EP0680258A1

EP0680258A1 - Aprotinin and synergistic combinations thereof with lectins as larvicides against insect pests of agronomic crops, harvested material thereof, and products obtained from the harvested material

Info

Publication number: EP0680258A1
Application number: EP94909462A
Authority: EP
Inventors: Thomas H. Czapla
Original assignee: Pioneer Hi Bred International Inc
Current assignee: Pioneer Hi Bred International Inc
Priority date: 1993-01-25
Filing date: 1994-01-14
Publication date: 1995-11-08
Also published as: AU6230194A; WO1994016565A1; CA2154576A1; BR9405668A; MX9400634A

Abstract

Aprotinin has been found to be larvicidal against a number of common insect pests of agricultural crops and stored grains, particularly in combination with an insecticidal or larvicidal lectin. In a preferred embodiment, plant resistance to these insects is produced by inserting into the cells of a plant a gene whose expression causes production of one or more of these proteinase inhibitors in combination with such a lectin, in larvicidal amounts.

Description

Aprotinin and Synergistic Combinations Thereof with Lectinε as Larvicides Against Insect

Pests of Agronomic Crops, Harvested Material Thereof, and

Products Obtained from the Harvested Material

Technical Field

This invention relates to materials and methods for killing insect larvae which are harmful to plants, and materials and methods for imparting insect resistance to plants, material harvested from the plants, and products derived from the harvested material.

Background of the Invention

Numerous insects are serious pests of common agricul¬ tural crops. One method of controlling insects has been to apply insecticidal organic or semiorganic chemicals to crops. This method has numerous, art-recognized problems. A more recent method of control of insect pests has been the use of biological control organisms which are typically natural predators of the troublesome insects. These include other insects, fungi (milky-spore) and bacteria (Bacillus thuringiensis cv., commonly referred to as "Bt"). However, it is difficult to apply biological control organisms to large areas, and even more difficult to have those living organisms remain and survive in the treated area for an extended period. Still more recently, techniques in recombinant DNA have provided the opportunity to insert into plant cells cloned genes which express insecticidal toxins derived from biological control organisms such as Bt. This technology has given rise to additional concerns about eventual insect resistance to well-known, naturally occurring insect toxins, particularly in the face of heavy selection pressure, which may occur in some areas. Thus, a continuing need exists to identify naturally occurring insecticidal toxins which can be formed by plant cells directly by translation of a single structural gene.

More recently, certain specific plant lectinε have come to be recognized as larvicidal and insecticidal agents of some merit, particularly since they are naturally produced by plant gene expression systems. However, some lectins are active at relatively high levels which limit flexibility in that they require maximal expression systems for most effective larval/insect control. Thus, a continuing need also exists for substances or combinations of substances which are effective in controlling larvae at lower concentrations. It would be particularly desirable to identify compounds or compositions which could be used to potentiate the larvicidal action of existing agents such as lectins. These and other objectives of this invention will be evident from the following disclosure.

Disclosure of the Invention

It has now been determined that the serine-specific proteinase inhibitor aprotinin has potent larvicidal activity when administered enterally to the larvae of insects such as European corn borer and corn rootworm. Thus, this invention provides a method for killing susceptible insect larvae, including larvae of European corn borer and corn rootworm comprising administering enterally to the larvae a larvicidal amount of aprotinin or a serine proteinase inhibitor which is at least 90% homologous to aprotinin by amino acid sequence. The terms "protease inhibitor" and "proteinase inhibitor" are considered equivalent. The terms "insect" and "larva", although not equivalent when used specifically, should be understood to include both adult and larval forms of a species when used generically. Thus, the term "insect resistance" should be understood to include resistance to larval forms as well as adults, and "larvicidal" materials should be considered insecticidal, particularly since killing larvae produces a corresponding absence of adults. The proteinase inhibitor can be effectively applied to plants, harvested materials or products consumed by the insects by spray, dust or other formulation common to the insecticidal arts. By "harvested plant material" herein is meant any material harvested from an agricultural or horticultural crop, including without limitation grain, fruit, leaves, fibers, seeds, or other plant parts. Products derived or obtained from such harvested material include flour, meal, and flakes derived from grain; and products in which such materials are admixed, such as, for example, cake, cookie, muffin, pancake and biscuit mixes. Alternatively, the larvicidal proteinase inhibitor can be incorporated into the tissues of a susceptible plant so that in the course of infesting and consuming the plant, its harvested material, or a product derived from harvested plant material, the larvae consume larvicidal amounts of the proteinase inhibitor. One method of doing this is to incorporate the proteinase inhibitor in a non-phytotoxic vehicle which is adapted for systemic administration to the susceptible plants. This method is commonly employed with insecticidal materials which are designed to attack chewing insects and is well within the purview of one of ordinary skill in the art of insecticide and larvicide formulation, but is a method which may not be as suitable for active enzyme blockers such as proteinase inhibitors. Alternatively, a dietary bait containing one or more of the selected proteinase inhibitors can be employed, with, optionally, an added pheromonal or other larval attractant material. However, the genes which code for these peptides can be isolated and cloned. Alternatively, they can be synthesized directly using a DNA sequence obtained by working backwards from the known amino acid sequence for aprotinin or a related proteinase inhibitor, preferably using plant-preferred codons. The resulting sequence can be inserted into an appropriate expression cassette and introduced into cells of a susceptible plant species, so that an especially preferred embodiment of this method involves inserting into the genome of the plant a DNA sequence coding for one or more insecticidal proteinase inhibitors selected from aprotinin and serine proteinase inhibitors having at least 90% homology to aprotinin by amino acid sequence, in proper reading frame relative to transcription initiator and promoter sequences active in the plant. Transcription and translation of the DNA sequence under control of the plant-active regulatory sequences causes expression of the larvicidal gene product at levels which provide an insecticidal amount of the proteinase inhibitor in the tissues of the plant which are normally infested by the larvae.

This method offers particular advantages when the potential for insects becoming resistant to these materials is considered. Insecticide-resistant insects become a problem as a result of application of strong selection pressure which highly favors naturally resistant individuals and any resistant mutants which occur. As a result, over the course of a few generations the resistant insects become the predominant type. Heavy application of insecticidal materials generally to a field or a geographical area by dust or spray or by soil incorporation tends to impose strong selection pressures of the kind described, since insects have no "safe havens" where non-resistant individuals can survive. However, many insect pests of crop plants also attack non- crop species. Limiting the insecticidal materials to the crop plants in the region by expressing the insecticidal materials only in those plants permits continued survival of non-resistant insects in associated weed plants which provide not only "safe havens" from the toxic compound but food for the insects. This reduces selection pressure significantly and thus slows development and spread of resistant insects.

This method also offers advantages from the standpoint of soil and groundwater contamination, since no application vehicle is required. The insecticidal components themselves are of natural origin and break down naturally when the plant is digested or decomposes. The method offers further advantages from the standpoint of cost, since no application expense is involved and the cost of the insecticidal materials is factored into the price of the seed or other reproductive material which the grower purchases. The plant should be a plant which is susceptible to infestation and damage, or whose harvested material or products are susceptible to infestation and damage by the larvae of European corn borer and corn rootworm. These include corn (Zea mays) , wheat (Triticum aestivum) and sorghum (Sorghum bicolor) ♦ However, this short list is not to be construed as limiting, inasmuch as these species are among the most difficult commercial crops to reliably transform and regenerate, and these insects (under other common names) also infest other crops. Thus the methods of this invention are readily applicable via conventional techniques to numerous plant species, if they are found to be susceptible to the plant pests listed hereinabove, including, without limitation, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manicot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Lolium, Triticum, and Datura.

Preferred plants that are to be transformed according to the methods of this invention are cereal crops, including maize, rye, barley, wheat, sorghum, oats, millet, rice, triticale, sunflower, alfalfa, rapeseed and soybean, fiber crops, such as cotton, fruit crops, such as melons, and vegetable crops, including onion, pepper, tomato, cucumber, squash, carrot, crucifer (cabbage, broccoli, cauliflower), eggplant, spinach, potato and lettuce.

The DNA sequence which when expressed imparts insecti¬ cidal activity is a structural gene which codes for aprotinin, or a proteinase inhibitor having at least 90% homology to aprotinin. It has been found that these proteinase inhibitors have sufficient insecticidal (larvicidal) activity to be operative in a plant cell expression system. That is, while certain other proteinase inhibitors such as cowpea trypsin inhibitors have some larvicidal activity at high concentrations in pure form, plant cell expression at such high concentrations is either not possible in a living plant cell system, or is not feasible if the commercially useful characteristics of the plant are to be preserved in terms of production of oils, starches, fibers, or other materials. A tissue-specific promoter can be used in any instance where it may be desirable to localize production of the proteinase inhibitor to an infested tissue or to a tissue which is efficient in production of the proteinase inhibitor.

In carrying out this invention, it will be appreciated that numerous plant expression cassettes and vectors are well known in the art. By the term "expression cassette" is meant a complete set of control sequences including initiation, promoter and termination sequences which function in a plant cell when they flank a structural gene in the proper reading frame. Expression cassettes frequently and preferably contain an assortment of restric¬ tion sites suitable for cleavage and insertion of any desired structural gene. It is important that the cloned gene have a start codon in the correct reading frame for the structural sequence. In addition, the plant expression cassette preferably includes a strong constitutive promoter sequence at one end to cause the gene to be transcribed at a high frequency, and a poly-A recognition sequence at the other end for proper processing and transport of the messenger RNA. An example of such a preferred (empty) expression cassette into which the DNA sequence of the present invention can be inserted is the pPHl414 plaεmid developed by Beach et al. of Pioneer Hi-Bred International, Inc., Johnston, IA and disclosed in U.S. Patent application No. 07/785,648, filed October 31, 1991. Highly preferred plant expression cassettes will be designed to include one or more selectable marker genes, such as kanamycin resistance or herbicide tolerance genes.

By the term "vector" herein is meant a DNA sequence which is able to replicate and express a foreign gene in a host cell. Typically, the vector has one or more endo- nuclease recognition sites which may be cut in a predictable fashion by use of the appropriate enzyme. Such vectors are preferably constructed to include additional structural gene sequences imparting antibiotic or herbicide resistance, which then serve as selectable markers to identify and separate transformed cells. Preferred selection agents include kanamycin, chlorosulfuron, phosphonothricin, glyphosate, hygromycin and methotrexate, and preferred markers are genes conferring resistance to these agents. A cell in which the foreign genetic material in a vector is functionally expressed has been "transformed" by the vector and is referred to as a "transformant" .

A particularly preferred vector is a plasmid, by which is meant a circular double-stranded DNA molecule that is not a part of the chromosomes of the cell.

As mentioned above, genomic, synthetic and cDNA encoding the gene of interest may be used in this invention. The vector of interest may also be constructed partially from a cDNA clone, partially from a synthetic sequence and partially from a genomic clone. When the gene sequence of interest is in hand, genetic constructs are made which contain the necessary regulatory sequences to provide for efficient expression of the gene in the host cell. According to this invention, the genetic construct will contain (a) a first genetic sequence coding for the proteinase inhibitor of interest and (b) one or more regulatory sequences operably linked on either side of the structural gene of interest. Typically, the regulatory sequences will be selected from the group comprising of promoters and terminators. The regulatory sequences may be from autologous or heterologouε sources. Promoters that may be used in the genetic sequence include nos, ocs, phaseolin, CaMV, FMV and other promoters isolated from plants or plant pests.

An efficient plant promoter that may be used is an overproducing plant promoter. Overproducing plant promoters that may be used in this invention include the promoter of the small sub-unit (ss) of the ribulose-1,5-biphosphate carboxylase from soybean (Berry-Lowe et al, J. Molecular and App. Gen., _l:483-498 (1982)), and the promoter of the cholorophyll a-b binding protein. These two promoters are known to be light-induced, in eukaryotic plant cells (see, for example, Genetic Engineering of Plants, An Agricultural Perspective, A. Cashmore, Pelham, New York, 1983, pp. 29-38, G. Coruzzi et al. , J. Biol. Che . , 258;1399 (1983), and P. Dunsmuir, et al. , J. Molecular and App. Gen., _2:285 (1983)).

The expression cassette comprising the structural gene for the proteinase inhibitor of interest operably linked to the desired control sequences can be ligated into a suitable cloning vector. In general, plasmid or viral (bacterio- phage) vectors containing replication and control sequences derived from species compatible with the host cell are used. The cloning vector will typically carry a replication origin, as well as specific genes that are capable of providing phenotypic selection markers in transformed host cells. Typically, genes conferring resistance to anti¬ biotics or selected herbicides are used. After the genetic material is introduced into the target cells, successfully transformed cells and/or colonies of cells can be isolated by selection on the basis of these markers. Typically, an intermediate host cell will be used in the practice of this invention to increase the copy number of the cloning vector. With an increased copy number, the vector containing the gene of interest can be isolated in significant quantities for introduction into the desired plant cells. Host cells that can be used in the practice of this invention include prokaryotes, including bacterial hosts such as E. coli, S . typhimurium, and ΣJ. marcescens. Eukaryotic hosts such as yeast or filamentouε fungi may alεo be used in thiε invention.

The isolated cloning vector will then be introduced into the plant cell using any convenient technique, includ- ing electroporation (in protoplasts), retroviruseε, microparticle bombardment, and microinjection, into cells from monocotyledonous or dicotyledonous plants, in cell or tissue culture, to provide transformed plant cells containing as foreign DNA at least one copy of the DNA sequence of the plant expression cassette. Preferably, the monocotyledonous species will be selected from maize, sorghum, wheat and rice, and the dicotyledonous specieε will be selected from soybean, sunflower, cotton, rapeεeed (either edible or industrial), alfalfa, tobacco, and Solanaceae such as potato and tomato. Using known techniques, protoplasts can be regenerated and cell or tissue culture can be regenerated to form whole fertile plants which carry and express the desired gene for the selected protein. Accordingly, a highly preferred embodiment of the present invention is a transformed maize plant, the cells of which contain as foreign DNA at least one copy of the DNA sequence of an expression caεεette of this invention.

Thiε invention alεo provideε methodε of imparting resistance to European corn borer and corn rootworm to plants of a susceptible taxon, comprising the stepε of: a) culturing cellε or tiεεueε from at leaεt one plant from the taxon, b) introducing into the cellε of the cell or tissue culture at leaεt one copy of an expreεεion caεεette compris¬ ing a εtructural gene coding for a proteinaεe inhibitor selected from aprotinin and serine proteinaεe inhibitors having at least 90% homology thereto by amino acid sequence, or a combination of such proteinase inhibitors, operably linked to plant regulatory sequenceε which cauεe the expression of the protein structural gene in the cellε, and c) regenerating insect-resistant whole plants from the cell or tissue culture. Once whole plants have been obtained in thiε manner, they can be sexually or clonally reproduced in any manner such that at leaεt one copy of the εequence provided by the expreεεion caεsette is present in the cells of progeny of the reproduction. Alternatively, once a single transformed plant has been obtained by the foregoing recombinant DNA method, conven¬ tional plant breeding methods can be used to transfer the protein structural gene and associated regulatory sequenceε via croεsing and backcrossing. Such intermediate methodε will compriεe the further steps of a) sexually crossing the insect-resistant plant with a plant from the insect-susceptible taxon; b) recovering reproductive material from insect- reεiεtant progeny of the cross; and c) growing insect-reεiεtant plantε from the reproductive material. Where desirable or necessary, the agronomic characteristics of the susceptible taxon can be subεtantially preserved by expanding thiε method to include the further steps of repetitively: a) backcrossing the insect-resistant progeny with insect-susceptible plantε from the susceptible taxon; and b) selecting for expression of insect resiεtance (or an associated marker gene) among the progeny of the back- croεε, until the desired percentage of the characteristicε of the susceptible taxon are present in the progeny along with the gene imparting insect resistance. Thiε will be important, for example, where the taxon iε a εubstantially homozygous plant variety, such as an inbred line of maize or a variety of a self-pollinated crop such as εoybeans. By "substantially homozygous" is meant homozygous within the limitε commonly accepted in the commercial production of certified seed of the species. For example, an inbred line of maize used in commercial seed production is typically 95% to 100% homozygous, and preferably 98% to 100% homozygous, as measured by RFLP analysis using 50 to 200 probes well distributed across the genome. If necessary, an RFLP-guided process of self-pollination and selection can be used to achieve thiε degree of genetic uniformity. By the term "taxon" herein is meant a unit of botanical classification of genus or lower. It thus includes genus, species, cultivars, varietieε, variantε, and other minor taxonomic groupε which lack a conεistent nomenclature. It will also be appreciated by those of ordinary skill that the plant vectors provided herein can be incorporated into Agrobacterium tumefaciens or Agrobacterium rhizogenes, which can then be used to transfer the vector into susceptible plant cells, primarily from dicotyledonous specieε. Thuε, thiε invention provideε a method for imparting insect resistance in Agrobacterium-suεceptible dicotyledonous plants in which the expression casεette is introduced into the cells by infecting the cellε with an Agrobacterium species, a plasmid of which haε been modified to include a plant expreεεion cassette of this invention.

Finally, it has now been determined that aprotinin and highly homologous serine proteinase inhibitors strongly potentiate the insecticidal activity of lectins such as wheat germ agglutinin. This effect is εurpriεing and unexpected, since many natural insecticides target the same structures or molecules within the insect and as a result many combinations of such natural insecticideε are at best additive and more often competitive, with little or no increase in insecticidal activity, as shown in the experimental results below. In addition to the increased level of activity which thiε result provides, there are also the practical benefits of increased technical flexibility and feasibility, since the εynergy or potentiation between theεe two groups of plant-expressible materials permits the attainment of effective insect or larval control with lower levelε of expression of each component. Since plant cell systems are more amenable to such lower levelε of expression, this offers the biotechnological entomologist greater flexibility in formulation and greater likelihood of achieving complete insect suppreεεion in hoεt plantε with lesε-than-maximal levelε of gene expression.

The combination of two different, synergistic inεecticidal materialε in a εingle εyεtem to provide effective inεect control alεo reduceε the potential for development of reεiεtant inεectε. Since εuch reεiεtanceε typically ariεe by spontaneous mutation, developing resistance to a binary syεtem εuch aε aprotinin plus wheat germ agglutinin would require two mutations, one in each target structure or molecule. The probability of such a double mutation is potentially (if there is no asεociation between the mutations) as low as the product of the probabilities of the individual mutationε, which would be quite low indeed — perhapε 1.0 x 10^"10, or less than fifty potentially survivable individuals per year in the entire United States. Also, the low or reduced selection pressure for each individual mutation further reduceε itε εpread within the population. Accordingly, this invention also provides a method for killing European corn borer and corn rootworm, comprising administering enterally to the larvae of those species a larvicidal combination of (a) aprotinin, a serine proteinase inhibitor having at leaεt 90% homology to aprotinin by amino acid sequence, or a combination thereof; and (b) an insecticidal lectin.

The following description further exemplifieε the compoεitionε of thiε invention and the methodε of making and uεing them. However, it will be understood that other methods, known by those of ordinary skill in the art to be equivalent, can also be employed.

In the following examples the wide variety of inεectε screened has resulted in several different bioasεayε being uεed to determine the effect of aprotinin and combinations of aprotinin pluε lectin on larval growth and εurvivorεhip. However, all of the bioaεεayε allow the teεt materialε to be enterally administered to the insect. In vitro bioaεsayε for the European corn borer (Ostrinia nubaliε) , and εouthern corn rootworm (Diabrotica undecimpunctata howardii) were done by incorporating the teεt protein into the artificial diet. Thiε is referred to herein as an "Incorporated Bioassay". This was accomplished by making up a standard artificial diet at 90% of the original water and adding a solution of the teεt protein to thiε mixture. Concentrationε of the protein in thiε diet are recorded as mg or μg of protein per ml of diet. Weight and mortality are recorded after seven days. Specific asεayε and variationε are deεcribed in the individual exampleε.

Example 1 EUROPEAN CORN BORER

Aprotinin waε the moεt effective proteaεe inhibitor againεt European corn borer with high mortality occurring during a replicated 7-day incorporated bioaεεay. The results are shown in Table 1.

Table 1. Effect of aprotinin and other protease inhibitors (PI) on European corn borer neonate larvae in incorporated bioassays

Treatment Weight Reduction (fold) %Mortality Control 5.2 — 0*

Aprotinin — — 100

Soybean PI (Bow an-Birk) 4.7 0

Soybean PI (Kunitz) 3.9 1.3 9

Chicken PI (Type IV) 2.9 1.8 0

*Corrected mortality

All materialε were tested at 20 mg Pi/ml of diet

SOUTHERN CORN ROOTWORM Aprotinin also showed effectiveness againεt Southern corn rootworm neonate larvae in incorporated bioaεεayε, as seen in Table 2. Table 2 Effect of aprotinin and other protease inhibitors (PI) on Southern corn rootworm neonate larvae in incorporated bioassays

Treatment Weight Reduction (fold) %Mortality

Control 3.3 — 0*

Aprotinin 0.4 8 60

Soybean PI (Bowman-Birk) 2.4 1.5 50 Cystatin 2.5 1.4 30

♦Corrected mortality All materials were tested at 20 mg Pi/ml of diet

Example 2 Combination of Aprotinin plus Wheat Lectin

Testε were performed employing Wheat Germ Agglutinin (WGA) , aprotinin, and combinations of the two in 7-day incorporated bioasεayε. The results are shown in Table 3.

Table 3 Effect of WGA and aprotinin on European corn borer neonate larvae in incorporated bioassays

Treatment Expected mortality from

%Mortality

Control 0

1. WGA 0.10 mg/ml 10

2. WGA 0.15 mg/ml 15

3. WGA 0.20 mg/ml 20

4. WGA 0.25 mg/ml 25

5. Aprotinin 0.25 mg/ml 15

6. Aprotinin 0.5 mg/ml 10

7. Aprotinin 1.0 mg/ml 25

8. Aprotinin 2.0 mg/ml 30

Combinations

4 + 8 90 55

3 + 7 80 45

2 + 7 75 40

3 + 5 70 35

3 + 6 70 30

2 + 6 55 25

When the whe<at lee :tin was replaced with Bauhinea purpurea lectin, similar resultε were lεo obtained.

Replicated 7-day bioassays were also performed to measure effects on growth. Results are shown in Table 4. Table 4 Effect of Aprotinin and WGA Combinations on ECB growth

Treatment Weight Reduction Weight (fold)

Control 10.5

1. Aprotinin 0.1 mg/ml 7.8 1.3

2. WGA 0.1 mg/ml 10.3

1 + 2 2.5 4.2 The weight and weight reduction is significantly different from all other weights and reductions at p< 0.05. The foregoing resultε indicate a synergy between aprotinin and insecticidal lectins in combination, both in terms of mortality and growth inhibition. Based on reεultε with other combinations of insecticidal compounds, an additive or neutral effect would have been expected.

Industrial Applicability

I. Isolation of the protein gene and insertion into bacteria

In order to isolate the coding sequence for the protein, it is necessary to have nucleotide sequence data which establishes an open reading frame (i.e., the correct triplet code for translation which should have only one "stop" signal at the very end of the gene.) It is also necessary to have an indication of where to look for the protease cleavage junction between the protein and the replicase which precedeε it in the εequence. Thiε can be determined from the peptide εequence of the N-terminal portion of the protein or by comparing the protein εequence with that of other homologouε proteinε. Thiε can generally be accomplished and the necesεary information obtained without sequencing the entire gene. Once the εequence at both endε of the gene haε been determined, the remainder of the gene can be cloned using restriction enzymes that flank the protein coding region or, more preferably, by cloning the precise protein coding region by oligonucleotide- directed amplification of DNA (polymerase chain reaction or PCR) .

Once the gene haε been iεolated, it can be cloned into a bacterial expreεεion vector with linkerε added to create all three reading frameε (uεing 8mer, lOmer, and 12merε each of which contain an ATG tranεlational εtart εite). The reεulting vectorε, containing the fragmentε of intereεt, can be inserted into, for example, BRL's Maximum Efficiency DH5 F' IQ tranεformation competent E_^ coli cellε. All three tranεformationε, one for each linker, are then εcreened via minipreps for the presence and orientation of insert. Appropriate clones are then chosen to test for expression of the protein gene.

Clones containing the properly oriented inserts are grown in culture medium conducive to the induction of the gene (LB medium with added IPTG) . The cells are lysed and bacterial proteinε are subjected to electrophoresiε in SDS polyacrylamide gels and then transferred to nitrocellulose. The resulting protein blots are easily screened for presence of protein using rabbit polyclonal and mouse monoclonal anti-protein antibody.

Having determined the proper reading frame, it is then necesεary to remove the gene from the bacterial expreεεion vector. The linker at the start of the gene region supplies the necessary start codon.

II. Expression of the Protein Gene in Plants

A plant expression caεεette, employing the regulatory sequences developed by Beach, et al . , and containing the protein gene, is constructed. The restriction map of the preferred plasmid, deεignated pPHl414, iε illuεtrated in Figure 1. Thiε plasmid contains an enhanced 35S promoter spanning nucleotideε - 421 to +2 of Cauliflower Moεaic Virus with the region from - 421 to - 90 duplicated in tandem, a 79 bp Hindlll Sail fragment from pJIIlOl spanning the 5' leader sequence of Tobacco Mosaic Viruε, a 579 bp fragment εpanning the first intron from maize AdHl-S, and a 281 bp fragment spanning the polyadenylation site from the nopaline synthase gene in pTiT37.

Another construct which can be used as an expression cassette is the pPHl412 plaεmid shown in Figure 2. it differs from pPHl414 in that it lacks the AdH intron segment. However, like pPHl414, it is constructed to have numerous restriction sites between the 0' segment and the NOS segment, which sites can be conveniently used for splicing any desired protein structural gene into position. This vector can be cotransformed with a similar plaεmid containing a selectable marker for antibiotic resistance into Black Mexican Sweet corn protoplaεtε by electroporation. Theεe protoplaεts can then be induced to regenerate cell walls and develop into callus by conventional techniques. Likewise, this callus can then be subjected to antibiotic selection to select for transformed colonies, and these colonies can be tested for expreεεion of protein with antisera for the appropriate protein using known methods. The efficiency of protection can be measured by infesting callus (or suspension cultures derived from callus) with the target insect and measuring survival percentages.

The protein gene can be introduced into embryogenic maize callus by methods similar to those used for Black Mexican Sweet. Embryogenic callus can be regenerated to whole fertile plants. The insect resistance imparted by the endogenous production of the protein is a simply inherited, dominant trait and can, if desired, be introduced into other plant varieties of the εpecieε by εimple croεεing or backcroεsing.

Using the foregoing techniques, aprotinin haε been expressed in maize suspension cells as determined by transient assays.

Claims

WHAT IS CLAIMED IS:

1. A method for killing inεectε or larvae of European corn borer and Southern corn rootworm, compriεing adminiεtering enterally to the larvae a larvicidal amount of aprotinin, a εerine proteinaεe inhibitor having at leaεt 90% homology to aprotinin by amino acid sequence, or a combination thereof.

2. A method according to Claim 1 wherein the proteinase inhibitor is administered enterally by incorporating it in the diet of the larvae.

3. A method according to Claim 2 wherein the diet of the larvae comprises the tissueε of a living plant.

4. A method according Claim 3 wherein the proteinaεe inhibitor iε not native to the plant.

5. A method according to Claim 1 for protecting a plant, harvested material from the plant, and products derived from the harvested material against infestation by insects or larvae of European corn borer and Southern corn rootworm, comprising inserting into the genome of the plant at least one εequence coding for aprotinin, a serine proteinase inhibitor having at least 90% homology thereto, or a combination of such proteinase inhibitors, in proper reading frame relative to transcription initiator and promoter sequences active in the plant to cause expression of the sequence or sequences at levels which provide a larvicidal amount of the gene product in the tisεueε of the plant or harvested material of the plant which are normally infested by the larvae.

6. A method according to Claim 5 wherein the plant is a monocotyledonous specieε εelected from corn, wheat, rice and εorghum.

7. A method according to Claim 5 wherein the plant iε a dicotyledonous species selected from soybean, sunflower, rapeseed, alfalfa, cotton and tomato.

8. A method according to Claim 5 further compriεing the steps of: a) culturing cells or tissues from the plant, b) introducing into the cells of the cell or tiεεue culture at leaεt one copy of an expression casεette compris- ing a sequence coding for the proteinase inhibitor or combination of proteinase inhibitorε, and c) regenerating resistant whole plantε from the cell or tisεue culture.

9. A method according to Claim 8 which compriεeε the further εtep of εexually or clonally reproducing the whole plant in εuch manner that at leaεt one copy of the sequence provided by the expression casεette iε preεent in the cellε of progeny of the reproduction.

10. A method according to Claim 8 in which the expreεsion casεette is introduced into the cells by electroporation.

11. A method according to Claim 8 in which the expreε¬ sion cassette is introduced into the cellε by microparticle bombardment.

12. A method according to Claim 8 in which the expres- sion casεette iε introduced into the cellε by microinjec- tion.

13. A method according to Claim 8 for providing reεistance to insectε in Agrobacterium tumefacienε-εuεcep- tible dicotyledonouε plantε in which the expression caεεette iε introduced into the cellε by infecting the cellε with Agrobacterium tumefacienε, a plaεmid of which haε been modi¬ fied to include the expression cassette.

14. A method of imparting resiεtance to European corn borer and Southern corn rootworm to plantε of a taxon susceptible to those insectε, and thereby to harveεted material from the plantε and productε obtained from the harvested material, comprising the εtepε of: a) εelecting a fertile, inεect reεiεtant plant pre¬ pared by the method of Claim 8 from a εexually compatible εpecieε; b) sexually crosεing the insect resiεtant plant with a plant from the insect susceptible taxon; c) recovering reproductive material from inεect resistant progeny of the crosε; and d) growing reεiεtant plantε from the reproductive material.

15. A method according to Claim 14 for imparting inεect resistance in a taxon consiεting of εubεtantially homozygous plants, and thereby to harvested material from the plants and products obtained from the harvested material, which compriseε the further εtepε of repetitively: a) backcroεεing the inεect resistant progeny with subεtantially homozygous, inεect εuεceptible plantε from the taxon; and b) εelecting for expreεεion of both inεect reεiεtance and the other characteristics of the susceptible taxon among the progeny of the backcross, until the desired percentage of the characteristicε of the susceptible taxon are present in the progeny along with insect resiεtance.

16. An iεolated DNA εequence which codeε εubεtantially εolely for aprotinin or a εerine proteinase inhibitor having at least 90% homology to aprotinin by amino acid sequence, or a combination of such proteinase inhibitors.

17. An expreεεion caεεette compriεing a DNA εequence according to Claim 16 operably linked to plant regulatory sequences which cause the expression of the DNA clone in plant cells.

18. An expresεion caεεette comprising a DNA sequence according to Claim 16 operably linked to bacterial expression regulatory sequenceε which cause the expresεion of the DNA clone in bacterial cells.

19. Bacterial cellε containing as a foreign plasmid at least one copy of an expresεion cassette according to Claim 18.

20. Transformed plant cells containing as foreign DNA at leaεt one copy of the DNA εequence of an expreεεion cassette according to Claim 17.

21. Transformed cells according to Claim 20, further characterized in being cells of a monocotyledonous εpecieε.

22. Tranεformed cellε according to Claim 21, further characterized in being maize, εorghum, wheat or rice cellε.

23. Tranεformed cells according to Claim 20, further characterized in being cells of a dicotyledonouε εpecieε.

24. Tranεformed cellε according to Claim 23, further characterized in being εoybean, alfalfa, εunflower, rapeεeed, cotton or tomato cellε.

25. A maize cell- or tiεεue-culture compriεing cells according to claim 23.

26. A transformed maize plant, the cells of which con¬ tain aε foreign DNA at least one copy of the DNA sequence of an expression casεette according to Claim 17.

27. A larvicidal compoεition, compriεing a larvicidal amount of aprotinin, a εerine proteinase inhibitor having at least 90% homology thereto, or a combination of such proteinase inhibitors in a non-phytotoxic vehicle.

28. A composition according to Claim 27 wherein the vehicle is adapted for systemic administration to a suεcep- tible plant.

29. A compoεition according to Claim 27 wherein the vehicle further compriεes a larval dietary bait for εuεcep- tible insects.

30. A composition according to Claim 29 wherein the bait further comprises a pheromonal larval attractant for εuεceptible inεectε.

31. A method of killing or controlling inεect peεtε of harvested plant material, comprising applying to the harvested material a compoεition comprising aprotinin, a proteinase inhibitor having at least 90% homology thereto, or a combination thereof.

32. A method of killing or controlling insect pests of harvested plant material, comprising incorporating into the harvested materials aprotinin, a serine proteinase inhibitor having at least 90% homology thereto, or a combination thereof.

33. A method for killing insectε or larvae of European corn borer and Southern corn rootworm, comprising administering enterally to the insects or larvae an larvicidal combination of

(a) aprotinin, a serine proteinase inhibitor having at leaεt 90% homology to aprotinin by amino acid εequence, or a combination thereof; and

(b) an inεecticidal lectin.

34. A method according to Claim 33 wherein the proteinase inhibitor and insecticidal lectin are administered enterally by incorporating them in the diet of the larvae.

35. A method according to Claim 34 wherein the diet of the larvae comprises the tissues of a living plant.

36. A method according Claim 35 wherein the proteinaεe inhibitor and lectin are not native to the plant.

37. A method according to Claim 33 for protecting a plant, harvested material from the plant, and products derived from the harvested material againεt infestation by inεects or larvae of European corn borer and Southern corn rootworm, compriεing inserting into the genome of the plant at least one εequence coding for

(a) aprotinin, a εerine proteinaεe inhibitor having at leaεt 90% homology thereto, or a combination of εuch proteinase inhibitors, and

(b) an insecticidal lectin, in proper reading frame relative to transcription initiator and promoter sequences active in the plant to cause expresεion of the sequence or sequences at levels which provide a larvicidal amount of the gene products in the tissueε of the plant or harvested material of the plant which are normally infested by the larvae.

38. A method according to Claim 37 wherein the plant is a monocotyledonous species selected from corn, wheat, rice and sorghum.

39 • A method according to Claim 37 wherein the plant is a dicotyledonous species selected from soybean, sunflower, rapeseed, alfalfa, cotton and tomato.

40. A method according to Claim 37 further comprising the steps of: a) culturing cellε or tiεεueε from the plant, b) introducing into the cells of the cell or tisεue culture at leaεt one copy of an expreεεion caεεette compriε¬ ing a εequence coding for (i) the proteinaεe inhibitor or combination of proteinaεe inhibitorε and

(ii) the lectin, and c) regenerating resistant whole plantε from the cell or tiεεue culture.

41. A method according to Claim 40 which compriεes the further εtep of sexually or clonally reproducing the whole plant in such manner that at least one copy of the sequence provided by the expression casεette iε present in the cellε of progeny of the reproduction.

42. A method according to Claim 40 in which the expression casεette iε introduced into the cellε by electroporation.

43. A method according to Claim 40 in which the expreε- sion cassette iε introduced into the cells by microparticle bombardment.

44. A method according to Claim 40 in which the expres¬ sion cassette iε introduced into the cellε by microinjec- tion.

45. A method according to Claim 40 for providing resistance to insects in Agrobacterium-susceptible dicotyledonouε plants in which the expresεion casεette iε introduced into the cells by infecting the cells with an Agrobacterium species, a plasmid of which haε been modified to include the expression casεette.

46. A method of imparting reεistance to European corn borer and Southern corn rootworm to plants of a taxon suεceptible to thoεe inεects, and thereby to harveεted material from the plantε and products obtained from the harveεted material, comprising the stepε of:

(a) εelecting a fertile, insect reεiεtant plant prepared by the method of Claim 40 from a εexually compatible species;

(b) sexually crosεing the insect resiεtant plant with a plant from the inεect susceptible taxon;

(c) recovering reproductive material from insect resiεtant progeny of the cross; and (d) growing resiεtant plantε from the reproductive material.

47. A method according to Claim 46 for imparting inεect reεistance in a taxon consisting of substantially homozygous plants, and thereby to harveεted material from the plantε and products obtained from the harvested material, which compriseε the further steps of repetitively:

(a) backcrosεing the insect resistant progeny with substantially homozygous, insect susceptible plants from the taxon; and

(b) selecting for expression of both insect resistance and the other characteristics of the suεceptible taxon among the progeny of the backcross,until the desired percentage of the characteristics of the suεceptible taxon are preεent in the progeny along with insect reεistance.

48. An isolated DNA sequence which codes subεtantially εolely for

(a) aprotinin or a εerine proteinaεe inhibitor having at leaεt 90% homology to aprotinin by amino acid εequence, or a combination of εuch proteinaεe inhibitorε, and

(b) an inεecticidal lectin.

49. An expreεsion casεette compriεing a DNA εequence according to Claim 48 operably linked to plant regulatory sequences which cause the expression of the DNA clone in plant cells.

50. An expreεεion caεεette compriεing a DNA εequence according to Claim 48 operably linked to bacterial expreεεion regulatory εequenceε which cauεe the expreεεion of the DNA clone in bacterial cellε.

51. Bacterial cellε containing aε a foreign plaεmid at leaεt one copy of an expreεεion caεεette according to Claim 50.

52. Tranεformed plant cellε containing aε foreign DNA at least one copy of the DNA sequence of an expreεεion cassette according to Claim 49.

53. Transformed cells according to Claim 52, further characterized in being cells of a monocotyledonous specieε.

54. Tranεformed cellε according to Claim 53, further characterized in being maize, sorghum, wheat or rice cells.

55. Transformed cells according to Claim 52, further characterized in being cellε of a dicotyledonouε εpecieε.

56. Tranεformed cellε according to Claim 55, further characterized in being soybean, alfalfa, sunflower, rapeseed, cotton or tomato cells.

57. A maize cell- or tisεue-culture compriεing cellε according to claim 54.

58. A tranεformed maize plant, the cellε of which con¬ tain aε foreign DNA at leaεt one copy of the DNA εequence of an expreεεion caεsette according to Claim 49.

59. A larvicidal compoεition, compriεing a larvicidal amount of compoεition conεiεting of

(a) aprotinin, a serine proteinase inhibitor having at least 90% homology to aprotinin by amino acid εequence, or a combination of εuch proteinaεe inhibitorε and (b) a larvicidal lectin; in a non-phytotoxic vehicle.

60. A compoεition according to Claim 59 wherein the vehicle further comprises a larval dietary bait for suscep¬ tible insectε.

61. A compoεition according to Claim 62 wherein the bait further comprises a pheromonal larval attractant for susceptible insects.