disease in (animals or humans) It can be used in any purity as long as it is. Appropriate decontamination methods , for example, after retention of the organism's E superorganism, these interferon lofts are It is useful for this invention even though it is rejected for floor applications.

Furthermore, the term "topically applied" is used to include the application of substances referred to as "inducing substances." Yes, the inducer is not the IFN molecule itself, but if the applied inducer is not present. in a relatively short period of time than would otherwise be required. It has the property of functioning as a biological trigger for producing IFN. The master of these What is important is the so-called “mismatched ds RNA J”. This substance is a strong IFN inducer, but has almost no toxic side effects in humans. does not indicate.

The biological aspects of the function of these materials are described in my U.S. Application No. 4,103.641. is fully described in the issue.

ds RNA is also active in members of the plant kingdom and is associated with IFN molecules or The biologically active fragments can be applied in the same manner as described above. Plant Things don't necessarily have to be genetically engineered. All you need is optional specific ds RNA has biological activity that generates an interferon-like response This means that it induces the production of molecules that have the essential (amino acid) sequence. vinegar As described in This means that it must contain a polynucleotide sequence. a particular plant species It can be determined by simple experimental means whether one or more ds Those who apply RNA to plants of interest and are known about antiviral growth factors Determined by testing according to the law. As an alternative to this method, IFN-inducing assays The inventors conducted the above-mentioned hybridization test for Nicotiana. This can be done using the Yon method.

Furthermore, intracellular mediators and 2/, 5/-olicoadenylic acid and/or functionally active derivatives thereof There is a series of substances represented by , which are also useful as topical agents. Ru. The exact biochemical mechanisms by which these substances function are unknown, but they , mimics the rFN effect produced by viral exposure and essentially obviates the need for IFN. It is known that it can be replaced by The present inventor has demonstrated that this mimicry can also extend to plants. Confirmed. Core (dephosphorylation) 2/-5/oligoadenylate, core 3'- 5' oligoadenylate and core 27-5/oligoadenylate codecipi Congeners containing (3'-deoxyadenon-7) can also be used.

ds RNA (e.g. Ampliken, I (EM) 1,1 Zarch trademark, Useful Moth Concentration Range for Applications of Bicycle, Maryland, or Polye/PolyC is an aqueous solution width between approximately 0.01 F/- and 1000 P/mlO. intracellular mediated The useful concentration range for the application of the substance is ml per ml in aqueous solution! 7 about 0. o1 and 1o　o,　o　Oo nanomoles.

The preferred embodiment for topical application is that IFN or its essential fragments or inducers of insects, especially aphids, perched on plants, and A physical substance that prevents the virus from acting as a carrier for spreading the virus This is a method of combining them as a mixture. In other words, insects like aphids are It feeds on many plants within a relatively short generation period and transfers contaminants to healthy plant tissues. replace it, pollute it, replace it with another healthy plant, and self-perpetuate As the cycle continues, it will play a major role in the transmission of the virus. Dear IFN Combining a substance that can cause a reaction with a substance that blocks (i.e. deters) insects. Together, there are effectively two attacks against virus spread.

That is, the first attack is to prevent insects from selecting treated plants. . Even if this attack fails, infected plants may be replaced by insects with healthy plants. (UlFN reaction product).

Particularly effective and chemical for blocking konzhong (and especially aphids) Comparable substances to IFH are pyrethrum and hormones called pheromones. Exclusion Chrysanthemum is made of known natural substances, such as derivatives from chrysanthemums. Pheromones are also wild It is a natural substance derived from potato.

Interferon mixed with either or both of these natural hormonal substances Interferon, applied separately but simultaneously, first kills the insect. Virus from insects that come into contact with plants that are continuously connected to plants or that remain intact despite blocking agents. form a combination that prevents the spread of Thus, blocking agents and antiviral agents go hand in hand. mutually reinforcing each other's biochemical levels and eliminating harmful rings in their final biological decomposition. does not leave any environmental residue or contaminants.

Interferon is present in the combination at the levels previously described (e.g. 0.0 001-100,000 IRU/ml solution). pyrethrum and/or pheromones are typically used at 0.005 to 10 pounds per sneaker. applied in the der. Therefore, one method is to obtain the desired level of IFN (or fragment, pyrethrum or pheromones and a suitable carrier (e.g. water or dry agricultural chemicals). commercially acceptable carrier) and apply these as a solution. Chiru by method.

Although a few illustrative embodiments of the present invention have been described in detail above, those skilled in the art will readily understand that new In the illustrative embodiments without departing materially from the fundamental teachings and advantages of this invention, It can be expected that many variations are possible. Therefore, all such changes The law is intended to be within the scope of this invention as defined in the following claims. Ru.

Engraving (no changes to the content) 2 Procedural amendment (formality) June 1980? Day Mr. Kazuo Wakasugi, Commissioner of the Patent Office 1 Display of incident PCT/US 83101198 2 Name of the invention Broad-spectrum plant protection from disease sources 3 Person making the amendment Relationship to the incident: Patent applicant L6 Carter, William Alvin 4 agents Address: 8-10-5 Toranomon-chome, Minato-ku, Tokyo 105 Date of amendment order 6 Target of correction translation of the drawing 7 Contents of amendment Catalog of 844-inch documents including engravings of translations of drawings (no changes in content) 1 engraving of the translation of the drawing

Claims (1)

[Claims] 1. Including producing an IFN-like response in plants. How to protect plants against pathogens. 2. The method according to claim 1, wherein the pathogen is a virus. 3. To produce at least one substance that causes the above-mentioned IFNtfi reaction. Claim 2 wherein the reaction occurs by genetically modifying the plant. The method described in section. 4. The substance is composed of a complete IFN molecule and a molecular fragment that has the ancestral amino acid sequence. 4. The method according to claim 3, wherein the method is selected from the group consisting of: 5. The genetic alteration described in @ contains at least one ancestral amino acid sequence. By inserting a human gene that codes for the production of one type of compound into plant cells. The method according to claim 3, which is carried out by: 6. Production of molecules containing ttf interferon or ancestral amino acid sequences Claims further comprising the step of inserting the encoding plant gene into said plant cell. The method described in scope item 4. 7. The reaction includes at least one compound containing the ancestral amino acid sequence. Claim 2 arises from the use of a substance consisting of Gosho-1 on said plant, also known as S. Method described. 8. The method of claim 7, wherein the method is stabilized. 9. Claim 7, wherein the substance comprises a complete IFN molecule as the compound. the method of. 10 Claims in which the substance includes a portion of a Hu IFN molecule as the compound The method described in Section 7. 11 The substance is present as a solution and from 0.0001 to 100,0001 RV/m 8. The method according to claim 7, applied in a concentration range of 1. 12. The method according to claim 11, wherein the solution is an aqueous solution. 13. The reaction comprises at least one non-IF capable of inducing this reaction. Claims arising from topical application of substances comprising N compounds to said plants The method described in box 2. 14. The compound induces the production of a second compound containing the ancestral amino acid sequence. 14. The method according to claim 13. 15. The method according to claim 14, wherein the inducing compound is da RNA. 16. The method of claim 14, wherein the second compound is all HuIFN. 17. The method according to claim 14, wherein the second compound is a HuIFN fragment. . 18. The method of claim 13, wherein said compound is an intracellular mediator. 19 The mediator is 27,5/-oligoadenylic acid or a derivative thereof. The method according to item 18. 20 A first compound that causes an IFN-like reaction in the plant, and a virus vector comprising topically applying in combination a second compound that blocks How to protect plants against pollutants. 21, the first compound is selected from the group consisting of ds RNA and intracellular mediators; 21. The method according to claim 20. 22. The claim wherein said second compound is selected from the group consisting of pyrethrum and pheromones. The method according to scope item 20. 23, mixed with a second compound selected from the group consisting of pyrethrum and pheromones. , dsRNA, and an intracellular mediator. A composition consisting of: 2. A plant protected by the method of claim 1. 25. A plant protected by the method of claim 20.