WO1994002513A2 - Derivatives of eranthis hyemalis lectin - Google Patents

Abstract

Compounds which are derived from Eranthis hyemalis lectin by modification at the position of one or more cysteine residues retain the larvicidal activity of the parent compound yet lack the inhibition of protein synthesis of the parent compound. The modification can be reduction of the cysteine residues and noncovalent linkage of the resulting peptide chains, or substitution of other amino acids for one or more of the cysteine residues.

Description

Derivatives of Eranthis hyemalis Lectin

Cross-Reference to Related Application

This is a continuation-in-part of prior copending U.S. Patent Application No. 07/919,464, filed July 24, 1992. 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 and virus resistance to plants, material harvested from the plants, and products derived from the harvested material. Background of the Invention

Numerous insects and viruses are . serious pests of common agricultural 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 r apply biological control organisms to large areas, and even more difficult to cause those living organisms to remain 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 structural gene. The lectin from Eranthis hyemalis (winter aconite) root tubers (EHL) has previously been purified and characterized by Cammue et al. as reported at Biochem. J. (1985) 227, 949- 955. The lectin was shown to be a glycoprotein of molecular weight (M_r) of about 60 kilodaltons (kda), consisting of two polypeptide chains with M_r of 30 kda and 28 kda, respectively, held together by disulfide bonds. Subsequently, the lectin was determined by Czapla et al. to have larvicidal activity, particularly against the difficult-to-kill Southern corn rootworm (SCRW), Diabrotica undecimpunctata howardi, as disclosed in the copending, commonly assigned U.S. patent application "Larvicidal Lectins and Plant Insect Resistance Based Thereon," filed September 20, 1991, Serial No. 07/763,100. One of us is also a coinventor of that application. However, the lectin is also a potent _in vivo inhibitor of protein synthesis in mammals which is believed to operate at the ribosomal level. Brief Description of the Drawing Figures

Figure 1 illustrates the gel electrophoretic pattern of EHL and EHL' .

Figure 2 illustrates an autoradiograph of iri vitro translation products obtained in the conduct of the protein synthesis inhibition assay of Example 9.

Figure 3 is a graphical representation of the circular dichroism measurements on EHL and EHL'. Disclosure of the Invention

It has now been discovered that the disulfide bonds in EHL can be completely reduced under native conditions in less than five minutes. This indicates that the disulfide bonds, which are hydrophobic in nature and therefore are normally buried within most peptides, are readily accessible in EHL. It has further been determined that when these groups are reduced and alkylated (to prevent reformation of the bonds) the resulting chains, referred to herein as A and B chains, reassociate spontaneously to form a new species EHL' which elutes at the same position as EHL during gel filtration and has the same M_r of 55 kda, in which the A and B chains, which apparently have some intrinsic affinity for one another, are noncovalently linked. This modified species retains the larvicidal activity of the parent compound.

From the foregoing it has been determined that the disulfide bridges present in the native lectin are not essential to its larvicidal activity. Further, since the two chains can noncovalently associate to form the biologically active species, it is possible to dispense with the disulfide bonds entirely by replacing one or more of the cysteine residues with any of the other 19 amino acids. From this it has also been determined that the modified compound can be formed intracellularly by expression of the two separate chains, which will spontaneously form the active compound because of their intrinsic affinity.

It has also been determined that the modified compounds of this invention do not exhibit the inhibition of protein synthesis in mammalian cells exhibited by the native lectin, thus solving a further problem associated with cellular implementation of a larval inhibition method using this lectin.

Accordingly, the present invention provides larvicidal compounds consisting of noncovalently linked first and second peptide chains having cysteine residues which are completely reduced, and having, respectively, at least 90% homology to the amino acid sequences of the A and B chains of Eranthis hyemalis lectin. The invention also provides a compound made from Eranthis hyemalis lectin by complete reduction of the disulfide bonds of the lectin and noncovalent reassembly of the resulting peptide chains, the compound having the larvicidal activity of the lectin but lacking the mammalian protein synthesis inhibitory activity of the lectin. This invention further provides larvicidal compounds having at least 90% homology to the amino acid structure of Eranthis hyemalis lectin but wherein one or more of the cysteine residues is replaced by an amino acid selected from Ala , Arg , Asn , Asp , Gin , Glu , Gly, His , lie , Leu , Lys , Met , Phe , Pro , Se r , Thr , Trp , Tyr , or Val .

It has also now been discovered that these compounds as well as native EHL provide antiviral activity against certain viral pathogens of plants. Accordingly, this invention also provides methods of treating or preventing viral infection, preferably in a plant, comprising the step of introducing into the environment of the viral pathogen a virucidal or virus-inhibiting amount of EHL or one of the compounds of this invention.

The compounds of this invention can be effectively applied to plants, harvested plant materials, or products consumed by the larvae 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, pancake and biscuit mixes. Alternatively, the larvicidal compound can be incorporated into the tissues of a susceptible plant so that in the course of infesting the plant, its harvested material, or a product derived from the harvested plant material, the larvae consume larvicidal amounts of the compound. One method of doing this is to incorporate the compound 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. However, genes which code for these compounds can also be synthesized directly using a DNA sequence obtained by working backwards from the known amino acid sequence and 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 or a suitable endophytic bacterium, so that an especially preferred embodiment of this method involves inserting into the genome of the plant or bacterium a DNA sequence coding for one or more insecticidal compounds according to this invention, in proper reading frame relative to transcription initiator and promoter sequences active in the plant or bacterium. Transcription and translation of the DNA sequence under control of the regulatory sequences causes expression, of the larvicidal gene product at levels which provide an insecticidal amount of the compound in the tissues of the plant which are normally infested by the larvae. Alternatively, a dietary bait containing one or more of the selected compounds can be employed, with, optionally, an added pheromonal or other larval attractant material.

The plant is preferably a plant susceptible, or whose harvested material or products are susceptible to infesta¬ tion and damage by the larvae of one or more insect larvae selected from European corn borer, corn rootworm and alfalfa weevil, sunflower moth, bollworm, tobacco budworm, and beet armyworm or whose harvested material is subject to attack by red flour beetle, confused flour beetle, black cutworm, lesser grain borer, sawtoothed grain beetle, rice weevil and Indian meal moth. These include corn (Zea mays) , wheat (Triticum aestivum) and sorghum (Sorghum bicolor) . However, this is not to be construed as limiting, inasmuch as these species have been among the most difficult commercial crops to reliably transform and regenerate, and these insects (under other common names) also infest certain 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 Allium, Antirrhinum, Arabidopsis, Arachis, Asparagus, Atropa, Beta, Brassica, Browallia, Capsicum, Cicer, Cicla, Citrullus, Citrus, Cucumis, Cucurbita, Datura Daucus, Digitalis, Fagopyrum, Fragaria, Geranium, Glycine, Gossypium, Helianthus, Hordeum, Hemerocallis, Lactuca, Lens, Lolium, Lotus, Lycopersicon, Majorana, Manihot, Medicago, Nasturtium, Nicotiana, Pelargonium, Persea, Petunia, Phaseolus, Pisum, Ranunculus, Raphanus, Ricinus, Senecio, Setaria, Solanum, Spinacia, Trifolium, Triticum, Bromus, Cichorium, Hyoscyamus, Linum, Nemesia, Panicum, Onobrychis, Pennisetum, Salpiglossis, Sinapis, Trigonella, and Vigna.

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 an EHL derivative compound according to this invention. It has been found that these compounds have sufficient insecticidal (larvicidal) activity to be operative in a plant cell expression system. That is, while certain other proteins 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 compound to an infested tissue or to a tissue which is efficient in production of the larvicidal compound.

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 plasmid developed by Beach et al. of Pioneer Hi-Bred International, Inc., Johnston, IA. 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, hygro- mycin and methotrexate, and preferred markers are genes conferring resistance to these compounds. 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 larvicidal compound 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 heterologous 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., _1:483-498 (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. Chem. , 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 compound of interest operably linked to the desired control sequences can be ligated into a suitable cloning vector. In general, plasmid or viral (bacteriophage) 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 antibiotics 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, £>. typhimurium, and Σ3. marcescens. Eukaryotic hosts such as yeast or filamentous fungi may also be used in this invention. The isolated cloning vector will then be introduced into the plant cell using any convenient technique, includ¬ ing electroporation (in protoplasts), retroviruses, 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 species will be selected from soybean, sunflower, cotton, rapeseed (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 cassette of this invention.

This invention also provides methods of imparting resistance to insects selected from European corn borer, corn rootworm, red flour beetle and alfalfa weevil, tobacco budworm, beet armyworm, bollworm, sunflower moth, confused flour beetle, black cutworm, lesser grain borer, sawtoothed grain beetle, rice weevil and Indian meal moth to plants of a susceptible taxon, comprising the steps of: a) culturing cells or tissues from at least one plant from the taxon, b) introducing into the cells of the cell or tissue culture at least one copy of an expression cassette compris¬ ing a structural gene coding for a larvicidal compound according to this invention, or a combination of such compounds, operably linked to plant regulatory sequences which cause the expression of the structural gene in the cells, and c) regenerating insect-resistant whole plants from the cell or tissue culture. Once whole plants have been obtained, they can be sexually or clonally reproduced in such manner that at least one copy of the sequence provided by the expression cassette 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 sequences via crossing and backcrossing. Such intermediate methods will comprise the further steps of a) sexually crossing the insect-resistant plant with a plant from the insect-susceptible taxon; b) recovering reproductive material from the progeny of the cross; and c) growing insect-resistant plants from the reproductive material. Where desirable or necessary, the agronomic characteristics of the susceptible taxon can be substantially preserved by expanding this method to include the further steps of repetitively: a) backcrossing the insect-resistant progeny with insect-susceptible plants from the susceptible taxon; and b) selecting for expression of insect resistance (or an associated marker gene) among the progeny of the back- cross, until the desired percentage of the characteristics of the susceptible taxon are present in the progeny along with the gene imparting insect resistance. By the term "taxon" herein is meant a unit of botanical classification of genus or lower. It thus includes genus, species, cultivars, varieties, variants, and other minor taxonomic groups which lack a consistent nomenclature.

It will also be appreciated by those of ordinary skill that the plant vectors provided herein can be incorporated into Agrobacterium tumefaciens, which can then be used to transfer the vector into susceptible plant cells, primarily from dicotyledonous species. Thus, this invention provides a method for imparting insect resistance in Agrobacterium tumefaciens-susceptible dicotyledonous plants in which the expression cassette is introduced into the cells by infect¬ ing the cells with Agrobacterium tumefaciens, a plasmid of which has been modified to include a plant expression cassette of this invention. Finally, insect pests of harvested material, including stored grain, such as the red flour beetle (Tribolium castanaeu ) can also be targets for the compositions and methods of this invention. In view of this, the invention also provides a methods and compositions for killing larvae of red flour beetle and confused flour beetle, black cutworm, lesser grain borer, sawtoothed grain beetle, rice weevil and Indian meal moth and other susceptible insect pests of harvested materials and products obtained from harvested materials, comprising applying to the grain or causing to be expressed in the grain a larvicidal compound of this invention.

The following description further exemplifies the compositions of this invention and the methods of making and using 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. Example 1 Extraction and Purification of the Native Lectin

The lectin from inter Aconite root tubers was extracted and purified by published methods. See, e.g., Cammue et al., Biochem. J. (1985) 227, 949-955. The first step in the published extraction method consists of cutting the tubers (20g) into small pieces and homogenizing in 250 ml ice cold 0.2 M sodium chloride solution containing lg/1 ascorbic acid (pH 5.0). The solution was decanted after 30 min. and the slurry was re-extracted with another 250 ml of ice cold sodium chloride solution. Both the fractions were combined and centrifuged at 10000 g for 10 minutes. The thick white fat layer was removed and NaOH was added to the supernatant to adjust the pH to 9.0. The solution was recentrifuged and the supernatant was neutralized. The supernatant was filtered through filter paper (Whatman 3M). The clear filtrate was applied to a column of asialofetuin immobilized on agarose. After washing the column with PBS (pH 7.2) to remove the unbound material, the lectin eluted with 50 mM tetraborate (pH 9.5) and the pH is adjusted to 7.0 by adding HCl. Solid Nacl was added to bring the solution to 0.2 M NaCl. The solution was again centrifuged and the affinity chromatography was repeated. After the second affinity purification the desorbed lectin was loaded on a Sepharose QFast Flow anion exchange column. The column was equilibrated with 20 mM Tris-HCl (pH 8.7). After washing the column with the Tris-buffer, the lectin was eluted with the buffer containing 0.4 NaCl, dialyzed against water and lyophilized. Example 2

Hemagglutination and inhibition of hemagglutination Hemagglutination of cells was done at room temperature in a final volume of 200 μl . One hundred microliters of the protein was serially twofold diluted into the wells of a microtiter plant followed by 100 μl (1 x 10⁷ cells per well) of rabbit red blood cells (Colorado Serum Co.) The plate was read after five hours. The ability of the following sugars to inhibit hemagglutination was tested. The concentration of sugars was varied from 1 M to 1 mM in the assay.

1. Glucose

2. Rhamnose 3. Threhalose

4. Arabinose

5. Fucoεe

6. Cellobiose

7. Xylose 8. Lactose

9. Mannose

10. Raffinose

11. Galactose

12. Maltose 13. N-Acetyllactosamine

14. N-Acetylchitobiose

15. N-Acetyl-D-galactosamine

16. N-Acetyl-D-Glucosamine

17. N,N' ,N"-Triacetylchitotriose 18. N,N' ,N",N"'-Tetraacetylchitotetrose

Binding of sugars was monitored by following the decrease in fluorescence of the fluorescent derivative 4 Methodiumbelliferyl-N,N'-diacetyl-β-chitobioside. 500 μl of the fluorescent ligand in PBS (pH 7.2) was placed in a thermostatted cuvette at 25°C and titrated with progressive additions of 10 μl aliquots of protein to the same buffer.. Fluorescence emission spectra were recorded at an excitation wavelength of 313 nm and corrected for dilution. Both EHL and EHL' caused agglutination at a minimum titer of 3 /g. Both EHL and EHL' reacted only with N-acetyl D- galactosamine. This sugar inhibited agglutination with EHL at a concentration of 400 /M, while it inhibited EHL' agglutination of cells at 200 /M.

Example 3 Tryptophan Determination

The number of tryptophans in EHL was determined by the oxidation with N-bromosuccinimide (NBS) according to published methods. See, e.g., Spande and Witkop (1967) Methods in Enzymology, pp. 498-506. 500 μl of the lectin solution having an absorbance of 0.5 at 280 nm was titrated with 10 μl aliquots of 4.5 mM NBS solution until the decreasing absorbance at 280 nm reached a constant value. The number of tryptophans were calculated using the equation published by Spande and witkop.

Example 4 Preparation of EHL' To 5 mg of lectin solution at 5°C was added 0.1 ml of β-mercaptoethanol. The contents of the solution were maintained under a nitrogen barrier. After 5 min., a freshly prepared solution of 0.268 g of iodoacetic acid in 1.0 ml of 1.0 N NaOH was added to the reaction mixture. After 30 minutes, the solution was dialyzed in PBS (pH 7.2). 1.0 ml of the sample was loaded on a Suρerose-12-HR-10/50 column (Pharmacia). EHL was eluted with PBS at a flow rate of 0.5 ml/min. It eluted at the same place as EHL. The peak fractions (monitored at 280 nm) were pooled.

Example 5 Molecular Weight Determination

Samples of EHL and EHL' prepared for sedimentation equilibrium experiments in the manner of Example 4, followed by passing them in PBS at pH 7.2 through a column of Superose-12-HR-10/50 column (Pharmacia) on an FPLC system and collecting the peak fractions. Fractions collected prior to the appearance of the peak, having no absorbance at 280 nm, were pooled and used as solvent blank.

Sedimentation equilibrium analysis of EHL' was carried out with AN-H rotor in a Beckman Pre-Scanner analytical ultracentrifuge. Low-speed sedimentation equilibrium in long columns (0.2 ml) was attained in 16 h by an initial 2h overspeed (28,000 rpm) and then equilibrated at 10,000 rpm. Scans (λ = 280 nm) were digitized and stored in a Bascom- Turner electronic recorder. Baselines were set to zero where necessary and the data were transferred to a PC-DOS file using a program supplied by On-Line instrument Systems. The data were analyzed as suggested by Johnson et al., Biophys. J. 36, 575-588 (1981) with a robust fitting procedure described by Matheson (1990) Comput. Chem. 14, 49. The fits were implemented in software supplied by On-Line Instrument systems. The partial specific volume of 0.75 was used to calculate the molecular weights. The SDS-PAGE gels are illustrated in Figure 1. Lane 1 contains EHL' and Lane 2 contains native EHL.

Example 6 Circular Dichroiεm Measurement Spectra (195-250 nm) were recorded at 25°C with a JASCO 600 spectropolarimeter using 0.1-cm cells. The lectin concentration was 0.03 mg/ml for both EHL and EHL'. Mean residue ellipticity was calculated using a mean residue weight of 110. The results are shown in Figure 3.

Example 7 Protein Sequencing and Amino Acid Analysis

EHL and EHL' were electroblotted into polyvinylidene diflouride membrane (Millipore) from SDS-acrylamide gel according to the published method of P. Matsudaira J. Biochem. 262, 10035 (1981) and sequenced on an applied biosystems 477A protein sequencer. Amino acid analysis was performed on an Applied Biosystems 420A derivatizer, where the free amino acids were converted to PTC amino acids followed by separation on a C-18 reverse phase column and detection at 254 nm. While complete sequence information has not yet been obtained, the A chain has an N-terminal end which begins Aspartic-Lysine-Asparagine and the b chain N- terminus begins Aspartic-Glutamic-Aspartic. Amino acid composition in residues per mole is shown in Table 1.

Table 1

P Y V M L F K I C w

Larval Bioaεεayε

Neonate larvae of 0. nubilalis were fed on 2 ml of standard diet into which 1 mg of EHL or EHL' had been microincorporated. Nine insects were used per treatment.

Weight and mortality were recorded at 7 days. Results are shown in Table 2.

Table 2

Protein Inhibition Analysis

A rabbit reticulocyte lysate kit supplied by Promega was used to determine the inhibitory action of EHL and EHL' on i_n vitro protein synthesis. The translation reaction ijn vitro was directed by brome mosaic virus (BMV). The components of the reticulocyte lysate assay were incubated at 30°C for 60 minutes in the presence of 10 nM EHL, 100 nM EHL and 100 nM EHL'. Ricin A chain served as the positive control. Following are the details of the translation according to the method of Pelham and Jackson (1976) Eur. J. Biochem. 67, 247 and adopted by the Promega Technical Bulletin. 1) 35 ul nuclease treated lysate

2) water (added to adjust the final volume to 50 ul )

3) 1 μl RNAsin

4) 1 μl ImM amino acid mixture (minus methionine) 5) 2 μl RNA substrate

6) 4 μl 35 S-methionine (1200 Ci/mMol)

The results of translation are assayed by radiography. As shown in Figure 2, the radiographs indicated that EHL inhibited protein synthesis and that EHL' did not inhibit protein synthesis in this assay. In this autorad, lane 1 is the unaltered assay which serves as a negative control (no protein synthesiε inhibition); lane 2 is the same assay done in the presence of 10 nM EHL; lane 3 contains the same assay in the presence of 100 nM EHL, lane 4 contains the same asεay in the presence of 100 nM EHL'; lane 5 contains the same assay in the presence of 100 nM Ricin A chain which serves as a positive control (strong protein synthesis inhibition) and lane 6 is the standard assay but without RNA to show any background.

A similar test run with the wheat germ translation system also supplied by Promega did not show inhibition of translation by EHL or EHL'. Thus, there is a differential specificity exhibited by EHL with respect to inhibition of in vitro translation in mammalian versus plant ribosomal syεtems. This same characteristic has been obεerved with ricin.

Example 10 Amino Acid Sequencing of Cyanogen Bromide Digests EHL' was digested with cyanogen bromide (CNBr) and the resulting digest was purified by reverse phase chromatography or SDS_PAGE followed by blotting onto a PVDF membrane. Resulting fragments yielded sequences as shown in SEQUENCE I.D. NO. 1 through SEQUENCE I.D. NO. 6. These are more compactly listed in single-letter code as follows: CNBr-1 (SEQUENCE I.D. NO. 1): DVRGSDVST?LG CNBr-2 (SEQUENCE I.D. NO. 2): DEDT?PLTETVTRSI?GR CNBr-3 (SEQUENCE I.D. NO. 3): MIYDTDSAVPDATV CNBr-3H (SEQUENCE I.D. NO. 4) RQIILYEPTGNPNQQ7LAYS CNBr-4 (SEQUENCE I.D. NO. 5) DEDT CNBr-4H (SEQUENCE I.D. NO. 6) VSEAARIRYIEHLVRRSTR

Compariεon of theεe sequences with protein sequence data bases shows homology to ricin as well as other ribosome inactivating proteins. Furthermore, the sequence SEAAR (SEQUENCE I.D. NO. 7) in CNBr-4H (SEQUENCE I.D. No. 6) forms part of the putative site of many proteins possessing the property of ribosome inactivation. Thus, this information establishes EHL and its derivatives as new members of a family of proteins that have this property.

Example 11 Anti-Viral Activity of EHL Antiviral activity of EHL was assayed by the appearance of local lesions in the leaves of cowpea plants (Vigna vigna) inoculated with the Alfalfa Mosaic Virus (AMV) according to the method of Ikeda et al. (Ikeda, T., Takanami, Y., imaizumi, S., Matsumoto, T., Mikami, Y., and Kubo, S., 1987 Plant Cell Rep. , 216-218). The sample was prepared as a εolution containing either the virus alone (50 μg/ml) or mixed with EHL. It waε then applied on the upper surface of the leaf by gentle rubbing with a piece of cheesecloth dipped in the sample. Inoculation of the virus was facilitated by the gentle abrasive action of the carborundum included in the sample preparation. Subsequently, the leaf was rinsed with distilled water. The number of local lesions which appeared on each inoculated leaf within two days after inoculation were counted and the degree of inhibition was determined by comparison with the control leaves which had no EHL. Six plants were used for each assay. EHL provided 80% inhibition of the lesions, reflecting the antiviral activity of the protein. SEQUENCE LISTING (1) GENERAL INFORMATION:

(i) APPLICANT: Rao, A. Gururaj ; Kumar, M. Arun

(ii) TITLE OF INVENTION: Derivativeε of Eranthis hyemalis Lectin and Their Uεe as Larvicides (iii) NUMBER OF SEQUENCES: 7 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Pioneer Hi-Bred International, Inc.

(B) STREET: 700 Capital Square, 400 Locust Street

Des Moines Iowa

United States

50309 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette, 3.5 inch, 1.44 Mb storage

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: MS-DOS, Microsoft Windows (D) SOFTWARE: Microsoft Windowε Notepad

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Roth, Michael J. (B) REGISTRATION NUMBER: 29,342

(C) REFERENCE/DOCKET NUMBER: 0210R US (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (515) 245-3594

(B) TELEFAX: (515) 245-3634 (2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acidε

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: lectin

(A) DESCRIPTION: (iϋ) HYPOTHETICAL: No (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE : (vi) ORIGINAL SOURCE:

(A) ORGANISM: Eranthis hyemalis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Asp Val Arg Gly Ser Asp Val Ser Thr Xaa Leu Gly (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: nucleotide

(C) STRANDEDNESS: εingle

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: lectin

(A) DESCRIPTION: (iϋ) HYPOTHETICAL: No (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Asp Glu Asp Thr Xaa Pro Leu Thr Glu Thr Val Thr Arg Ser lie Xaa Gly Arg (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acidε

(B) TYPE: nucleotide

(C) STRANDEDNESS: εingle (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: lectin

(A) DESCRIPTION: (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Met lie Tyr Aεp Thr Aεp Ser Ala Val Pro Aεp Ala Thr Val (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids

(B) TYPE: nucleotide

(C) STRANDEDNESS: εingle

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: lectin (A) DESCRIPTION: (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Arg Gin lie lie Leu Try Glu Pro Thr Gly Aεn Pro Aεn Gin Gin Xaa Leu Ala Tyr Ser (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acidε (B) TYPE: nucleotide

(C) STRANDEDNESS: εingle

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: lectin

(A) DESCRIPTION: (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Aεp Glu Aεp Thr

(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: nucleotide

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: lectin

(A) DESCRIPTION: (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Val Ser Glu Ala Ala Arg lie Arg Tyr lie Glu His Leu Val Arg Arg Ser Thr Arg

(2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids

(B) TYPE: nucleotide

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: lectin (A) DESCRIPTION: (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Ser Glu Ala Ala Arg

Claims

WHAT IS CLAIMED IS:

1. A compound conεisting of noncovalently linked first and εecond peptide chainε having cyεteine residues which are completely reduced, and having, respectively, at least 90% homology to the amino acid sequenceε of the A and B chainε of Eranthis hyemalis lectin.

2. A compound made from Eranthis hyemalis lectin by complete reduction of the disulfide bonds of the lectin and noncovalent reassembly of the resulting peptide chains, the compound having the larvicidal activity of the lectin but lacking the protein synthesis inhibitory activity of the lectin.

3. A compound having at least 90% homology to the amino acid structure of Eranthis hyemalis lectin but wherein one or more of the cysteine residueε iε replaced by an amino acid εelected from Ala, Arg, Aεn, Aεp, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.

4. A method of killing or inhibiting insect larvae, comprising administering enterally to the larvae a larvicidal or feeding inhibitory amount of a compound according to Claim 1.

5. A method according to Claim 4 wherein the compound is administered enterally by incorporating the compound in the diet of the larvae.

6. A method according to Claim 5 wherein the diet of the larvae comprises the tisεueε of a living plant.

7. A method according to Claim 4 for protecting a plant, harvested material from the plant, and products derived from the harvested material against infestation by inε»ct larvae compriεing inεerting into the genome of the p3 ; at leaεt one sequence coding for a peptide compound ac :ding to claim 3 or a combination of such compounds in proper reading frame relative to tranεcription initiator and promoter sequences active in the plant to cause expresεion of the εequence or εequences at levels which provide a larvicidal amount of the gene product in the tisεueε of the plant or harveεted material of the plant which are normally infeεted by the larvae.

8. A method according to Claim 7 wherein the plant iε a monocotyledonouε species selected from corn, wheat, rice, oats, rye, and sorghum.

9. A method according to Claim 7 wherein the plant is a dicotyledonous species selected from soybean, εunflower, rapeseed, alfalfa, cotton, melon, cucumber, lettuce, pepper and tomato.

10. A DNA sequence which codes for a compound according to Claim 3 or a combination of such compounds.

11. An expression casεette comprising a DNA sequence according to Claim 10 operably linked to plant regulatory sequenceε which cauεe the expreεsion of the DNA clone in plant cells.

12. An expression casεette compriεing a DNA sequence according to Claim 10 operably linked to bacterial expression regulatory sequences which cause the expression of the DNA clone in bacterial cells.

13. Bacterial cellε containing as a foreign plasmid at leaεt one copy of an expreεεion caεsette according to Claim 12.

14. Transformed plant cellε containing aε foreign DNA at leaεt one copy of the DNA sequence of an expresεion cassette according to Claim 11.

15. Transformed cells according to Claim 14, further characterized in being cells of a monocotyledonous species.

16. Transformed cells according to Claim 15, further characterized in being maize, sorghum, wheat, oat, rye or rice cellε.

17. Tranεformed cellε according to Claim 14, further characterized in being cellε of a dicotyledonouε εpecies.

18. Transformed cells according to Claim 17, further characterized in being εoybean, alfalfa, εunflower, rapeεeed, cotton, melon, cucumber, pepper, lettuce or tomato cellε.

19. A tranεformed maize plant, the cellε of which contain aε foreign DNA at leaεt one copy of the DNA εequence of an expreεεion caεεette according to Claim 11.

20. A larvicidal compoεition, compriεing a larvicidal amount of a compound according to Claim 1, 2 or 3 or a combination of εuch compoundε in a non-phytotoxic vehicle.

21. A method of killing or controlling inεect pests of harvested plant material, comprising applying to the harvested material a composition according to claim 20.

22. A method of killing or controlling insect pests of harvested plant material, comprising incorporating into the harvested materialε a compound according to Claim 1, 2 or 3 or a combination thereof.

23. A method of killing or inhibiting viral plant pathogens, comprising the step of introducing into the environment of the pathogen a viral-inhibitory amount of Eranthiε hyemaliε lectin or a derivative thereof according to claim 1, 2 or 3.