NL8320256A - WIDE SPECTRUM PLATE PROTECTION AGAINST PATHOGEN. - Google Patents

Description

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Title: Broad spectrum plant protection against pathogens.

The invention relates to the protection of members of the plant kingdom from diseases and in particular to the use of complete interferon (IFN) molecules from any phylogenetic source, and / or ancestor components, in order to prevent or prevent virus infections of the plants. to bring.

The ubiquity of plant viruses contributes to the global problem of food supply in a cost-effective manner. No part of this earth is spared the devastation that plant viruses wreak on the food harvests and even the ornamental plants. Consider, for example, the fact that virally-induced plant pathology is capable of a lethal disease of coconut palms in the tropics as well as destruction of wheat, barley and other grains in Northern Canada. Currently, there is no known therapeutic approach or cure other than the primitive art of "selecting" where the farmer or grower removes the infected plant material before an infectious spread to previously uninfected plants occurs. When one considers the microscopic nature of the infecting viral organisms and thus the obvious impossibility of observing the infection with the naked eye before it is too late to "select" it becomes immediately apparent that a fundamentally different approach is needed.

It is necessary (1) an opportunity to change plant seed plasma in a specific manner in such a way that the intrinsic resistance of the plant to viruses is enhanced, and (2) an opportunity to exogenously supply a safe substance or substances to already infected plant cell material, which provide broad protection of the plant cell against a whole spectrum of different plant viruses, thereby preventing further damage to the crop and halting the continued spread of the infectious agents. The present invention meets these needs by providing methods by which the beneficial physiological effects of IFN can be realized in members of the plant kingdom.

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Studies regarding the placement of exogenous human interferon (HuIFN) in plants have been conducted, see Orchansky et al,

Proc. Natl. Acad. Sci. USA _79, 2278 (1982). A similar study has also been conducted regarding the exogenous placement of a so-called intracellular mediator (2'-5 'oligoadenylate synthetase) in plants, see Devash et al, Science 216, 1415 (1982).

The invention is a consequence of the inventor's finding that certain essential amino acid sequences or biochemical fragments of a naturally occurring class of proteins, referred to as interferons (IFNs), are capable of both plant virus growth and cell damage once a plant has been infected, as well as prevent the spread of various chronic or latent plant viruses to normal uninfected plants, i.e., to prevent infection initially. This dual capability is referred to in the claims as "protecting" a plant against viruses.

The essential amino acid sequences referred to above are also referred to as "ancestor" sequences in the description and in the claims. An ancestor sequence is a sequence that has been preserved throughout development history, that is, over a period of at least millions and probably even tens or hundreds of millions of years without change. For purposes of this invention, an "ancestor sequence" or "ancestor fragment" is defined as an amino acid sequence that does not necessarily normally exist freely in nature, and which in and of itself has the ability to give a plant an anti-viral response (response). to show. The inventor has determined that progenitor sequences are at least partially responsible for imparting IFN properties to complete molecules or larger fragments containing the progenitor sequence (s).

The invention can be practiced in many different ways, from (a) the genetic level, ie incorporation of the genetic information encoding (programming) biosynthesis within the plant cell of an ancestor sequence or a molecule (eg 13 2 0 2 5 6 .............................._ __ ............ ...........

- 3 - complete (HuIFN) that contains one or more ancestor sequences; or (b) the mechanical level, ie the physical supply of IFN (s) or fragments thereof in an exogenous manner, ie topically to already infected plant tissues or plant tissue requirement, which threatens the risk of a viral infection.

In addition, double-stranded RNAs (dsRNAs) of natural (any phylogenetic source) or synthetic origin can manipulate the same result (provided the target plant cells contain a programmable IFN genetic element) and indeed enhance the therapeutic result under various plant cell virus interactions.

One of the main findings that led to this invention is that incomplete IFN molecules are sufficient to provide an anti-viral response. In other words, the inventor has determined, as will also be explained in detail below, that certain amino acid sequences within an IFN molecule give an anti-viral response that is virtually equivalent to that which would be given by the total IFN molecule. The inventor has further determined that the sequences are ancestral - that, as mentioned, they probably evolved over the centuries and are further inherent in molecules in many organisms, both plants and animals.

This observation quite simply implies that the present invention can be practiced well beyond the scope of total IFN molecules. That is, any molecule containing an (ancestral) amino acid sequence as will be described below can be applied topically to provide an anti-viral response. Furthermore, any plant containing a gene encoding the production of an ancestral sequence, or encoding a molecule containing an ancestral sequence, can be "induced" to produce the slide sequence or molecule. This means that some plants, without being genetically modified to produce total IFN molecules, can nevertheless be induced to react in an IFN-like manner, i.e. as if the plant was genetically programmed to produce its own IFN, or as if he had been topically treated with total IFN molecules. It is noted that "IFN-like" has this 8320256-4 meaning throughout the description and claims. If a plant has been genetically modified to contain a gene encoding (total molecules of) IFN, the plant can of course also be artificially induced to produce total IFN.

Furthermore, the inventor has determined that plants themselves produce substances containing ancestral sequences, that is, plants supply plant interferon (P1IFN), an unprecedented finding. Therefore, the invention also encompasses genetically altering plant seed plasma so that it contains a working gene that brings plant interface expression even though the gene is of plant origin.

The present invention therefore provides for the arrest or prevention of viral infections in plants by causing the plants to develop an IFN-like response. The modus operandi to realize this can follow one of three different tracks.

15 1. Genetically modifying the plant seed plasma so that it contains one or more copies of IFN genes that actually make the plant produce its own IFN. The implanted genes can either be turned on de novo or turned on using inducers. Genetic modification is particularly useful in plants that have no IFN genes at all or only have deficient IFN genes.

2. Topically applying molecules containing an ancestral sequence to the plant.

3. Inducing a plant, which is not necessarily genetically modified, to produce a molecule containing an ancestral sequence. This mode "turns on" or activates P1IFN genes that are otherwise latent or dormant.

Plant topical agents useful in the invention as typically applicable sources of viral protection (ie, useful for producing an IFN-like response) therefore include several total IFNs, ancestral (ie essential) molecular fragments thereof, or IFN inducers (including, but not limited to, dsRNAs), as well as the specific oligonucleotides ("mediation") substances, which are the molecular changes within the plant cell, associated with virus resistance specifically mediated by cell exposure to the ancestral. 83 2 0 2.56 ...........................

- 5 - can perform IFN bioactive fragments.

The. topical application or treatment can be performed according to a wide variety of approaches, due to the biological potency of the agents. Such treatment approaches 5 include, but are not limited to, dipping plants or seeds, spraying, or pollinating crops.

"IFN" includes Ca) all natural forms of animal IFN, including human interferon alpha, beta or gamma, (b) "synthetic" forms of animal interferon produced in non-human organisms bearing animal (eg human) interferon genes or pieces thereof (resulting in hybrid genes) have been added, and (c) chemical derivatives of such interferons with, for example, modified polypeptide chains or modified glycosyl units.

The invention, of course, has a significant and broad applicability, the most immediate and economically important of which is to protect main crops produced on a national and even world scale. Furthermore, the invention, for example by strengthening the intrinsic defense mechanism of the plants, even makes it possible for the plants to be pressured by the environment without succumbing to their viral infection, something which generally occurs when, for example, the farmer has a natural geographic growth zone for plant cell material. Consider that many tropical plants cannot be grown in non-tropical regions because the associated strain apparently increases susceptibility to pathogens including viruses; for example, papaya plants, which are normally relatively virus-free in the tropics, contract lethal viral infections almost uniformly when attempted to grow them in California. The invention is therefore not only of direct applicability, but will also result in additional agricultural advantages - which are apparent even to the trained observer in the field of agronomic biotechnology - over time as the invention becomes increasingly is put into practice.

Furthermore, phatogenic organisms other than viruses, the life cycle of which involves the use of intracellular products from plant cells, are also halted by IFN as disclosed in this invention, 8320256-6. The other pathogenic organisms include certain bacteria and protO2. See "Selective Inhibitors of Viral Functions", W.A. Carter, Ed .; Chemical Rubber Company Press; Cleveland, Ohio; 1973; that contains an extensive discussion of several non-virus pathogenic .5 agents sensitive to the IFN effect, particularly at a high IFN concentration, and which publication is to be considered incorporated herein by reference.

Figures 1a and 1b show a comparative three-dimensional structure of human IFN beta one (HuIFN SI) and human IFN gamma 10 (HuIFN-γ).

I. Detection and therapeutic exploitation of the essential ancestral IFN bioactive regions and resulting ancestral IFN fragments.

The determination of IFN structural homology (ie that most if not all IFNs have common structural elements in the form of amino acid sequences) over the giant evolutionary time can be proven as illustrated by the new construction of three-dimensional models as shown in Fig. 1. The models show strong tertiary (three-dimensional) similarities that ultimately reveal the generality of regions encompassing the bioactive site, ie, that part of the IFN molecule that imparts the biological activity of the total molecule to the target cell, be it a mammalian cell or a cell of plant origin.

Figure 1 is a two-dimensional representation of three-dimensional models created using the data shown in Table A.

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TABLE A

STRUCTURAL CHARACTERISTICS OF IFN. ^ AND

[Pfc] [P] [P]

Reverse turns 5 γ3-6 1.29 0.88 0.99 69-12 1.35 0.77 0.75 γ27-30 1.38 1.01 0.86 632-35 1.14 0.92 1, 00 γ43-46 1.21 0.91 0.79 10 γ65-68 1.33 0.98 0.84 871-74 1.44 0.84 0.74 Y86-89 1.39 0.83 0.69 6110-113 1.12 0.80 0.91 Y132-135 1.08 0.86 0.81 15 8 136-139 1.18 0.85 1.01

Alpha helix Y8-16 1.13 1.01 816-22 1.27 1.01 γ31-42 1.23 0.93 20 640-55 1.29 0.92 Y51-65 1.03 1.10 859- 68 1.17 1.18 Y95-100 1.10 0.89 891-107 1.22 0.87 25 8 116-121 1.09 0.89

Beta plate Y103-121 1.14 1.15 6123-135 0.89 1.15 6141-161 0.86 1.16 30 Y47-60 1.04 1.25 859-68 1.17 1.18 § t % 2. ^ - 8 -

Four amino acids were taken simultaneously and the probability for the existence of certain conformation structures within the total IFN molecule was calculated. For example, the existence of reverse twists was calculated using the formulas of Chou and Fasman (Annual Reviews 5 of Biochemistry, vol. 47, biz. 251-276, 1978). The existence of alpha helices and beta-pleated plates was deduced for average pieces of ten to twenty contiguous amino acids using the same formulas, p indicates the probability of a reverse turn; P indicates the probability of alpha helix formation and P indicates the probability of beta-pleated plate formation. The numbers in the left column indicate the relative position of the amino acid residues, starting from the amino endpoint of the IFN molecule.

The total and far-reaching deduction of IFN structural homology has never been proclaimed or published before as it entails a creative fusion of certain previously known and isolated facts plus additional insight due to the inventor's new approach *. approach using comparative nucleotide sequence data, comparative protein sequence data, mutation frequency data, conformation data, and Markov chain analysis (all for different forms of IFN) has led to the determination of IFN's ancestral or progenitor ancestry sequences that may provide an anti-viral activity in all contemporary life forms, including dispersed species of plant life. From these ancestor sequences, a further determination was made into the constituent amino acid components. To emphasize it again, these ancestor (ancestral) sequences, which are found within larger molecules, are not found isolated in nature and may exhibit biological activity in and of themselves and confer viral resistance to plants, i.e. resistance to viral multiply 30. The inventor has determined that the tertiary structure in the sequences is retained even when certain amino acids are mutated upon reaching the ancestral or ancestor IFN molecule that no longer exists in nature, of course, since it was millions of years ago. mutated. Only the rudiments thereof 35 in the form of the biologically active essential amino acid sequences 83 2 0 2 5 6 .............................. ...........

- 9 - exist in contemporary plant and animal life, including humans.

The desired molecular sites (ie, the biologically active amino acid sequences) from IFNs can be easily isolated by a range of available techniques and sequenced in either the genomic form (J .. Bresser and D. Gillespie in Analytical Biochemistry, vol. 129, p. 357, 1983) or the protein form in which the genomic product has been expressed on nitrocellulose paper (Bresser et al. in Proceedings National Academy Science, US, 10 in press, 1983; Bresser et al., DNA, in press, 1983). Thereafter, many available variations in the solid phase synthesis of peptides can be undertaken to produce the active fragments of IFN having the therapeutic value described herein · The synthetic production by any route (solid phase synthesis or recombinant DNA technology in bacteria, yeast or animal cells) of such amino acid sequences for the purpose of favorably affecting plant cell function and terminating viral pathology is therefore certainly within the scope of the invention.

The essential amino acid sequences are typically about 10 to 15% of the normal molecular length of IFNs, i.e., more or less 25-45 amino acids in length, rather than the usual about 162 constituent amino acids. The identification of these essential amino acid (peptide) stretches was made possible by the inventors' deductions and facilitated by computer analysis of tens of thousands of the theoretically possible sequences from certain regions of many different IFNs that have been preserved during vertebrate evolution. The absolute location of these regions will vary from IFN molecule to IFN molecule because some IFNs (e.g. human IFN-) are shortened in length by about 10-15%. Important to the active fragments is their three-dimensional activity, not their relative location to the amino end of the intact molecule.

In addition to desirable physicochemical properties, the IFN pieces or sequences are also useful from a cost-effectiveness standpoint as their small size will allow large-scale production at a fraction of the cost of the full IFN

8320256-10 molecules, as well as by various preparation methods, such as by recombinant DNA methodology, solid phase synthesis, etc., as noted above.

The invention can of course also be practiced with all naturally occurring or synthetic (recombinant DNA technology in bacteria, yeast or animal cells) complete IFN molecules, provided that they contain the necessary biologically active regions and are also free of any molecular frills (usually sugar or carbohydrate groups) that can hide the biologically active site from the plant cell surface or otherwise, for example, by disruption of conformation, render the region of the biologically active site relatively or completely inert on plant cell material. Usually, the presence of bulky side chains of core or peripheral oligosaccharide groups will prevent transspecies expression of IFN activity.

15 by such a direct or conformational mechanism (see, for example, W. Carter, in Life Sciences, vol. 25, p. 717-728, 1979 and also in Pharmacology and Therapeutics, vol. 8, p. 359,355, 1980).

II. Active interferon areas

Genes encoding interferon have been donated (eg, Derynk, et al, Nature, Volume 285, pp. 542-547, 1980). These genes have been sequenced (Goeddel et al, Nature, Volume 290, pp. 20-26, 1980; Derynk et al, Nature, Volume 285, pp. 542-547, 1980) and the primary structure of nine of the several interferons have been identified. With the aid of computer programs, the inventor has identified areas that are necessary for certain biological properties (Gillespie and

Carter, Handbook of Experimental Pharmacology of Interferon, in press.) Of the interferon system.

Ancestral interferon amino acid sequences evoking the anti-viral response include sequences 25-40 and 115-141. By analysis of published gene sequences (Derynk et al and Goeddel et al, supra) it is possible to determine the existence of at least these two ancestral sequences in each of human IFN .. and ... Similar analyzes as above are applicable to other animal IFNs.

The interferon region, which was determined to be essential 8320256-11 for binding to specific cell receptors, includes amino acids 115-141 (Gillespie and Carter, 1982). The position of this region is determined or facilitated in part by homology to a region of the B subunit of cholera toxin (Lai, Journal Biol. Chem. Volume 252 5 pp. 7249-7256, 1977), a substance that competes with interferon for the binding to cell receptors.

It is emphasized that other ancestral fragments different from the above-identified fragments exist in nature, and the invention should not be construed to be limited to only the ancestral fragments described herein. The inventive concept lies in the treatment of plants with molecules containing ancestral fragments or with the fragments themselves. Identification of additional fragments is possible by an expert.

15 III. Preparation of Interferon Pieces with Selected Areas.

Interferon pieces can be prepared in various ways. By way of example, one suitable method will now be described. Other means, such as chemical modification of natural interferon or chemical modification of donated (synthetic) complete interferon, interferon inducers, etc., are also readily known and are within the scope of the invention.

Donated interferon genes can be cut at specific sites with specific restriction endonucleases. The resulting interferon gene fragment, encoding a stretch of the interferon polypeptide, can be fused to an expression vector containing RNA polymerase promoter, ribosome binding site, initiation codon and any other necessary signals. This recombinant DNA can be introduced into suitable host cells for the purpose of synthesis of the interferon stretch.

As an example, the following procedure for expression in a host of a HuIFN gene fragment was performed. 5 g of the human interferon beta gene, equipped with a polyA.dT tail, and donated in pBR322, were incubated with 50 units of EndoR'P 1 in 20 mM tris, pH 7.4, 10 mM for two hours at 37 ° C. MgC ^, 50 mM (NH ^^ SO ^ 35 and 100 mg / ml. Bovine serum albumin to cut the interferon gene, 8320256-12 - leaving a single stranded end of bases 203-206. The DNA was at 0, Brought 3 M in potassium acetate and precipitated from ethanol The DNA was dissolved in 100 ml of 50 mM potassium phosphate, pH 7.4, 6.7 mM MgC 2 · 1 mM mercaptoethanol, 35 mM deoxyadenosine and deoxycytidine, 5 and 25 units of the Klenow fragment end of Escherichia coli DNA polymerase The DNA was placed at 0.3 mM in potassium acetate and precipitated from ethanol The precipitate was dissolved in 100 ml of 30 mM sodium acetate, pH 4.6, 50 mM NaCl, 1 mM ZnSO 5% glycerol and 25 units of SI nuclease and incubated for 1 hour at 37 ° C. This gave a blunt end interferon gene fragment completely to nucleotide 204, the first based in the code word for leucine at position 45. Sodium acetate was added to 0.3 M and the DNA was precipitated from ethanol.

Separately, 5 g of the human interferon 15 beta gene (fitted with a polydAldT tail and donated in pBR322) were incubated with 50 units of Endo R MboII in 10 mM tris, pH 7.9, 6 mM KCl for two hours at 37 ° C. .10 mM MgCl 3, 1 mM dithiothreitol and 100 mg / ml bovine serum albumin. The DNA was precipitated from ethanol and treated with SI nuclease as described above. The 288 base-20 pairs blunt-ended fragment containing nucleotides 401-688 of the interferon gene was purified by electropheresis through 3% agarose. A 0.25 mg portion of this fragment was taken up with 5 g of Pst 1 -treated, DNA polymerase -treated and SI nuclease -treated DNA.

The recombinant DNA thus formed contained an interferon gene that lacked nucleotide 205-400, which gene will encode an interferon polypeptide lacking amino acids 46-111.

This recombinant interferon fragment can be inserted into many suitable vectors for expression in bacterial or mammalian cells by conventional techniques. Interferon or pieces of interferon can be purified by standard methods, although the invention can be practiced with interferons that are less than 1% pure, provided no other harmful substances are present. For example, proteins in solutions containing interferon are bound in Cibachrom 35 blue sepharose in the absence of ethylene or propylene glycol.

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The column is washed with various solutions that remove most proteins, except interferon, and the interferon is eluted with solutions containing ethylene or propylene glycol or other suitable eluents (Carter et al, Pharmacology and Therapeutics, 5 Volume 8 pp. 359-377 1980). If desired, further purification of Cibachrom blue fractions or other materials can be carried out by passing interferon-containing solutions over column matrices containing anti-interferon antibodies using conventional binding and elution conditions.

The above-described recombinant DNA can be prepared by using some of the same enzymes or all the same enzymes under different conditions or in different sequences or by variants of the above-mentioned enzymes or interferon genes. Other suitable recombinant DNAs can be generated with other enzymes and other protocols. Finally, the use of recombinant DNA to obtain pieces of interferon is just one example of a way of producing a piece of interferon. Other ways such as solid synthesis, proteolytic cleavage of intact material or genetic donation of interferon prepared in bacteria and yeast can be used in an equivalent manner.

IV. Alteration of plant seed plasma to obtain plants with hereditary resistance to viral infections.

The inventor has determined that plants producing increased concentrations of plants or humans interferon will be resistant to a wide spectrum of pathogens. Therefore, the invention provides a method of forming such a plant. The new plant created by the following method is genetically new.

The method comprises A. donating human and plant interferon genes into recombinant DNA; B. inserting this recombinant DNA into protoplasts and isolating cells that synthesize increased concentrations of plants or humans interferon; and G. producing a mature plant from the protoplasts.

8 3-2025-6 ............-........... - -.....-............ ...........................-..... ............

- 14 -

For example, the Nicotiana tobacco plant has been used and is described below as a prototype.

A. Donating human and plant interferon.

As previously mentioned, human interferon genes have been donated (eg Derynck et al, supra). Several conventional procedures can be used to similarly donate plant interferon genes; what is needed is a suitable "probe" for finding the recombinant DNA. Bar probe that can be used for this purpose is the highly preserved part of the human interferon or. gene contained within, but not limited to, nucleotides 345-423 (Gillespie and Varter 1982, Handbook of Experimental Pharmacology of Interferon, in press). Other suitable probes may also exist. Importantly, the invention does not stem from a choice of methods used for donating the plants to interferon genes, but rather stems from the genetically unique and enhanced desirability of the final plant.

B. Inserting the recombinant DNA into Nicotiana protoplasts and isolating cells that synthesize increased concentrations of plants and human interferon.

The analytical processes described herein for HuIFN allow one to easily identify comparable ancestral sequences in various animal or vegetable IFNs. The methodologies described herein for the isolation of gene fragments and resulting insertion and expression will use a methodology similar and in some cases identical to that described for ancestral sequences in HuIIF .. and .. Protoplasts can be generated and DNA can be introduced therein according to conventional techniques (Bourgin et al 1979. Physiol. Plant. 45: 288-292; Caboche, M.1980. Planta 149: 7-18; Cocking et al 1981. Nature 293: 265-270) . The difficulty is that little. recombinant DNAs insert themselves into plant chromosomes, with the result that they do not become a stable part of the plant's genetic material. The Ti DNA plasmid from Agrobacter (Davey et al 1980. PI. Sci. Lett. 18: 307-313; Wullems et al 1979. In Adv. Protoplast Res. Proc. Sth. Symp: 407-424) is an exception to this. .................................................. ........................-......-.................. ...............

- 15 - but this DNA plasmid has several unfavorable properties. Problems include that the Ti plasmid DNA is too large, the DNAs inserted into this plasmid do not express the correct proteins, and cells infected by this plasmid do not develop properly. Agrobacter's Ri DNA plasmid allows plants to develop well, but retains the other unwanted properties.

To insert foreign DNA into Nicotiana chromosomes, the following plasmid can be used: pBR 322 plasmid from E. coli can be modified to contain two inverted tandem copies of a Nicotiana short, scattered, repeated DNA. This plasmid will contain only one Eco R1 site: located on the boundary between the two Nicotiana repeated sequences. The plasmid will be opened with Eco R1, then plants and / or human interferon genes can be inserted into the plasmid. On the other hand, other plant sequences containing pBR322. or other vectors such as Ti and Ri plasmids from Rhizobium are used.

Many copies of the plasmid containing a plant interferon can be introduced into Nicotiana protoplasts by microinjection, calcium phosphate precipitation, Agrobacter infection, protoplast fusion, etc. 20 Transformed protoplasts can be propagated as plant cells and by in situ molecular hybridization to chromosome DNA with a pBR 322 probe are detected. Such cells, induced with poly I: C, can be tested for increased concentrations of plant interferon mRNA production by in situ hybridization, using a pure plant interferon gene as probe. Such cells producing increased concentrations of plant mRNA can be tested for increased plant interferon synthesis by the conventional biological assay for anti-viral growth factor.

Nicotiana cells producing increased concentrations of plant inter-feron can be reconverted into protoplasts and transformed as above by said vectors now containing human interferon genes. Transformed protoplasts can be propagated as plant cells and tested for increased concentrations of plant interferon plus human interferon mRNA and protein 35 by in situ molecular hybridization and the biological 8320256-16 assay for anti-viral growth factor, respectively. These transformed protoplasts can then be grown into mature tobacco plants.

More specifically, the method can be performed as follows: Protoplasts can be isolated from Nicotiana spp. according to 5 Chupeau et al (C.R. Acad. Sci. Ser. D (Paris) 278: 1656-1568, 1974). Sterilized leaves can be peeled and incubated in Tq medium 10.3 mM NH ^ NO ^, 9.4 and mM KNO ^, 1.5 mM CaCl2.7H20, 0.75 mM MgS04.7H20, 0.62 mM KH2PC> 4 0.1 mM FeSC> 4.7H20, 0.1 mM Na2EDTA, 16 mM H3B03> 0.6 mM MnS04.H20, 3.5 mM ZnS04.7H20, 0.2 mM CuS04.5H20, 0.22 mM 10 AlCly 0 , 13 mM NiCl2.6H2O, 8 μM Nicotinic Acid, 2 μM Ca Panthothenate, 0.04 mM Biotin, 16.1 μM Naphthalene Acetic Acid, 4.4 μM 6-Benzyladenine, 58 mM Sucrose, 440 mM Mannitol) without Sucrose, and with 0, 02% Macerozyme R10, 0.1% Cellulase Onozuka R10 and 0.05% Driselase. The released protoplasts can be washed in medium Tq 15 by low speed centrifugation. Other methods of protoplast isolation can be substituted for such that viable single cells can be isolated without significant amounts of their cell wall.

Nicotiana spp protoplasts can be incubated with transformation vector: DNA carrying interferon genes, or with bacteria containing such DNA, using one of a number of standard transformation procedures (Davey et al 1980. PI. Sci. Lett. 18: 307 -313; Willems et al 1979. In Adv Protoplast Res Proc Proc 5th symp .: 407-424 Such vector DBA may be the E. coli plasmid containing repeated plant sequences as described above or any other vector DNA , which can be multiplied in a microorganism and incorporated into protoplast chromosomes.

Transformed protoplasts can be grown in Tq medium at 25 ° C in the dark for four days and then transferred under fluorescent lamps (2500 lx, 16 hours per day).

After 30-60% of the protoplasts have shared once, they can be collected by centrifugation and washed once in medium AG (10 mM KNO3, 3 mM CaCl2.2H20, 3 mM MgS04-7H20, 1 mM KH2PO4, 0.1 mM FeSO4.7H20, 0.1 mM Na2EDTA, 49 mM H3BC> 3.1, 1.8 mM MnSC> 4.H20, 10.4 mM ZnSO ,,. 7H „0.04 mM CoCl„ .6Η „0, 0.36 uM CuSO .5H_0, 0.41 uM NaMoO .2EL0, 4 l 2. IQ 2.4 Δ 35 0.66 uM A1C13, 0.40 mM NiCl2.6H20, 0.18 uM KI, vitamins as in Tq medium -.f 3 2..0 k S t) .............................-..... -------------------------------------------------; --------------------- - 17 - 0.53 µM naphthalene acetic acid, 4.4 µM 6-benzyladenine, 58 mM sucrose, 440 mM mannitol, 1 mM glutamine. Cells can be plated on petri dishes in medium AG (Caboche, Planta 149: 7-18, 1980). The dishes can then be incubated under standard light conditions at 25 ° C in tightly closed transparent boxes under which conditions the transformed protoplasts form colonies.

Individual colonies can then be picked, reconverted into suspensions of some protoplasts and seeded on replica petri dishes in AG medium. Replica plates can be examined for vector or recombinant interferon gene expression by analyzes of: • 1) the presence of specific DNA sequences by molecular hybridization (Robins et al 1981 J. Molec. Appl. Gen. 1: 191-203,

2) the presence of specific RNA transcripts by molecular .15 hybridization (Brahic and Haase. 1978. Proc. Nat. Acad. Sci., USA

75: 6125-6129 and / or 3) the presence of excess interferon protein by immunological or biological analyzes (Sela. 1982. Interferon Sci. Memo., Memo number Iall34, January). Other analyzes for excess interferon production 20 may be suitable.

C. Producing a mature tobacco plant from genetically engineered plant cells.

Colonies showing the presence and expression of new interferon genes can be grown in AG medium for 1-2 months, then plated on solid R4 medium for bud regeneration (Bourgin et al. 1979. Physiol. Plant. 45: 288-292). The buds produced can be transferred to seedling medium B to form plantlets (Bourgin et al. 1979. Physiol. Plant, 45: 288-392). Plants with roots can be potted and grown to maturity in a greenhouse. Other methods of converting some transformed plant cells into mature plantlets can be used instead.

The plants can then be tested for their production of interferon and for their resistance to tobacco mozalek virus, -Jy Ο f- ·. (j'1 't-. · i.- - 18 - (Sela. 1981. Adv. Vir. Res. 26: 201-234) or against other foreign agents using standard evaluation methods. Disease resistant strains with high interferon production can be selected and sexually propagated in any convenient and effective manner.

The above protocol is given by way of illustration of the feasibility of the method of constructing this genetically novel plant with desirable properties. It will be appreciated that in this rapidly changing region many modifications of this method may be successfully expected without departing from the scope of the invention.

V. Topical application

As mentioned, the therapeutic agents of the invention can be applied topically by myriad techniques ranging from the earthly (eg using a plant watering can in a greenhouse) to the complicated (eg crop pollination of hundreds or thousands of hectares).

The preferred way to practice the invention will depend on biological and mechanical factors when treatment of the plant is desired; for example, permanent protection of a seed crop will preferably be carried out by seed plasma change via an increased IFN copy number (plants and / or animal IFN genes). On the other hand, to protect plants already in the field that occupy a large area, crop pollination would be a desirable embodiment. Here, for example, special topological conditions (think of the molecular stability, the wind shear effect, etc., when parental IFN components are released from low-flying aircraft) as well as biochemical conditions (think of the uptake efficiency by young versus mature or aging leaf cell structures). ) are taken into account. In such cases, the preferred embodiment will be low molecular weight components as they generally provide high thermal and physical stability and increased cellular uptake / receptor binding. The ancestral IFN-targeted oligonucleotides and IFN fragment peptides are examples of 83 2 0 2 5 6 ...... .......................... ... ................

- 19 - Relatively heat-resistant and swirl-resistant active parts derived from the basic invention.

For topical application, at least one IFN or other substance containing an ancestral sequence (or inducer) will be dispersed in a suitable agricultural acceptable carrier such as water, ie the application is in the form of a solution. The concentration can range from about 0.0001 to 100.000 IRU (International Reference Unit) per milliliter of solution.

If it is desired that IFN be spread as a dry powder to be spray applied, a concentration range by weight from about 1 ppm (parts per million) to about one part 13 16 out of 10 -10 parts is effective when the interferon becomes added to a dry, agriculture-acceptable carrier. Suitable carriers are known in the field of crop pollination. The interferon 15 can be mixed or combined with other plant treatment agents - pesticides, herbicides, fertilizers, etc., provided that none of the additional agents affect the biological activity of the ancestral sequence.

The final concentration of IFN or other ancestral agent will depend on the crop to be protected, the virus to be protected against, and the field conditions. The final concentration can also vary depending on the molecular fraction (fraction of the molecule), which is not ancestral. For example, in the synthetic IFN fragment, which mainly consists only of the amino acid sequence 115-141, the molecular fraction of non-ancestral material would approach zero, whereas in complete IFN molecules the molecular fraction of non-ancestral material is approximately 0, 6, i.e. about 60% of a total IFN / e.g. HuIFN) molecule is not ancestral.

For example, a typical application of IFN to crops would be at a concentration of one part per trillion (dpb; it is noted that for HuIFN 1 mg is about 10 IRU). For surface application (which requires close to 100 gallons per acre, i.e. 378.5 liters per 4047 m2), typically less than half a milligram of complete HuIFN per acre would be required. Crop pollination, for which f320256 .............._.........................__..... ..........__...............................

Typically one fifth to one tenth of the volume for surface application is required would require comparable amounts of interferon, or perhaps depending on the effectiveness of the topical application.

In some instances, however, it may be desirable to add an additional stability to IFN or its ancestral fragments so that it is more resistant to application loads and possibly prolonged exposure to the elements in the environment.

This will now be described.

Interferon, like other proteins, is a relatively unstable chemical. It depends on a very specific three-dimensional orientation of its various linearly arranged amino acids for solubility in aqueous solutions and for biological activity (see Fig. 1). In particular, regions of the interferon molecule responsible for binding to cells and evoking complex biological responses must be properly presented and arranged. However, there is also a large amount of alternative non-functional orientations that can be freely formed. These alternative states are promoted by heating, certain external chemicals and long-term storage. A solution aimed at preventing the formation of inactive alternative states is to build molecules that exhibit IFN activity, but which have only a limited number of alternative states, such as the interferon fragments described above. Another solution developed by the inventor is to limit the formation of alternative states by immobilizing the interferon on a carrier molecule or structure.

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

To investigate the feasibility of such an approach, the inventor constructed several column matrices containing different ligands which could be effective stabilizers (see Figure 2, Carter et al, 1980, Pharmac. Ther. 8: 359-377. The column was characterized with attached albumins. 100 ml of a 35 non-dialyzed interferon preparation, containing 11,500 units and 0.99 mg 83 2 0 2 5 6 ................... ...... _.............................

Protein per ml was applied to a column using a peristaltic pump at a flow rate of 60 ml per cm2 per hour.

The albumin column was equilibrated with a 0.02 M sodium phosphate (pH 7.4) solution containing 0.15 M NaCl. The eluent from the column was partitioned by a 1: 9 flow divider.

The 10% portion of the eluent was collected in 1ml of a 1% bovine serum albumin solution containing 0.02M sodium phosphate and 0.15M NaCl and used to analyze the interferon activity.

The 90% portion of the eluent was used to measure the protein-10 concentration. The breakthrough fractions contained about 48% of the applied protein and less than 1% of the applied interferon activity. Further elution of the column was performed with 50% (v / v) ethylene glycol and 50% 0.04 M phosphate (pH 7.4) and 0.30 M NaCl. The remainder (86%) of the interferon activity was recovered with very little (less than 2%) of the original protein. Some of these experiments have been recently published (Carter, W.A., Methods in Enzymology 78: 576-582, 1981).

The above experiment showed that interferon can be coupled with serum albumin whereas most other cellular proteins could not. Experiments with other immobilizing proteins such as cytochrome C showed that interferon had a general property of combining with proteins. An inventor hypothesis is that the highly hydrophobic nature of interferon compels interactions with commonly sequenced hydrophobic pockets of other proteins, and this experiment suggests that the hypothesis is correct. Whatever the mechanism, the above experiment spurred the development of complexes with proteins to stabilize interferon.

The procedure developed by the inventor for this purpose is as follows: human interferon can be prepared in any conventional manner from any biological source τ from human cells, from recombinant microorganisms, etc. After purification, human serum albumin or other protein carrier added in excess, usually up to 3 mg / ml. The solution can be dialyzed against phosphate buffered saline or other suitable buffer and the material can be lyophilized. In a typical example, 1 million units 83 2 0 2 5 6 ............................. ....... .................................................. ...

- 22 - purified interferon lyophilized with 3.46 mg of sodium phosphate.

Several changes to this procedure are acceptable in this rapidly changing area. Carrier proteins other than albumin or cytochrome C are acceptable. Other carrier molecules or structures can also be acceptable, such as lipids, small hydrophobic ligands, membrane fragments, or even solid particles. Other fasteners such as ionic or covalent bonds may also be suitable. Other salts or buffers or other concentrations of salt or buffer (including the absence of salt or buffer) may also be acceptable. However, the procedure clearly stabilizes IFN for external conformational changes, including denature ring against the effects of, for example, wind friction and environmental influences.

Topical application of HuIFN to plants is generally effective in controlling the spread of virus from one plant to another. To demonstrate this, young potato leaves were systemically infected with potato virus N or tobacco leaf roll virus.

Discs were punched from the leaves and soaked at 25 ° C for 7-10 days in a nutrient medium lacking or containing human interferon alfa. After this incubation, the leaves were ground in 1% Brij 35 and particulate matter was removed by centrifugation. Viral proteins were quantitated by immunoassay (SLISA) using optical density as a measure of the virus. High optical density values correspond to increased amounts of virus. The results are shown in Table B below.

Table B - halting the spread of potato virus by HuIFN - number of days units / ml HuIFN virus Incubation 0_ 100 1000 30 potato virus N 7 0.519 0.342 0.184 potato virus N 10 1.331 0.702 0.838 leaf roll virus 10 0.673 0.298 0.254 8320 ^ 56 - 23 -

As the data show, IFN exhibits a pronounced anti-viral effect in plants, although it appears that virus arrest has reached a plateau, i.e. virus arrest has not increased linearly over the range tested 5 with an increasing interferon concentration. In addition, while topical application produces a dramatic virus slowdown, this impact decreases somewhat over time, indicating that repeated applications are needed at regular intervals, for example, every 10 to 15 days. Importantly, the inventor has determined that a periodic application of interferon to virus infected plants will prevent the spread of viruses in those plants and also prevent the transmission of viruses from these plants to uninfected plants. The inventor obtained photo series of the leaves during the above mentioned HuIFN experiments and noted that the leaves were growing and blooming at the same time that the virus life cycle was stopped.

Given the high yields of crops due to topical application coupled with the fact that very low purities of IFN are sufficient (ie, no expensive purification procedures are required), repeated applications are economically very feasible. For example, based on his experiments, the inventor estimates that a greenhouse of about 9.3 m2 can be protected at common prices for about ten dollars per month. The IFN of any purity can be administered, provided it meets the criterion of being free from contaminating living organisms (bacteria, etc.) or intact viral agents that could induce plant cytopathology or pathology in the ultimate users, or now that they are humans or animals. After appropriate fumigation procedures such as filtering living organisms, such IFN lots would be useful for the invention, although they would be rejected for human clinical applications.

The term "topical application" further encompasses the application of substances referred to as "inducers", which, although not themselves IFN molecules, have the property of serving as a biological stimulus whereby an IFN producing organism is forced to produce its own IFN in an interval short of the interval that would be required in the absence of applied inducer. Important of these are the so-called "mismatched dsRNAs", which are potent IFN inducers, but show few toxic side effects in humans. The biological mode of action of these materials, together with a list thereof, is fully disclosed in U.S. Pat. No. 4,103,641 to the present inventor.

10 dsRNAs are also effective in members of the plant kingdom, and can be applied in the same manner as noted above for IFN molecules or biologically active fragments thereof. The plant does not necessarily have to be genetically engineered. All that is required is that a given dsRNA induce the formation of a molecule that has a biologically active essential (amino acid) sequence that elicits an interferon-like response. As stated previously, this requirement means that the plant must contain a polynucleotide sequence encoding a biologically active amino acid sequence. Whether a particular plant species meets this requirement can be determined by simple experiments, ie topically applying one or more dsRNAs to plants of interest and testing the anti-viral growth factor in a known manner . Alternatively, a test of the inducibility of IFN can be performed using the in situ hybridization technique illustrated by the inventor above for Nicotiana.

In addition, there exist a series of substances referred to as intracellular mediators, of which 2'f5'-oligoadenylic acid and / or a functionally active derivative thereof are typical examples, which substances are also useful as topical application agents. The exact biochemical mechanism by which these compounds function is not known, but they are known to mimic the IFN effect caused by virus exposure, essentially replacing the need for IFN. The inventor has determined that this imitation can be extended to plants. Analogs 35 can also be used, including core (dephosphorylated) 2'-5 'oligo-13 2 0 2 5 1 __.

Adenylate, core 3'-5 'oligoadenylate, and core 2'-5' oligoadenylate cordecypine (3'-deoxyadenosine).

The concentration range useful in applying dsRNAs (such as Ampligen, a branded product of HEM Research, Rockville, Maryland, or Poly I. Poly C) is between about 0.01 g / ml and 1000 g / ml in aqueous solution. The useful concentration range for application of the intracellular mediators is between about 0.01 and 100,000 nanomoles per ml in aqueous solution.

A preferred embodiment with respect to topical application lies in the combination, generally as a physical mixture, although separate applications will also suffice, of an IFN, or an essential fragment or inducer thereof, with a substance containing insects, in especially lice, keeps from landing on the plant and acting as a vector that spreads the virus. Insects such as -15 lice may represent an important way of transmitting viruses, feeding on many plants within relatively short spaces of time and exchanging infected material for healthy plant tissue which then re-infects the louse, exchanges it with other healthy plants, etc. an ever-repeating cycle.

By combining a substance that can produce an IFN-like response with a substance that blocks insects (ie, deterred), a two-prong attack against virus spread can be launched effectively. The first way of attack is to prevent insects from picking out the treated plant. Should this fail, then all virus-infected plant material exchanged by the insect with a healthy plant is counteracted by the substance that produces a HuIFN response.

In particular, substances which are particularly effective in blocking insect (and in particular lice) vectors and which are chemically compatible with IFNs, hormone substances, are referred to as pyrethrums and pheromones. Pyrethrums are known natural substances, which are, for example, derived from chrysanthemums. Pheromones are also natural substances derived from wild potatoes.

Interferon mixed with or separately but applied simultaneously with one of these natural hormonal substances or both forms ΐ: Cj ΐ: o __ - 26 - a combination that effectively blocks insects in the first place, or the spread of virus from insects which, despite the blocking agent, prevents contact with healthy plants. The blocking agent and the anti-viral agent thus reinforce each other at a biochemical level and do not leave any environmentally harmful residues or contaminants in their final biodegradation.

Interferon can be administered in combination at the aforementioned levels (e.g. 0.0001-100.000 IRU / ml solution).

Pyrethrums and / or pheromones are administered at levels typically of the order of 0.005-10 pounds / acre. One procedure is to simply combine the desired levels of IFN (or fragment, inducer, etc.) with the pyrethrum or pheromone and a suitable carrier (e.g., water or a dry agricultural acceptable carrier) and apply it in the form of a solution .

Although only a few examples of embodiments of the invention have been described in detail above, those skilled in the art will recognize that many modifications can be made in the exemplary embodiments without departing from the invention and its advantages. It is therefore contemplated that all such modifications will fall within the scope of the invention as defined in the following claims.

8320256

Claims (25)

A method of protecting plants from pathogens, wherein said plants are caused to exhibit an IFN-like response. The method of claim 1, wherein said pathogen is a virus. The method of claim 2, wherein said response is obtained by genetically modifying said plants to produce at least one substance that elicits said IFN-like response. The method of claim 3, wherein said substance is selected from the group consisting of complete IFN molecules and molecular fragments containing ancestral amino acid sequences. A method according to claim 3, wherein said genetic modification is processed by inserting into human cells a human gene encoding the production of at least one compound containing at least one ancestral amino acid sequence. The method of claim 4, further comprising the step of inserting into said plant cells a plant gene encoding the production of plant interferon or a molecule containing an ancestral amino acid sequence. The method of claim 2, wherein said response is elicited by topical application to said plants of a substance comprising at least one compound containing an ancestral amino acid sequence. The method of claim 7, wherein said at least one compound is stabilized against denaturation. The method of claim 7, wherein said substance as said compound comprises complete IFN molecules. The method of claim 7, wherein said substance as said compound comprises part of a HuIFN molecule. The method of claim 7, wherein said substance is applied in the form of a solution and in a concentration range equivalent to 0.0001 to 100.000 IRU / ml. The method of claim 11, wherein said solution is an aqueous solution. 8 3 2 0 2 5 § ......................... .............. <·> V - 28 - The method of claim 2, wherein said response is elicited by topically applying to said plants a substance comprising at least one non-IFN compound capable of inducing said response. The method of claim 13, wherein said compound induces the production of a second compound containing an ancestral amino acid sequence. The method of claim 14, wherein said inducing compound is a dsRNA. The method of claim 14, wherein said second compound is complete HuIFN. The method of claim 14, wherein said second compound is a HuIFN fragment. 18. The method of claim 13, wherein said compound is an intracellular mediator. The method of claim 18, wherein said mediator is 2'5'-oligodenylic acid or a derivative thereof. 20. A method of protecting plants from viruses, comprising a topical application in combination of a first compound, which causes said plant to exhibit an IFN-like response, and a second compound, which blocks virus vectors. The method of claim 20, wherein said first compound is selected from the group consisting of dsRNAs and intracellular mediators. The method of claim 20, wherein said second compound is selected from the group consisting of pyrethrums and pheromones. 23. A composition comprising a first compound selected from the group consisting of dsRNAs and intracellular mediators mixed with a second compound selected from the group consisting of pyrethrums and 30 pheromones. Plant protected by the method of claim 1. 25. Plant protected by the method of claim 20. 8320256