GB2177702A - Broad spectrum plant protection from pathogens - Google Patents

Description

1

SPECIFICATION

Broad spectrum plant protection from pathogens GB 2 177 702 A 1 Field of the invention

This invention relates to protecting members of the plant kingdom from disease and, in particular, to the use of whole interferon OFN) molecules from any phylogenetic-source, and/or ancestral components thereof, to prevent or arrest plant viral infections.

Background of the invention

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. Consider, for example, that viraHnduced plant pathology is capable of lethal disease of coconut palms in the tropics as well as ravaging wheat, barley and other grains in the 16 Canadian North. At this time, there is no known therapeutic approach or remedy save the primitive art of 15 "culling" whereby the farmer or nurseryman removes the infected plant material before contagious spread occurs to previously uninfected plants. When one considers the microscopic nature of the infect ing viral organisms and thus the self-evident inability to detect the infection by naked eye before it is too late to "cull out", it becomes immediately clear that a fundamentally different approach is needed.

What is needed is (1) an ability to alter plant germ plasm ins a specific manner such that the plants' 20 intrinsic resistance to viruses is strengthened, and (2) an ability to add exogenously to already infected plant cell material a safe substance, or substances, which is (are) broadly protective of the plant cell against an entire spectrum of different plant viruses, thereby preventing further damage to the crop and arresting the continual spread of the infectious agents. This invention fulfills these needs by providing methods whereby the beneficial physiological effects of IFN may be implemented in members of the 25 plant kingdom.

Studies relating to placement of exogenous human interferon (HuIFN) in plants have been con ducted, 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).

11 Summary of the invention

This invention derives from the inventor's determination that 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 35 of preventing the spread of various chronic or latent plant viruses to normal uninfected plants, i.e. pre venting infection in the first instance. This dual capability is referred to as "protecting" a plant against viruses in the claims.

The essential amino acid sequences referred to above are also referred to in the specification and in the claims as "ancestral" sequences. An ancestral sequence is one which has been conserved through 40 evolutionary history, i.e. a period of at least millions and, probably, even tens of hundres of millions of years without change. For purposes of this invention, an "ancestral sequence" or "ancestral fragment" is defined to be any amino acid sequence which is not per se norm-ally encountered free in nature, and which in and of itself possesses the capbility of causing a plant to- exhibit an anti-viral response. The inventor has determined that ancestral sequence are, at least in part, responsible for imparting IFN prop- 45 erties 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:

(a) 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 M the mechanical level, i.e. physical addition of IFN(s) or fragments thereof exogenously, i.e. topi cally to already infected plant tissue or plant tissues at imminent risk of viral infection.

Additionally, double-stranded RNAs (dsRNAs), of natural (any phylogenetic source) or synthetic origin can effect the same result (provided the target plant cells contain a programmable IFN genetic element) and indeed even augment the therapeutic outcome under a variety of plant cell-virus interactions.

One of the most important determinations leading to. this- invention is that less than whole IFN mole cules are sufficient to produce an anti-viral response. In other words, and as will subsequently be ex plained in detail, 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 molelcule. The inventor has further determined that the sequences are ancestral -- that, as mentioned, they likely have evolved through the ages and, furthermore, that they are common to mole cules 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 molecules containing an (ancestral) amino acid sequence as hereinafter detailed may be applied topically to produce an anti-viral response. Further, 65 2 GB 2 177 702 A 2 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. That is, some plants, without being genetically altered to produce whole IFN molecules, can nevertheless be induced to respond in an IFN-like manner, i.e. as though the plant had been genetically programmed to produce its own IFN, molecules, can nevertheless be induced to respond in an IFN-like manner, i.e. as though the plant had been genetically programmed to produce its own IFN, or as though it had been treated topically with whole IFN molecules. "IFINI-like" is understood to having this meaning throughout the specification and claims. Of course, if 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.

1() Further, the inventor has determined that plants themselves produce substances containing ancestral 10 sequences, i.e. that plants provide plant interferon (PIIFN), a determination which heretofore has not been made. Thus, genetically altering plant germ plasm to contain a functioning gene which expresses a plant interferon, even though the gene is of plant origin, is also within the scope of the invention.

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! 15 1. Genetically altering the plant germ plasm to contain one or more copies of IFN genes so that the plant actually produces its own IFN. The implanted genes may either be turned on de novo or turned on by the use of inducing substances. Genetic alteration is particularly useful in plants not having IFN genes at all or having only defective IFN genes.

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

3. Inducing a plant, which has not necessarily been genetically altered, to produce a molecule con taining an ancestral sequence. This mooe "turns on" or activates PIIFN genes which otherwise are latent or dormant.

External plant treatment agents useful in the invention as typically applicable sources of viral protec tion (i.e. useful in producing an IFN-like response) thus include various whole IFNs, ancestral (i.e. essen25 tial) molecular fragments thereof, IFN inducers (including, but not limited to, dsRNAs), as well as the specific oligonucleoticle ("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 bioactivo fragments.

The topical cipplication or treatment may be carried out by a great variety of approaches due to the 30 biological potency of the agents. Such treatment approaches include, but are not limited to, dipping of plants or seeds, spraying, or crop dusting.

"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. hu man) interferon genes or pieces thereof (resulting in hybrid genes) have been added, and (c) chemical 35 derivatives of such interferons with, for example, modified polypepticle chains or modified glycosyl units.

The invention possesses, of course, significant and wide utility, the most immediate and econimically important of which is the protection of staple crops produced on national and even global scales. Fur ther, 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 occur- 40 rence happening, for example, when the farmer attempts to alter a natural geographic growth zone for plant cell material. Consider that many tropical plants cannot be grown in non-tropical areas since the attendant "stress" apparently increases susceptibility to pathogens including viruses; for example, pa paya plants, normally relatively virus-free in the tropics, almost uniformly acquire lethal viral infections if their growth is attempted in California. Hence, the invention not only is of immediate utility but also will 45 derive additional agricultrual advantages - not apparent even to the trained observer in agronomic bio technology -over time as it is progressively practiced.

Further, 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 50 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.

Brief description of the Figure

Figures la and 1b illustrate comparative 3-dimensional structure of human Iffi beta one (HuIFN 1) and human 1M gamma (HuIFN-); Detailed description

1. Detection and therapeutic exploitation of the essential ancestral IFN bioactive domains & resultant 60 ancestral IFN fragments.

Determination of IFN structural homology (i.e. that most, if not all, IFNs possess common structural elements in the form of amino acid sequences) over vast evolutionary time can be proven as illustrated by the novel construction of 3-climensional models as shown in Figure 1. The models establish close tertiary (3-dimensional) similarities which utimately reveal the commonality of domains (regions) com- 65 3 GB 2 177 702 A 3 prising the bioactive site, i.e. that portion of the WN 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:

TABLE 1

Structural features of IFIV 1 and M] [P 1 [P 1 1() Reverse Turns 10 ^13-6 1.29 0.88 0.99 P9-12 1.35 0.77 0.75 y27-30 1.38 1.01 0.86 P32-35 1.14 0.92 1.00 y43.46 1.21 0.91 0.79 15 y65-68 1.33 0.98 0.84 071-74 1.44 0.84 0.74 y86-89 1.39 0.83 0.69 P1 10-113 1.12 0.80 0.91 y132-135 1.08 0.86 0.81 20 P136-139 1.18 0.85 1.01 Alpha Helix y8-16 1.13 1.01 P16-22 1.27 1.01 25 ^131-42 1.23 0.93 P40-55 1.29 0.92 y51-65 1.03 1.10 P59-68 1.17 1.18 y95-100 1.10 0.89 30 P91-107 1.22 0.87 P1 16-121 1.09 0.89 Beta Sheet -V103-121 1.14 1,15 35 P123-135 0.89 1.15 P141-161 0.96 1.16 y47-60 1.04 1.25 P59-68 1.17 1.18 40 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. For example, 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 eixstence 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. Pt refers to the probability of 45 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.

The overall and far reaching deduction of IFN structural homology has never been previously declared or published since it involves the creative coalescence of certain previously known and isolated facts plus 50 additional insight derivative from the inventor's novel approach. This approach using comparative nu cleoticle sequence data, comparative protein sequence data, mutational frequency data, conformational data, and Markov chain analysis (all for different forms of IFN), led to the determination of the ancestral or primordial progenitor sequences of IFN which are capable of providing antiviral activity in all present day life forms, including disperse species of plant life. From within these progenitor sequences, further 55 determination has been made of the component amino acid constituents. To reemphasize, these progeni tor (ancestral) sequences, which are found within larger molecules, are not encountered isolated in na ture 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 sequence is preserved even when certain amino acids are mutated in arriving at the ancestral or progenitor IFN molecule which 60 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, in cluding man.

The desired molecular sites (i.e. the bioactive amino acid sequences) of lFNs can be readily isolated by a series of available techniques and sequenced in either the genomic form (J. Bresser and D. Gillespie in 65 4 GB 2 177 702 A 4 Analvtical Biochemistry, vol. 129, p. 357f 1983), or protein form wherein the genomic produce is expressed on nitrocellulose paper (Bresser et al. in Proceedings National Academy Science, U.S., in press 1983; Bresser et al., DNA, in press, 1983). Thereupon, many available variations in solid- phase synthesis of peptides can be undertaken to generate the active fragments of IFN with the therapeutic value de- scribed herein. Thus, 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 beneficially affecting plant cell function and stopping viral pathology is well within the scope of the invention.

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 1() 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 impor- tant about the active fragments is their 3-climensional activity, not their relative location from the amino 15 end of the intact molecule.

In addition to desirable physico-chernical properties, 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. Commonly, the presence of bulky side-chains of core or peripheral oligosaccaricle 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 writing also in Pharmacology and Therapeutics, vol. 8, pp. 359-355, 1980).

11. Active interferon domains Genes which code for interferon have been cloned (e.g., 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 none of the various interferons determined. Using computer programs, the inventor has determined domains necessary for certain bio35 logical properties (Gillespie and Carter, Handbook of Experimental Pharmacology of Interferon, in press.) of the interferon system.

The interferon ancestral amino acid sequences which elicit the antiviral response include the 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 sequencws in each of human IFN and. 40 Similar analyses as above are applicable to other animal IFNs.

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.

It is to be emphasized that other ancestral fragments different from those identified above exist in na ture, and the invention is not to be construed as being limited to only the ancestral fragments disclosed herein. The inventive concept resides in treating plants with molecules containing ancestral fragments or with the fragments themselves. Identification of additional fragments is well within the scope of one skilled in the art.

111. Preparation of pieces of interferon with select domains 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 modifi cation of cloned (synthetic) entire interferon, interferon inducers, etc., are also readily apparent and are 55 well within the scope of the invention.

Cloned interferon genes can be cut in specific places with specific restriction enclonucleases. The re sulting interferon gene fragment, coding for a piece of the interferon polypepticle can be fused with an expression vector containing RNA polymerase promotor, ribosome binding site, initiation cocion 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.

As an example, 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 370 for 2 hours with 50 units of Endor-P 1 in 20 mM tris, pH 7.4 10mM MgCl,, 50mM (NH,),SO, and 100 mg/ml of bovine serum albumin to cleave the interferon gene, leaving a single- c GB 2 177 702 A 5 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 5OmM potassium phosphate, pH 7.4, 6.7mM MgCl,, 1 mM mercaptoethanol, 35mM deoxyadenosine and deoxycytidine, and 25 units of the Kienow fragment end of Escherichia coli DNA polymerase. The DNA was made 0.3mM in potassium acetate and precipitated from 5 ethanol. The precipitate was dissolved in 100 ml of 30mM sodium acetate, pH 4.6, 5OmM NaCl, 1 mM ZnSO,, 5% glycerol and 25 units of SI 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.

Separately, 5 g of the gene for human interferon Beta ("tailed" with polydA-dT and cloned in pBR322 was incubated at 37' for 2 hours with 50 units of Endo R Mboll in 10mM tris, pH 7.9, 6mM KCL, 10mM 10 MgC12r 1 mM dithiothreitol and 100 mg/ml of bovine serum albumin. The DNA was precipitated from ethanol and treated with S1 nuclease as described above. The 288 base pair, blunt end fragment contain ing nucleotides 401-688 of the interferon gene was purified by electrophoresis through 3% agarose. An aliquot of 0.25mg of this fragment was incorporated with 5 g of Pst 1 treated, DNA polymerase - treated and S1 nuclease - treated DNA.

The recombinant DNA so formed contained an interferon gene lacking nucleotides 205-400 which will code for an interferon polypeptide lacking amino acids 46-111.

This recombinant interferon fragment can be inserted into many suitable vectors for expression in bac terial or mammalian cells using conventional techniques. Interferon or pieces of interferon can be puri fied by standard methodology although the invention can be practiced with interferons less than 1% pure, provided no otherwise harmful substances are present. For example, proteins in solutions contain ing 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). If desired, further pu- 25 rification 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 30 genes. Other suitable recombinant DNAs can be formed using other enzymes and other protocols. Fi nally, the use of recombinant DNA to obtain pieces of interferon is merely illustrative of a means of gen erating 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.

is IV. Alteration of plant germ plasm to yield a plant having heritable resistance to viral infections The inventor has determined that plants producing elevated levels of both plant and human interferon will have resistance to a broad spectrum of pathogens. Accordingly, there is provided a process for creat ing such a plant. The new plant created by the following process is genetically novel.

The process comprises:

A. Cloning human and plant interferon genes into recombinant DNA; B. Inserting said recombinant DNA into protoplasts and isolating cells which synthesize elevated lev els of plant and human interferon; and C. Producing a mature plant from the protoplasts.

As an example, the tobacco plant Nicotiana has been used and is hereinbelow described as a proto- 45 type.

A. Cloning human and plant interferon. As previously mentioned, human interferon genes have been cloned (e.g. Derynck et al, supra). Several conventional procedures can be used to similarly clone plant interferon genes; what is needed is a suitable probe for finding the recombinant DNA. One probe which can be used for this purpose is the highly conserved portion of the human interferon or gene contained 50 within but not limited to nucleotides 345-423 (Gillespie and Carter 1982, Handbook of Experimental Phar macology of Interferon, in press). Other suitable probes may exist as well. The important point is that the invention does not derive from the choice of methods used to clone the plant interferon genes, but rather derives from the genetic uniqueness and increased desirability of the final plant.

B. Instering the recombinant DNA into Nicotiana protoplasts and isolating cells which synthesize ele- vated levels of plant and human interferon. By the 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 method ology comparable, and in some instances identical, to that described for ancestral sequences in HulIF and 60 Protoplasts may be formed and DNA may be introduced into them by conventional techniques (Bourgin et a[ 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 a[ 1980. Pl. Sci. Lett. 18:307-313; Wullems et al 1979. In Adv. Protoplast Res. Proc. Sth. 65 6 GB 2 177 702 A 6 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.

To insert foreign DNA into Nicotiana chromosomes the following plasmid can be used: pBR 322 plas- 5 mid of E. co/i can be modifiecl to contain two inverted tandem copies of a Nicotiana short, interspersed repeated DNA.This plasmid will contain oniyone Eco R1 site: located atthejunction between thetwu Nicotiana repeated sequences, The plasmid will be opened with Eco RI, then plant and/or human inte, feron genes can be inserted into the plasmid. Alternatively pBR322 containing other plant sequences or other vectors such as Ti and Ri plasmids from Rhizobium can be used.

Many copies of the plasmid containing a plant interferon gene can be introduced into Nicotiana proto plasts 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 hy bridization to chomosone DNA with a pBR 322 probe. Such cells induced with poly I:C can be tested for increased levels of plant interferon mRNA production y in situ hybridization using a pure plant inter- 15 feron gene as a probe. Such cells producing elevated levels of plant mRNA can be tested for increased synthesis of plantinterferon 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 proto plasts can be propagated as plant cells and tested for increased levels of plant interferon plus human 20 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.

More specifically, the process can be accomplished as follows:

Protoplasts can be isolated from Nicotiana spp. according to Chupeau et al (C.R. Acad. Sci. Ser. D 25 (Paris) 278:1565-1568, 1974). Sterilized leaves can be peeled and incubated in T. medium (10.3 mM NH,NO,, 9.4 and mM KNO, 1.5 mM CaCl,.7H2010.75 mM MgSO,,-7H,O, 0.62 mM KH2PO4, 0.1 MM FeSO,-7H,O, 0.1 mM Na,EDTA, 16 mM H,,BO,, 0.6 mM MnSO,-H20,3.5 mM ZnSO,,- 7H2010.2 mM CUS04-51-120,0.22 mM AICI, 0.13 mM N'Cl2,6H2018 uM Nicotinic acid, 2 uM Ca panthothenate, 0.04 mM Biotin, 16.1 uM naphthalenacetic acid, 4.4 uM 6-benzy[adenine, 58 mM sucrose, 440 mM mannitol) lack- 30 ing sucrose and containing 0.02% Macerozyme R10, 0.1% cellulase Onozuka R10 and 0.05% Driselase.

The liberated protoplasts can be washed by low speed centriguation in medium T.. 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 35 with bacteria containing such DNA using any of a number of standard transforming procedure (Davey et a[ 1980. Pl. 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. cofl plasmid containing plant repeated sequences as desribed 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. medium at 25' in the dark for four days and then trans ferred under fluorescent lamps (2500 Ix, 16 h per day). After 30-60% of the protoplasts have divided once, they can be collected by centrifugation and washed once in medium AG (10 mM KNO., 3 mM CaC'2-2H,O, 3 mM MgS04-7HO, 1 mM KH2P041 0-1 mM FeSO,-7H,, 0.1 mM Na2EDTA 49 mM H2BO,, 1.8 mM MnS04-H20,10.4 mM ZnS04-7H,O, 0.04 mM CoCl2-6H,O, 0.36 uM CuSO,-5H20,0.41 uM NaMo04.2H,O, 45 0.66 uM AICI,,, 0.40 mM NiCl2.6H,O, 0.18 uM KI, vitamins as in L medium, 0.53 uM naphthaleneacetic acid, 4.4 uM 6-benzyl adenine, 58 mM sucrose, 440 mM mannitol, 1 mM glutamine. Cells can be plated on petri dished in medium AG (Caboche, Planta 149:7-18, 1980). The dishes can then be incubated in stand ard 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 inter feron 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. 55 1978. Proc. Nat. Acad. Sci., USA 75:6125-6129 and/or 3) the presence of excess interferon protein by im munological or biological assays (Sela. 1982. Interferon Sci. Memo., Memo number la1134, January).

Other assays for excess interferon production may be suitable.

C. Producing a mature tobacco plant from genetically engineered plant cells. Colonies exhibiting pres ence and expression of new interferon genes can be cultured in AG medium for 1-2 months, then plated 60 on solid R4 medium for bud regeration (Bourgin et al. 1979. Physiol. Plant. 45:288-292). The buds pro duced can be transferred to rooting medium 8 to form plantlets (Bourgin et al. 1979. Physiol. Plant.

45:288-392). Rooted plantlets can be plotted 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 vi- 65 c 7 GB 2 177 702 A 7 rus (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 con venient and effective means.

The above protocol is given only by way of illustration of the feasibility of the process for constructing this genetically novel plant with desirable characteristics. Obviously, in this rapidly changing field, many 5 modifications of this process can be expected to be successful without departing from the scope of the invention.

V. Topical application As mentioned, the therapeutic agents of this invention can be topically applied by myriad techniques 10 ranging from the mundane (e.g. by means of a watering can in a greenhouse) to the sophisticated (e.g.

crop dusting hundreds of thousands of acres).

The preferred mode of practicing the invention will depend upon biological as well as mechanical fac tors 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 15 animal IFN genes). Alternatively, to protect plants already in the field occupying wide acreage, crop dust ing would be a desired embodiment. Here, for example, special topological conditions, (consider the mo lecular 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 ma ture or senescent leaf cell structures). In such instances, the preferred embodiment will consist of lower 20 molecular weight components as they generally provide greater thermal and physical stability and en hanced 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.

For topical application, at least one IFN or other substance containing an ancestral sequence (or indu cer) will be dispersed in a suitable, agriculturally acceptable vehicle or carrier such as water, i.e. applica tion will occur as a solution. The 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 1015-10"i parts is efficacious when the interferon is added to a dry, agriculturally accepta ble 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 nonancestral. For example in the syn thetic IFN fragment which consists substantially only of amino acid sequence 115-141 the molecular frac tion of non-ancestral material would approach zero. In whole IFN molecules, by contrast, 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 108 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.

In some circumstances, however, it may be desirable to add additional stability to IFN or its ancestral fragments so that it can better withstand the rigors of application and possible extended exposure to environmental elements. This will now be described.

Interferon, like other proteins, is a relatively labile chemical. It depends on a very specific three-dimen sional 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 display ing IFN activity, but which have only a limited number of alternate states, such as the interferon frag ments 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, nu cleic acids, proteins, etc., and the enzymes are usually more stable in the complexed state. To evaluate the feasibility of such an approach the inventor constructed. several column matrices containing various ligands which might be effective stabilizers (see Figure 2, Carter et a[, 1980, Pharmac. Ther. 8:359-377).

The column was characterized with albumin attached to it. 100 ml of an undialyzed interferon prepara 65tion, containing 11,500 units and 0.99 mg protein per ml was applied to a column by means of a peristal- 8 GB 2 177 702 A 8 tic 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) containing 0.15 M NaCl. The eluent from the column was divided by a stream-splitting device in a ratio of 1:9. The 10 percent portion of the eluent was collected into 1 ml of a 1 percent solution of bovine serum albumin, containing 0.02 M sodium phosphate and,0.15M NaCl, and used to assay interferon activity. The 90 percent portion of the eluent was used to measure the protein 5 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 7A 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 pro tein. Some of these experiments have been recently published (Carter, W.A. , Methods in Enzymology 10 78:576-582, 1981).

The above experiment showed that interferon could be coupled with serum albumin while most other cellular proteins could not be. Experiments with other immobilized proteins such as cytochrome C showed that interferon has a general property of combining with proteins. The inventor had hypothe sized that the strong hydrophobic nature of interferon forces interactions with ordinarily sequestered hy- 15 drophobic pockets of other proteins, and this experiment suggested the accuracy of the hypothesis.

Whatever the mechanism, the above experiment encouraged the development of complexes with pro teins to stabilize interferon.

The procedure developed by the inventor for this purpose is as follows: Human interferon can be pre pared by any conventional means from any biological source -- from human cells, from recombinant micro-organisms, etc. After purification, 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.

Various alterations of this procedure are acceptable in this rapidly changing field. Carrier proteins other 25 than albumin or cytochrome C are acceptable. Other carrier molecules or structures may also be accepta ble, 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 (including no salt or buffer) may also be acceptable. Clearly, how ever, the procedure stabilizes IFN to external conformational changes, including denaturation from the 30 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. 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 cultured for 7-10 days at 250 in nutrient medium lacking or containing human interferon alpha. After this incubation, leaves were ground 35 in 1% Brij 35 and particulate material was removed by centrifugation. Viral proteins were quantiitated by an immunoassay (ELISA), using optical density as a measure of virus. High optical density values corre spond to increased amounds of virus. The results are shown in Table 2 following:

Table 2 - Arrest of potato virus spread by HuIFN- days of units/mi HulFW virus incubation 0 100 1000 Potato virus N 7 0.519 0.342 0.184 Potato virus N 10 1.331 0.702 0.838 45 leaf roll virus 10 0.673 0.298 0.254 As the data show, 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 concentra tion. Moreover, although topical application does dramatically slow the virus, the effect drops off some- so what with time, indicating that repeated applications may be necessary at regular intervals e.g. every 10 to 15 days. Importantly, the inventor has determined that periodic application of interferon to virus-in fected plants will prevent the spread of viruses in those plants and also prevent the transmission of vi ruses from those plants to uninfected plants. The inventor obtained serial photographs of the leaves during the Hu]FN experiments cited above and noted that the leaves grew and thrived at the same time 55 that the virus life cycle was arrested.

Given the increased yields of crops due to topical application coupled with the fact that very low pu rities of IFN are sufficient (i.e. expensive purification procedures are not required), repeated applications are very economically feasible. For example, the inventor, based on his experiments, estimates that a size greenhouse of about 100 sq. ft. can be protected for about ten dollars per month at current prices. 60 The Iffi of whatever purity may be administered provided it meets the criterion of having no contaminat ing living organisms (bacteria, etc.) or intact viral agents which might induce plant cytopathology or in duce pathology in ultimate consumers, be they animal or man. Following appropriate decontamination procedures such as filter retention of living organisms, such IFN lots would be useful for this invention, even though they might be rejected for human clinical applications.

A z 9 A GB 2 177 702 A 9 Further, the term "topical application" encompasses the application of substances, termined "inducers", which, though not IFN molecules themselves, have the property of serving as a biological trigger to force an IFIN-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. Chief among these are the so-called "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 numbers of the plant kingdom also, and may be applied in the same fashion as cited above for IFN molecules or bioactive fragments thereof. The plant does not necessarily have to be genetically engineered. All that is required is that any particular dsRNA induce the formation a molecule 10 having a bioactive essential (amino acid) sequence which generates an interferon-like response. As men tioned previously, this requirement means that the plant must contain a polynucleotide sequence encod ing 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-induceabil- 15 ity can be conducted using the in-situ hybridization technique which the inventor illustrated above with Nicotinia.

Additionally, there exist a series of substances termed intracellular mediators and typified by 2',5'-oli goadenylic 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 20 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 (such as Ampligen, a trademark of HEM Re search, Rockville, Maryland, or Poly I-Poly Q is between about 0.01 g/ml and 1000 g/mI in aqueous solu tion. 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 physi cal mixture although separate applications will also suffice, of an IFN, or an essential fragment or inducer 30 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 in fected matter for healthy plant tissue which the aphid then reinfects, exchanges with other healthy plants, and so forth in a self-perpetuating cycle. By combining a substance capable of producing an IFN- 35 like response with a substance with blocks (i.e. deters) insects, a two- pronged attach 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.

In particular, substances which are particularly effective for blocking insect (and particularly aphid) vec- 40 tors, and which are chemically compatible with IFNs, are hormone substances termed pyrethrums and pheromones. Pyrethrums are known natural substances derivative, for example, from chrysanthemums.

Pheromones are also natural substances derivable from wild potatoes.

Interferon admixed with, or applied separately but contemporaneously with either or both of these nat ural hormonal substances forms a combination with either effectively blocks insects in the first instance, 45 or prevents the spread of virus from any insects which do contact health 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. 0.0001100,000 IRU/ml of solution) pre viously mentioned. Pyrethrums and/or pheremones are administered at levels typically on the order of 50 0.005-10 pounds per acre. Thus one procedure is simply to combine the desired levels of IFN (or frag ment, inducer, etc.) with the pyrethrum or pehremone and a suitable carrier (e.g. water or dry agricultur ally acceptable carrier) and apply them to a solution.

Although only a few exemplary embodiments of this invention have been described in detail above, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. Ac cordingly, all such modifications are intended to be included within the scope of this invention as defined in the following claims.

GB 2 177 702 A

Claims (21)

1. A method of protecting plants against pathogens comprising causing said plants to exhibit an IFNlike response.

2. The method of Claim 1 wherein said pathogen is a virus.

3. The method of Claim 2 wherein said response is generated by topically applying to said plants a substance comprising at least one compound containing an ancestral amino acid sequence.

4. The method of Claim 3 wherein said at least one compound is stabilized against denaturation.

5. The method of Claim 3 wherein said substance includes, as said compound, entire WN molecules.

6. The method of Claim 3 wherein said substance includes, as said compound, a portion of an HuIFN 10 molecule.

7. The method of Claim 3 wherein said substance is applied in solution and in a concentration range equivalent to 0.0001 to 100,000 IRL1/m].

8. The method of Claim 7 wherein said solution is aqueous.

9. ' The method of Claim 2 wherein said response is generated by topically applying to said plants a is substance comprising at least one non-IFN compound capable of inducing said response.

10. The method of Claim 9 wherein said compound induces the production of a second compound containing an ancestral amino acid sequence.

11. The method of Claim 10 wherein said inducing compound is a dsRNA.

12. The method of Claim 10 wherein said second compound is whole HuIFN.

13. The method of Claim 10 wherein said second compound is a HuWN fragment.

14. The method of Claims 9 wherein said compound is an intracellular mediator.

15. The method of Claim 14 wherein said mediator is 2'-5'-oligadenylic acid or a derivative thereof.

16. A method of protecting plants against viruses, comprising topically applying, in combination, a first compound which causes said plant to exhibit an WN-like response and a second compound which 25 blocks virus vectors.

17. The method of Claim 16 wherein said first compound is selected from the groups consisting of dsRNAs and intracellular mediators.

18. The method of Claim 16 wherei said second compound is selected from the group consisting of pyrethums and pheremones.

19. A composition of matter comprising a first compound, selected from the group consisting of ds RNAs and intracellular mediators, admixed with a second compound selected from the group consisting of pyrethums and pheremones.

20. A plant protected by the method of Claim 1,

21. A plant protected by the method of Claim 16.

Printed for Her Majesty's Stationery Office by Croydon Printing Company (UK) Ltd, 12/86, D8817356.

Published by The Patent Office, 25 Southampton Buildings, London WC2A 1AY, from which copies may be obtained.

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