Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
  • A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
  • A01N57/16—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds containing heterocyclic radicals
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/50—Isolated enzymes; Isolated proteins
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/60—Isolated nucleic acids
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
  • A01N65/08—Magnoliopsida [dicotyledons]
  • A01N65/12—Asteraceae or Compositae [Aster or Sunflower family], e.g. daisy, pyrethrum, artichoke, lettuce, sunflower, wormwood or tarragon
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
  • A01N65/08—Magnoliopsida [dicotyledons]
  • A01N65/38—Solanaceae [Potato family], e.g. nightshade, tomato, tobacco or chilli pepper
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/555—Interferons [IFN]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• This invention relates to protecting members of the plant kingdom from disease and, in particular, to the use of whole interferon (IFN) molecules from any phylogenetic source, and/or ancestral components thereof, to prevent or arrest plant viral infections.
• IFN whole interferon
• the ubiquity of plant viruses contributes to the global problem in generating food supplies in a cost-effective manner. No hemisphere of this earth is spared the ravages of plant viruses on its food crops and even its ornamental plants.
• viral-induced plant pathology is capable of lethal disease of coconut palms in the tropics as well as ravaging wheat, barley and other grains in the Canadian North.
• there is no known therapeutic approach or remedy save the primitive art of "culling" whereby the farmer or nurseryman removes the infected plant material before contagious spread occurs to previously uninfected plants.
• interferons certain essential amino acid sequences or biochemical fragments from within a naturally occurring class of proteins, termed interferons (IFNs), are capable of arresting both plant viral growth and cell damage once a plant has become infected, and also of preventing the spread of various chronic or latent plant viruses to normal uninfected plants, i.e. preventing infection in the first instance.
• IFNs interferons
• an ancestral sequence is one which has been conserved through evolutionary history, i.e. a period of at least millions and, probably, even tens or hundreds of millions of years without change.
• an "ancestral sequence” or “ancestral fragment” is defined to be any amino acid sequence which is not per se normally encountered free in nature, and which in and of itself possesses the capability of causing a plant to exhibit an anti-viral response.
• the inventor has determined that ancestral sequences are, at least in part, responsible for imparting IFN properties to whole molecules or larger fragments which contain the ancestral sequence (or sequences).
• the invention can be practiced in a multiplicity of different modes which stem either from:
• the genetic level i.e. incorporation of the genetic information which encodes for (programs) the biosynthesis within the plant cell of an ancestral sequence, or a molecule (e.g. whole HuIFN) containing one or more ancestral sequences; or
• dsRNAs double-stranded RNAs
• dsRNAs double-stranded RNAs
• the inventor has determined that certain sequences of amino acids within an IFN molecule produce an anti-viral response substantially equivalent to that which would be provided by the entire IFN molecule.
• sequences are ancestral -- that, as mentioned, they likely have evolved through the ages and, furthermore, that they are common to molecules in many organisms, both plant and animal.
• the implication of this determination is, quite simply, that the present invention may be practiced widely beyond the scope of whole IFN molecules. That is, any molecule containing an (ancestral) amino acid sequence as hereinafter detailed may be applied topically to produce an anti-viral response. Further, any plant containing a gene encoding for the production of an ancestral sequence, or encoding for a molecule containing an ancestral sequence, can be "induced" to produce that sequence or molecule.
• IFN-like is understood to have this meaning throughout the specification and claims.
• a plant has been genetically altered to contain a gene encoding for (whole molecules of) IFN, the plant can also be artificially induced to produce whole IFN.
• This invention thus provides for arresting or preventing viral infections in plants by causing the plants to exhibit an IFN-like response.
• the modus operandi to achieve this can take one of three different tracks!
• External plant treatment agents useful in the invention as typically applicable sources of viral protection thus include various whole IFNs, ancestral (i.e. essential) molecular fragments thereof, IFN inducers (including, but not limited to, dsRNAs), as well as the specific oligonucleotide ("mediating") substances which can carry out the molecular changes within the plant cell associated with virus resistance brought about specifically by cell exposure to the ancestral IFN bioactive fragments.
• IFN includes (a) all natural forms of animal IFN, including human interferon alpha, beta, or gamma, (b) "synethetic" forms of animal interferon produced in non-human organisms to which animal (e.g. 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 possesses, of course, significant and wide utility, the most immediate and economically important of which is the protection of staple crops produced on national and even global scales. Further, by strengthening the plants' intrinsic defense mechanism, for example, the invention even allows plants to be "stressed” environmentally without then succumbing to viral infection, a common occurrence happening, for example, when the farmer attempts to alter a natural geographic growth zone for plant cell material.
• the invention not only is of immediate utility but also will derive additional agricultural advantages - not apparent even to the trained observer in agronomic biotechnology - over time as it is progressively practiced.
• pathogenic species other than viruses, whose life cycle involves utilizing intracellular products of plant cells, are also arrested by IFN as taught in this invention.
• the other pathogenic species include certain bacteria and protozoans. See “Selective Inhibitors of Viral Functions”, W.A. Carter, Ed.; Chemical Rubber Company Press; Cleveland, Ohio; 1973; which contains a comprehensive discussion of various non-virus pathogenic agents susceptible to the IFN effect, particularly at high IFN concentration, and which is herein incorporated by reference.
• FIGURES 1a and 1b illustrate comparative 3-dimensional structure of human IFN beta one (HuIFN 1) and human IFN gamma (HuIFN- ); DETAILED DESCRIPTION
• FIGURE 1 3-dimensional models as shown in FIGURE 1.
• the models establish close tertiary (3-dimensional) similarities which ultimately reveal the commonality of domains (regions) comprising the bioactive site, i.e. that portion of the IFN molecule which confers the biological activity of the entire molecule upon the target cell, be it a cell of mammalian or plant origin.
• FIGURE 1 is a two-dimensional representation of three-dimensional models generated using the data shown in Table 1, following:
• Amino acids were taken four (4) at a time and the probability for existence of certain conformational structures within the entire IFN molecule was calculated.
• the existence of reverse turns was calculated using the formulas of Chou and Fasman (Annual Reviews of Biochemistry, vol. 47, pp. 251-276, 1978).
• the existence of alpha helices and beta pleated sheets was deduced for stretches, on average, of ten (10) to twenty (20) consecutive amino acids using the same formulae.
• P t refers to the probability of a reverse turn;
• P refers to the probability of alpha helix formation and P refers to the probability of formation of a beta pleated sheet.
• the numbers in the left-hand column refer to relative position of amino acid residues starting from the amino terminal end of the IFN molecule.
• these progenitor (ancestral) sequences which are found within larger molecules, are not encountered isolated in nature and can, in and of themselves, display bioactivity and confer viral resistance to plants, i.e. resistance to viral multiplication.
• the inventor has determined that tertiary structure in the sequences is preserved even when certain amino acids are mutated in arriving at the ancestral or progenitor IFN molecule which self-evidently no longer exists in nature, having mutated away millions of years ago. Only the vestiges, in the form of the bioactive essential amino acid sequences, exist in modern day plant and animal life, including man.
• the desired molecular sites (i.e. the bioactive amino acid sequences) of IFNs can be readily isolated by a series of available techniques and sequenced in either the genomic form (J. Bresser and D. Gillespie in Analytical Biochemistry, vol. 129, p. 357, 1983), or protein form wherein the genomic product is expressed on nitrocellulose paper (Bresser et al. in Proceedings National Academy
• the essential amino acid sequences are typically approximately 10 to 15% of the normal molecular length of IFNs, being more or less 25-45 amino acids in length rather than the usual approximately 162 component amino acids.
• the determination of these essential amino acid (peptide) pieces was made possible by the inventor's deduction and facilitated by computer analysis of tens of thousands of the theoretically possible sequences of certain domains or regions of many different IFNs conserved during vertabrate evolution. The absolute location of these regions will vary from IFN molecule to IFN molecule since some IFNs (e.g. human IFN- ) have been foreshortened about 10-15% in their length. What is important about the active fragments is their 3-dimensional activity, not their relative location from the amino end of the intact molecule.
• the IFN pieces or sequences are also useful from a cost-effectiveness point of view in that their small size will allow their large scale production at a fraction of the cost of the whole IFN molecules, and by a variety of manufacturing modes, such as recombinant DNA methodology, solid phase synthesis, etc., as noted above.
• the invention may also be practiced, of course, with any naturally occurring or synthetic (recombinant DNA technology in bacteria, yeast or animal cell) whole length IFN molecules provided they contain the necessary bioactive domains and also lack any molecular embellishment (commonly sugar or carbohydrate moieties) which may conceal the bioactive site from the plant cell surface or otherwise, as through conformational distortion, render the bioactive site domain relatively or completely inert on plant cell material.
• any molecular embellishment commonly sugar or carbohydrate moieties
• the presence of bulky side-chains of core or peripheral oligosaccaride moieties will prevent transpecies expression of IFN activity by such a direct or conformational mechanism (see, for example, W. Carter, in Life Sciences, vol. 25, pp. 717-728, 1979 and writings also in Pharmacology and Therapeutics, vol. 8, pp. 359-355, 1980).
• the interferon ancestral amino acid sequences which elicit the antiviral response include the sequences 25-40 and 115-141.
• the interferon domain determined to be essential for binding to specific cell receptors includes amino acids 115-141 (Gillespie and Carter, 1982). The position of this domain is determined in part or facilitated by the homology with a region of the B subunit of cholera toxin, (Lai, Journal Biol. Chem. Volume 252 pp. 7249-7256, 1977) a substance which competes with interferon for binding to cell receptors.
• Pieces of interferon can be prepared in several ways. By way of illustration, one suitable process will now be described. Other means, such as chemical modification of natural interferon or chemical modification of cloned (synthetic) entire interferon, interferon inducers, etc., are also readily apparent and are well within the scope of the invention.
• Interferon genes can be cut in specific places with specific restriction endonu ⁇ leases.
• the resulting interferon gene fragment, coding for a piece of the interferon polypeptide can be fused with an expression vector containing RNA polymerase promotor, ribosome binding site, initiation codon and whatever other signals may be necessary.
• This recombinant DNA can be introduced into suitable host cells for the purpose of synthesis of the piece of interferon.
• the following procedure for the expression in a host of a HuIFN gene fragment was carried out. 5 g of the gene for human interferon Beta when "tailed" with polyA. dT and cloned in pBR322 was incubated at 37° for 2 hours with 50 units of EndoR'P 1 in 20 mM tris, pH 7.4 10mM MgCl 2 , 50mM (NH 4 ) 2 SO 4 and 100 mg/ml of bovine serum albumin to cleave the interferon gene, leaving a single-stranded end from bases 203-206. The DNA was made 0.3M in potassium acetate and precipitated from ethanol.
• the DNA was dissolved in 100 ml of 50mM potassium phosphate, pH 7.4, 6.7mM MgCl 2 , 1mM mercaptoethanol, 35mM deoxyadenosine and deoxycytidine, and 25 units of the Klenow fragment end of Escherichia coli DNA polymerase.
• the DNA was made 0.3mM in potassium acetate and precipitated from ethanol.
• the precipitate was dissolved in 100 ml of 30mM sodium acetate, pH 4.6, 50mM NaCl, 1mM ZnSO 4 , 5% glycerol and 25 units of S1 nuclease and incubated at 37° for 1 hour. This produced an interferon gene fragment with a blunt end complete up to nucleotide 204, the first based in codeword for leucine at position 45.
• Sodium acetate was added to 0.3M and the DNA precipitated from ethanol.
• proteins in solutions containing interferon are bound in Cibachrom blue sepharose in the absence of ethylene or propylene glycol.
• the column is washed with a variety of solutions which remove most proteins, except interferon and the interferon is eluted with solutions containing ethylene or propylene glycol or other suitable eluting agents (Carter et al, Pharmacology and Therapeutics, Volume 8 pp. 359-377, 1980).
• further purification of Cibachrom blue fractions or other materials can be achieved by passing interferon-containing solutions over column matrices containing anti-interferon antibodies, using conventional conditions for binding and elution.
• the recombinant DNA described above can be prepared using some or all of the same enzymes under different conditions or in different sequences or using variants of the above stated enzymes or interferon genes.
• Other suitable recombinant DNAs can be formed using other enzymes and other protocols.
• the use of recombinant DNA to obtain pieces of interferon is merely illustrative of a means of generating a piece of interferon.
• Other means, such as solid state synthesis, proteolytic cleavage of intact material or genetically cloned interferon prepared in bacteria and yeast can be equivalently used.
• the process comprises: A. Cloning human and plant interferon genes into recombinant DNA;
• the tobacco plant Nicotiana has been used and is hereinbelow described as a prototype.
• Nicotiana protoplasts and isolating cells which synthesize elevated levels of plant and human interferon.
• analytic processes described herein for HuIFN one can readily determine comparable ancestral sequences in various animal or plant IFNs.
• the methodologies described herein for isolation of gene fragments and resultant insertion and expression will use methodology comparable, and in some instances identical, to that described for ancestral sequences in HuIIF and .
• Protoplasts may be formed and DNA may be introduced into them by 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 few recombinant DNAs insert themselves into plant chromosomes, hence they do not become a stable part of the plant genetic material.
• the Ti DNA plasmid of Agrobacter (Davey et al 1980. P1. Sci. Lett. 18:307-313; Wullems et al 1979. In Adv. Protoplast Res. Proc. Sth. Symp: 407-424) is an exception to this but this DNA plasmid has several unfavorable characteristics. Among the problems are that the Ti plasmid DNA is too large, DNAs inserted into said plasmid do not express the appropriate proteins and cells infected by said plasmid do not develop properly.
• the Ri DNA plasmid of Agrobacter allows plants to develop properly but retains the other undesirable properties.
• the following plasmid can be used: pBR 322 plasmid of E. coli can be modified to contain two inverted tandem copies of a Nicotiana short, interspersed repeated DNA. This plasmid will contain only one Eco R1 site: located at the junction between the two Nicotiana repeated sequences. The plasmid will be opened with Eco R1, then plant and/or human interferon genes can be inserted into the plasmid.
• pBR322 containing other plant sequences or other vectors such as Ti and Ri plasmids from Rhizobium can be used.
• plasmid containing a plant interferon gene can be introduced into Nicotiana protoplasts by microinjection, calcium phosphate precipitation, infection by Agrobacter, protoplast fusion, etc.
• Transformed protoplasts can be propagated as plant cells and can be detected by in situ molecular hybridization to chromosome DNA with a pBR 322 probe.
• Such cells induced with poly I:C can be tested for increased levels of plant interferon mRNA production by in situ hybridization using a pure plant interferon gene as a probe.
• Such cells producing elevated levels of plant mRNA can be tested for increased synthesis of plant interferon by the conventional biological test for antiviral growth factor.
• Nicotiana cells producing elevated levels of plant interferon can be reconverted to protoplasts and transformed as above by said vectors which now contain human interferon genes.
• Transformed protoplasts can be propagated as plant cells and tested for increased levels of plant interferon plus human interferon mRNA and protein by in situ molecular hybridization and the biological test for antiviral growth factor, respectively. These transformed protoplasts can then be cultured into mature tobacco plants.
• Protoplasts can be isolated from Nicotiana spp. according to Chupeau et al (C.R. Acad. Sci. Ser. D (Paris) 278:1565-1568, 1974). Sterilized leaves can be peeled and incubated in T o medium (10.3 mM NH 4 NO 3 , 9.4 and mM KNO 3 , 1.5 mM CaCl 2 .7H 2 O, 0.75 mM MgSO 4 ⁇ 7H 2 O, 0.62 mM KH 2 PO 4 , 0.1 mM FeSO 4 ⁇ 7H 2 O, 0.1 mM Na 2 EDTA, 16 mM H 3 BO 3 , 0.6 mM MnSO 4 ⁇ H 2 O, 3.5 mM ZnSO 4 ⁇ 7H 2 O, 0.2 mM CuSO 4 ⁇ 5H 2 O, 0.22 mM AICI 3 , 0.13 mM NiCl 2 ⁇ 6H 2 O, 8 uM Nicotinic acid
• the liberated protoplasts can be washed by low speed centrifugation in medium T o .
• Other methods of protoplast isolation can be substituted as long as viable single cells lacking substantial portions of their cell wall can be isolated.
• Nicotiana spp protoplasts can be incubated with transforming vector DNA carrying interferon genes or with bacteria containing such DNA using any of a number of standard transforming procedure (Davey et al 1980. P1. Sci. Lett. 18:307-313; Willems et al 1979. In Adv. Protoplast Res. Proc. 5th. symp.:407-424).
• Such vector DNA can be the E. coli plasmid containing plant repeated sequences as described above or any other vector DNA which can be propagated in a microorganism and can integrate into protoplast chromosomes.
• Transformed protoplasts can be cultured in T o medium at 25° in the dark for four days and then transferred under fluorescent lamps (2500 1x, 16 h per day). After 30-60% of the protoplasts have divided once, they can be collected by centri fugation and washed once in medium AG (10 mM KNO 3 , 3 mM CaCl 2 ⁇ 2H 2 O, 3 mM MgSO 4 ⁇ 7H 2 O, 1 mM KH 2 PO 4 , 0.1 mM FeSO 4 ⁇ 7H 2 O, 0.1 mM Na 2 EDTA, 49 mM H 3 BO 3 , 1.8 mM MnSO 4 ⁇ H 2 O, 10.4 mM ZnSO 4 ⁇ 7H 2 O, 0.04 mM CoCl 2 ⁇ 6H 2 O, 0.36 uM CuSO 4 ⁇ 5H 2 O, 0.41 uM NaMoO 4 ⁇ 2H 2 O, 0.66 uM
• AICI 3 0.40 mM NiCl 2 ⁇ 6H 2 O, 0.18 uM KI , vitamins as in T o medium, 0.53 UM naphthaleneacetic acid, 4.4 uM 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 in standard light conditions at 25° in tightly closed, transparent boxes under which conditions the transformed protoplasts form colonies. Individual colonies can then be picked, reconverted to suspensions of single protoplasts and seeded on replicate petri plates in AG medium.
• Replicate plates can be evaluated for vector or recombinant inteferon gene expression by assays 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 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 assays (Sela. 1982. Interferon Sci. Memo., Memo number Ia1134, January). Other assays for excess interferon production may be suitable.
• C Producing a mature tobacco plant from genetically engineered plant cells.
• Colonies exhibiting presence and expression of new interferon genes can be cultured 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 rooting medium B to form plantlets (Bourgin et al. 1979. Physiol. Plant. 45:288-392). Rooted plantlets can be potted and grown to maturity in the greenhouse. Other methods for converting single, transformed plant cells to mature plantlets may be substituted.
• Plantlets can then be tested for their production of interferon and for resistance to tobacco mosaic virus (Sela. 1981. Adv. Vir. Res. 26:201-234) or to other foreign agents using standard evaluation methods.
• High interferon-producing disease-resistant strains can be selected and propagated sexually by any convenient and effective means.
• the above protocol is given only by way of illustration of the feasibility of the process for consructing this genetically novel plant with desirable characteristics. Obviously, in this rapidly changing field, many modifications of this process can be expected to be successful without departing from the scope of the invention.
• the therapeutic agents of this invention can be topically applied by myriad techniques ranging from the mundane (e.g. by means of a watering can in a greenhouse) to the sophisticated (e.g. crop dusting hundreds or thousands of acres).
• the preferred mode of practicing the invention will depend upon biological as well as mechanical factors at the time plant treatment is desired; for example, permanent protection of a seed crop will be conducted preferably by the mode of germ plasm alteration via increased IFN copy number (plant and/or animal IFN genes). Alternatively, to protect plants already in the field occupying wide acreage, crop dusting would be a desired embodiment.
• special topological conditions (consider the molecular stability, windshear effect, etc., if ancestral IFN components are dropped from low flying aircraft) must also be considered as well as biochemical ones (consider efficiency of uptake by young versus mature or senescent leaf cell structures).
• the preferred embodiment will consist of lower molecular weight components as they generally provide greater thermal and physical stability and enhanced cellular uptake/receptor binding.
• the ancestral IFN-directed oligonucleotides and IFN fragment peptides are examples of relatively thermal-resistant and vortical-resistant active moieties which derive from the basic invention.
• At least one IFN or other substance containing an ancestral sequence (or inducer) will be dispersed in a suitable, agriculturally acceptable vehicle or carrier such as water, i.e. application will occur as a solution.
• Trie concentration may range between about 0.0001 and 100,000 IRU (International Reference Unit) per milliliter of solution. If it is desired to spread IFN as a dry powder for purposes of application by dusting, a weight concentration range of about 1 ppm (part per million) to about 1 part in 10 15 -10 16 parts is efficacious when the interferon is added to a dry, agriculturally acceptable carrier. Suitable carriers are well known to the crop dusting art.
• the interferon may be admixed or combined with other plant treatment agents - pesticides, herbicides, fertilizers, etc. provided that none of the additional agents affects the bioactivity of the ancestral sequence.
• the final concentration of IFN or other ancestral substance will depend on the crop being protected, the virus being protected against, and field conditions.
• the final concentration may also vary depending on the molecular fraction (i.e. fraction of the molecule) which is non-ancestral.
• the molecular fraction of non-ancestral material would approach zero.
• the molecular fraction of non-ancestral material is approximately 0.6, i.e. approximately 60% of a whole IFN (e.g. HuIFN) molecule is non-ancestral.
• a typical application of IFN to crops would be, for example, at a concentration of 1 part per billion (ppb; note, for HuIFN, that 1 mg is approximately 10 8 IRU) .
• ppb part per billion
• HuIFN that 1 mg is approximately 10 8 IRU
• For surface application (which requires in the neighborhood of 100 gallons per acre), less than half a milligram of whole HuIFN would be required per acre.
• Crop dusting requiring typically one-fifth to one-tenth as much volume as surface application, would require comparable amounts of interferon, or perhaps less depending on the efficacy of topical application.
• Interferon like other proteins, is a relatively labile chemical. It depends on a very specific three-dimensional orientation of its various linearly-arrayed amino acids for solubility in aqueous solutions and for biological activity (see FIGURE 1). Specifically, domains of the interferon molecule responsible for binding to cells and for eliciting complex biological responses must be properly exposed and aligned. However, a vast array of alternate non-functional orientations are also possible and can freely form. These alternate states are encouraged by heat, certain external chemicals and prolonged storage.
• One solution toward preventing the formation of inactive alternate states is to build molecules displaying IFN activity, but which have only a limited number of alternate states, such as the interferon fragments described supra.
• Another solution, developed by the inventor is to restrict the formation of alternate states by immobilizing the interferon on a carrier molecule or structure.
• Active enzymes are often found as part of complex structures, being attached to cell membranes, nucleic acids, proteins, etc., and the enzymes are usually more stable in the complexed state.
• the inventor constructed several column matrices containing various ligands which might be effective stabilizers (see FIGURE 2, Carter et al, 1980, Pharmac. Ther. 8:359-377). The column was characterized with albumin attached to it.
• the 90 percent portion of the eluent was used to measure the protein concentration.
• the breakthrough fractions contained about 98 percent of the applied protein and less than 1 percent of the applied interferon activity. Further elution of the column was done with 50 percent (v/v) ethylene glycol and 50 percent 0.04 M phosphate, (pH 7.4) and 0.30 M NaCl. The remainder (86 percent) of the interferon activity was recovered with very little (less than 2 percent) of the original protein.
• human serum albumin or other proteinaceous carrier can be added in excess, usually to 3 mg/ml.
• the solution can be dialyzed against phosphate buffered-saline or another appropriate buffer, then the material can be freeze-dried. In a typical example 1 million units of purified interferon was freeze-dried with 3.46 mg sodium phosphate.
• Carrier proteins other than albumin or cytochrome C are acceptable.
• Other carrier molecules or structures may also be acceptable, such as lipids, small hydrophobic ligands, membrane fragments, or even solid particles.
• Other means of attachment such as ionic or covalent bonds may also be suitable.
• Other salts or buffers or other concentrations of salt or buffer may also be acceptable.
• the procedure stabilizes IFN to external conformational changes, including denaturation from the effects, e.g. of windshear and environmental influences.
• Topical application of HuIFN to plants is generally effective in combatting the spread of virus from one plant to another.
• IFN exhibits a pronounced antiviral effect in plants, albeit the viral arrest appears to level, i.e. over the range tested viral arrest did not increase linearly with increasing interferon concentration.
• topical application does dramatically slow the virus, the effect drops off somewhat with time, indicating that repeated applications may be necessary at regular intervals e.g. every 10 to 15 days.
• the inventor has determined that periodic application of interferon to virus-infected plants wili prevent the spread of viruses in those plants and also prevent the transmission of viruses from those plants to uninfected plants.
• the inventor obtained serial photographs of the leaves durinq the HuIFN experiments cited above and noted that the leaves grew and thrived at the same time that the virus life cycle was arrested.
• topical application encompasses the application of substances, termed “inducers”, which, though not IFN molecules themselves, have the property of serving as a biological trigger to force an IFN-producing organism to produce its own IFN in an interval that is short relative to that which would be required in the absence of applied inducer.
• inducer substances, termed "inducers”, which, though not IFN molecules themselves, have the property of serving as a biological trigger to force an IFN-producing organism to produce its own IFN in an interval that is short relative to that which would be required in the absence of applied inducer.
• inducer a biological trigger to force an IFN-producing organism to produce its own IFN in an interval that is short relative to that which would be required in the absence of applied inducer.
• mismatched dsRNAs which are potent IFN inducers but which exhibit few toxic side effects in humans.
• the biological mode of operation of these substances along with a listing of them is fully set forth in my U.S. patent 4,103,641.
• dsRNAs are effective in members
• the plant does not necessarily have to be genetically engineered. All that is required is that any particular dsRNA induce the formation a molecule having a bioactive essential (amino acid) sequence which generates an interferon-like response. As mentioned previously, this requirement means that the plant must contain a polynucleotide sequence encoding for a bioactive amino acid sequence. Whether any particular plant species satisfies this criterion can be determined by means of simple experiments, i.e. topically applying one or more dsRNAs to plants of interest and testing as known in the art for antiviral growth factor. Alternatively, a test of IFN-induceability can be conducted using the in-situ hybridization technique which the inventor illustrated above with Nicotinia.
• intracellular mediators there exist a series of substances termed intracellular mediators and typified by 2',5'-oligoadenylic acid and/or a functionally active derivative thereof which are also useful as topical application agents.
• the exact biochemical mechanism by which these compounds function is not known, but it is known that they mimic the IFN effect brought about by exposure to a virus, in essence replacing the need for IFN.
• the inventor has determined that this mimicry can be extended to plants.
• Analogues may also be used, including core (dephosphorylated) 2' -5' oligoadenylate, core 3' -5' oligoadenylate, and core 2' -5' oligoadenylate-cordecypin (3'-deoxyadenosine).
• the range of concentration useful in applying dsRNAs 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 milliliter in aqueous solution.
• a preferred embodiment regarding topical application resides 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 which deters insects, particularly aphids, from landing on the plant and acting as the vector which spreads the virus. That is, insects such as aphids represent perhaps a major mode for viral transmission, feeding at many plants within relatively short temporal spans and exchanging infected matter for healthy plant tissue which the aphid then reinfects, exchanges with other healthy plants, and so forth in a self-perpetuating cycle.
• a substance capable of producing an IFN-like response with a substance which blocks (i.e.
• a two-pronged attack against virus spread may be effectively mounted. That is, the first prong of the attack is to prevent insects from selecting the treated plant. Should this prong fail, any virus-infected plant matter exchanged by the insect with a healthy plant is then counteracted by the HuIFN response-producing substance.
• pyrethrums are known natural substances derivative, for example, from chrysanthemums.
• Pheromones are also natural substances derivable from wild potatoes.
• Interferon admixed with, or appplied separately but contemporaneously with either or both of these natural hormonal substances forms a combination which either effectively blocks insects in the first instance, or prevents the spread of virus from any insects which do contact healthy plants in spite of the blocking substance.
• the blocking agent and antiviral agent thus reinforce each other on a biochemical level and in their ultimate biodegradation leave behind no noxious environmental residue or contaminants.
• Interferon can be administered in combination in the levels (e.g. 0.0001-100,000 IRU/ml of solution) previously mentioned.
• Pyrethrums and/or pheremones are administered at levels typically on the order of 0.005-10 pounds per acre.
• a suitable carrier e.g. water or dry agriculturally acceptable carrier

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• 1983
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