CA2358161A1 - Organisms containing insecticidal proteins - Google Patents

Abstract

Organisms containing insecticidal proteins for controlling pests, particularly insect pests, are provided.
The organisms comprise proteins isolated from plants of the genus Pentaclethra.

Description

ORGANISMS CONTAINING INSECTICIDAL PROTEINS
FIELD OF THE INVENTION
The invention relates to organisms which have been genetically transformed with the compositions for controlling insect species.
BACKGROUND OF THE INVENTION
Numerous insect species are serious pests to common agricultural crops such as corn, soybeans, peas, cotton, and similar food and fiber crops. The primary method of controlling such pests has been through the application of synthetic chemical compounds. However, the widespread use of chemical compounds poses many problems with regard to the environment because of the non-selectivity of the compounds and the development of insect resistance to the chemicals.
Other approaches to pest control have been tried including the use of biological organisms which are typically "natural predators" of the species sought to be controlled.
Such predators may include other insects, fungi, and bacteria such as Bacillus thuringiensis. Alternatively, large colonies of insect pests have been raised in captivity, sterilized and released into the environment in the hope that mating between the sterilized insects and fecund wild insects will decrease the insect population. While these approaches have had some success, they entail considerable expense and present several major difficulties.

For example, it is difficult both to apply biological organisms to large areas and to cause such living organisms to remain in the treated area or on the treated plant species for an extended time. Predator insects can migrate and fungi or bacteria can be washed off of a plant or removed from a treated area by rain. Consequently, while the use of such biological controls has desirable characteristics and has met with some success, in practice these methods seem severely limited.
Advances in biotechnology in the last two decades have presented new opportunities for pest control through genetic engineering. In particular, advances in plant genetics coupled with the identification of insect growth factors and naturally-occurring plant defensive compounds or agents offer the opportunity to create transgenic crop plants capable of producing such defensive agents and thereby protect the plants against insect attack.
Transgenic plants that are resistant to specific insect pests have been produced using genes encoding Bacillus thuringiensis (Bt) endotoxins or plant protease inhibitors (PIs). Transgenic plants containing Bt endotoxin genes have been shown to be effective for control of some insects. Effective plant protection using transgenically inserted PI genetic material has not yet been demonstrated in the field. While cultivars expressing Bt genes may presently exhibit resistance- to some insect pests, resistance based on the expression of a single gene might eventually be lost due to the evolution of Bt resistance in the insects. Thus, the search for additional genes which can be inserted into plants to provide protection from insect pests is needed.
Scientists have identified some specific plant components or compounds which act as defensive agents to protect a plant from attack by insect pests and pathogens. While such components are usually present at only low levels in various plant tissues, some of them are also capable of being induced to higher levels upon attack by an insect pest or a pathogen.
Examples of such defensive compounds include alkaloids, terpenes, and various proteins such as enzymes, enzyme inhibitors, and lectins. Of particular interest are plant-derived compounds which can block or alter normal biomolecular activity and thus inhibit insect growth or kill the insect.

The corn rootworm (CRW) complex in the United States consists of three species, Diabrotica barberi Smith and Lawrence (Northern), D. undecimpunctata howardi Barber (Southern) and D. virgifera virgifera LeConte (Western). The western and northern species contribute the most to the economic damage to maize. The economic damage and control costs are estimated to exceed one billion dollars a year. As noted above, the major concerns of pesticide use in controlling CRW damage are its negative effect on the environment and the development of resistance by the insect. Crop rotation is becoming less effective as a CRW control method due to extended diapause in the northern CRW and the development of modified egg laying behavior in western CRW. The generation of transgenic plants with resistance to CRW could have a major economic impact. Unfortunately there are relatively few, if any, genes available that can control CRW in transgenic plants.
Thus, there is a need for additional insecticidal principles, particularly those active against CRW.
SUMMARY OF THE INVENTION
The present invention provides an organism which has been transformed with a vector comprising a DNA sequence encoding a substantially purified protein having insecticidal activity, wherein said protein comprises the amino acid sequence set forth in Figure 1 and SEQ ID N0:2.
The present invention also provides a plant, a seed or a plant cell which has been stably transformed with an expression cassette comprising a promoter that drives expression in a plant cell operably linked to a nucleotide sequence that encodes an insecticidal protein wherein said protein is selected from the group consisting of: a) the amino acid sequence set forth in SEQ ID N0:2; b) residues 22-409 of the sequence set forth in SEQ ID N0:2; c) residues 29-409 of 3a the amino acid sequence set forth in SEQ ID N0:2; d) a polypeptide comprising a variation of (a), (b) or (c), wherein said variation comprises at least one amino acid substitution, truncation, internal deletion or insertion;
and e) a polypeptide comprising at least 60% amino acid sequence identity to the sequence of (a), (b), or (c). The promoter may be a root preferential promoter.
One aspect of the invention provides a plant or seed or plant cell stably transformed as described above, wherein the nucleotide sequence that encodes the insecticidal protein is selected from: (a) the sequence set forth in Figure 1 and SEQ ID N0:1; (b) nucleotide sequences which encode a polypeptide having insecticidal activity and which hybridize to sequences of (a) above under stringent conditions defined by a wash stringency of 0.3M NaCl, 0.03M
sodium citrate, 0.1% SDS at 70°C; (c) nucleotide sequences which encode a polypeptide having insecticidal activity and which differ from the sequence of (a) due to the degeneracy of the genetic code; and (d) nucleotide sequences comprising at least 70% sequence identity to the sequence of (a). The promoter may be a root preferential promoter.
In one embodiment, the plant, seed or cell described above is a monocot, e.g. maize.
Compositions and methods for the control of insects and other pests are provided. The compositions comprise proteins having pesticidal activities which can be isolated from plants of the genus Pentaclethra. Purified protein, as well as amino acid and DNA sequence information is provided for proteins having rootworm activity. The DNA
sequences encoding the pesticidal proteins can be used to transform plants, bacteria, fungi, yeasts, and other organisms for the control of pests.

3b The compositions and methods of the invention may be used in a variety of systems for controlling plant and non-plant pests.
According to one aspect of the present invention, there is provided a substantially purified protein isolated from the genus Pentaclethra, which has insecticidal properties, said protein isolated by a process comprising:
preparing a crude extract from at least one seed of a plant from the genus Pentaclethra, said extract comprising said protein; and purifying said protein by conventional chromatography.
Another aspect of the invention provides a substantially purified lipid acyl hydrolase having insecticidal properties, said lipid acyl hydrolase isolated by a process comprising: preparing a crude extract from at least one seed of a plant from the genus Pentaclethra, said extract comprising said lipid acyl hydrolase; and purifying said lipid acyl hydrolase by chromatography.
According to another aspect of the present invention, there is provided a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of: a) the amino acid sequence set forth in SEQ ID N0:2; b) residues 22-409 of the sequence set forth in SEQ ID N0:2; c) residues 29-409 of the amino acid sequence set forth in SEQ ID N0:2; d) a pesticidally active fragment of the amino sequence set forth in SEQ ID N0:2; and e) an amino acid sequence of a polypeptide substantially similar to the polypeptide of any one of a), b), c), and d) which has insecticidal properties.
According to still another aspect of the present invention, there is provided a polypeptide having insecticidal activity against corn rootworm, wherein said 3c polypeptide comprises a variation of an amino acid sequence selected from the group consisting of: a) the amino acid sequence set forth in SEQ ID N0:2; b) residues 22-409 of the sequence set forth in SEQ ID N0:2; and c) residues 29-409 of the amino acid sequence set forth in SEQ ID N0:2; wherein said variation comprises at least one amino acid substitution, truncation, internal deletion or insertion.
Another aspect of the invention provides a substantially purified protein having insecticidal activity, wherein said protein comprises the amino acid sequence set forth in Figure 1 and SEQ ID N0:2.
According to yet another aspect of the present invention, there is provided an isolated nucleotide sequence encoding a polypeptide having insecticidal activity for corn rootworm, wherein said nucleotide sequence encodes a polypeptide selected from the group consisting of: a) the amino acid sequence set forth in SEQ ID N0:2; b) residues 22-409 of the sequence set forth in SEQ ID N0:2; c) residues 29-409 of the amino acid sequence set forth in SEQ ID N0:2;
d) a polypeptide comprising a variation of (a), (b) or (c), wherein said variation comprises at least one amino acid substitution, truncation, internal deletion or insertion;
and e) a polypeptide comprising at least 60% amino acid sequence identity to the sequence of (a), (b), or (c).
According to a further aspect of the present invention, there is provided an isolated nucleotide molecule encoding a polypeptide having insecticidal activity, said nucleotide molecule having a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence set forth in Figure 1 and SEQ ID NO: l; (b) nucleotide sequences which encode a polypeptide having insecticidal activity and which hybridize to sequences of (a) above under 3d stringent conditions defined by a wash stringency of 0.3M
NaCl, 0.03M sodium citrate, 0.1% SDS at 70°C; (c) nucleotide sequences which encode a polypeptide having insecticidal activity and which differ from the sequence of (a) due to the degencracy of the genetic code; and (d) nucleotide sequences comprising at least 70% sequence identity to the sequence of (a).
According to yet a further aspect of the present invention, there is provided a plant which has been stably transformed with an expression cassette comprising a promoter that drives expression in a plant cell operably linked to a nucleotide sequence that encodes an insecticidal protein wherein said protein is selected from the group consisting of: a) the amino acid sequence set forth in SEQ
ID N0:2; b) residues 22-409 of the sequence set forth in SEQ
ID N0:2; c) residues 29-409 of the amino acid sequence set forth in SEQ ID N0:2; d) a polypeptide comprising a variation of (a), (b) or (c), wherein said variation comprises at least one amino acid substitution, truncation, internal deletion or insertion; and e) a polypeptide comprising at least 60% amino acid sequence identity to the sequence of (a), (b), or (c).
According to still a further aspect of the present invention, there is provided a method for controlling corn rootworm said method comprising: transforming a plant cell with an expression cassette comprising a promoter that drives expression in a plant cell operably linked to a nucleotide sequence that encodes a protein having insecticidal activity wherein said protein is selected from the group consisting of: a) the amino acid sequence set forth in SEQ ID N0:2; b) residues 22-409 of the sequence set forth in SEQ ID N0:2; c) residues 29-409 of the amino acid sequence set forth in SEQ ID N0:2; d) a polypeptide 3e comprising a variation of (a), (b) or (c), wherein said variation comprises at least one amino acid substitution, truncation, internal deletion or insertion; and e) a polypeptide comprising at least 60% amino acid sequence identity to the sequence of (a) , (b) , or (c) .
According to yet another aspect of the present invention, there is provided a substantially purified polypeptide having insecticidal activity against corn rootworm, wherein said polypeptide comprises an amino acid sequence with at least 60% sequence identity to an amino acid sequence selected from the group consisting of: a) the amino acid sequence set forth in SEQ ID N0:2; b) residues 22-409 of the sequence set forth in SEQ ID N0:2; and c) residues 29-409 of the amino acid sequence set forth in SEQ
ID N0:2.
Another aspect of the invention provides a plant cell or a seed cell which has been stably transformed with an expression cassette comprising a promoter that drives expression in a plant cell operably linked to a nucleotide sequence that encodes a protein having insecticidal activity, wherein said protein is selected from the group consisting of: a) the amino acid sequence set forth in SEQ
ID N0:2; b) residues 22-409 of the sequence set forth in SEQ
ID N0:2; c) residues 29-409 of the amino acid sequence set forth in SEQ ID N0:2; d) a polypeptide comprising at least 60% amino acid sequence identity to the sequence of (a), (b) , or (c) .

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 provides the amino acid and nucleotide sequence of the cDNA
sequence of the corn rootworm active principle, Pentin-1, from Pentaelethra SEQ ID NOS:1 and 2.
Figure 2 provides the amino acid and nucleotide sequence of the CDNA sequence of Pentin-1, optimized for enhanced expression SEQ ID
NOS:3 and 4.
Figure 3 provides the amino acid sequence of the Pentin-1 protein with the underlined portion representing the putative signal sequence. The AFS
residues immediately following the signal sequence are the first three residues of the mature protein. The ASK residues beginning five residues from the AFS start of the mature protein designates the region of apparent mature amino terminus of pentin-1 expressed as full length protein and proteolyzed in maize roots.
Figure 4 provides the expression cassette for expression of Pentin-1 sequences.
DETAILED DESCRIPTION OF THE INVENTION
Compositions and methods for controlling pests, particularly plant pests, are provided. In particular, novel pesticidal proteins are provided.
The proteins are purified from members of the family Leguminosae, particularly the Leguminous genus Pentaclethra, more particularly the species P.
macrophylla and P. macroloba.
In accordance with the invention, the pesticidal proteins produced by members of the genus Pentaclethra can be isolated by methods known in the art. Methods for protein isolation include conventional chromatography, including gel-filtration, ion-exchange, and immunoaffinity chromatography, by high-performance liquid chromatography, such as reversed-phase high-performance liquid chromatography, ion-exchange high-performance liquid chromatography, size-exclusion high-performance liquid chromatography, high-performance chromatofocusing and hydrophobic -S-interaction chromatography, etc., by electrophoretic separation, such as one-dimensional gel electrophoresis, two-dimensional gel electrophoresis, etc.
See for example Current Protocols in Molecular Biology Vols. I and 2, Ausubel et al. (eds.), John Wiley & Sons, NY (1988) , Once purified protein is isolated, the protein, or the polypeptides of which it is comprised, can be characterized and sequenced by standard methods known in the art. For example, the purified protein, or the polypepddes of which it is comprised, may be fragmented as with cyanogen bromide, or with proteases such as papain, chymotrypsin, trypsin, lysyl-C
endopeptidase, etc. (Oike et al. (1982) .I. Biol. Chem. 257:9751-9758; Liu et al.. (1983) Int. J. Pept. Protein Res. 21:209-215). The resulting peptides are separated, preferably by HPLC, or by resolution of gels and electroblotting onto PVDF membranes, and subjected to amino acid sequencing. To I S accomplish this task, the peptides are preferably analyzed by automated sequenators. It is recognized that N-terminal, C-terminal, or internal amino acid sequences can be determined. From the amino acid sequence of the purified protein, a nucleotide sequence can be synthesized which can be used as a probe to aid in the isolation of the gene encoding the pesticidal protein.
In the same manner, antibodies raised against partially purified or purified peptides can be used to determine the spatial and temporal distribution of the protein of interest. Thus, the tissue where the protein is most abundant, and possibly more highly expressed can be determined and expression libraries constructed. Methods for antibody production are known in the art. See, for example Antibodies, A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988), and the references cited therein. See also, Radka et al. (1983) J. Immunal. 128:2804;
and Radka et al. (1984) Immunogenetics 19:63. Such antibodies can be used to isolate proteins with similar binding domains and the proteins tested for activity against insect pests of interest_ It is recognized that any combination of methods may be utilized to purify proteins having pesticidal properties. As an isolation protocol is being determined, the pesticidal activity can be tested for each fraction of material obtained after each purification step.
Such purification protocols will result in a substantially purified protein fraction. By "substantially purified" or "substantially pure" is intended protein which is substantially free of any compound normally associated with the protein in its natural state. "Substantially pure" preparations of protein can be assessed by the absence of other detectable protein bands following SDS-PAGE as determined visually. or by densitometry scanning.
Alternatively, the absence of other amino-terminal sequences or N-terminal residues in a purified preparation can indicate the level of purity. Purity can be verified by rechromatography of "pure" preparations showing the absence of other peaks by ion exchange, reverse phase or capillary electrophoresis.
The terms "substantially pure" or "substantially purified" are not meant to exclude artificial or synthetic mixtures of the proteins with other compounds.
1 S The terms are also not meant to exclude the presence of minor impurities which do not interfere with the biological activity of the protein, and which may be~present, for example, due to incomplete purification.
From fragments of the protein, the entire nucleotide sequence encoding the protein can be determined by PCR experiments. Likewise, fragments obtained from PCR experiments can be used to isolate cDNA sequences from expression libraries. See, for example, Molecular Cloning, A Laboratory Manual, Second Edition, Vols. 1-3, Sambrook et al. (eds.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY ( 1989), and the references cited therein.
In this manner, proteins and the nucleotide sequences encoding such proteins can be isolated which are inhibitory or toxic to particular insect species. Such proteins and nucleotide sequences of the invention can be utilized to protect plants from pests, including insects, fungi, bacteria, nematodes, viruses or viroids, and the like, particularly insect pests. In particular, proteins and nucleotide sequences which are inhibitory or toxic to insects of the order Coleoptera can be obtained.
Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera. Insect pests of the invention for the major crops include: Maize: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zea, corn earworm; Spodoptera frugiperda, fall armyworm; Diatraea grandiosella, southwestern corn borer;
Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea saccharalis, surgareane borer; Diabrotica virgifera, western corn rootworm; Diabrotica barberi, northern corn rootworm; Diabrotica undecimpunctata howardi, spotted cucumber beetle, Melanotus spp., wireworms; Cyclocephala borealis, northern masked chafer (white grub); Cyclocephala immaculata, southern masked chafer (white grub); Popillia japonica, Japanese beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug;
Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis, corn root I S aphid; Blissus leucopterus, chinch bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratory grasshopper; Delia platura, seedcorn maggot; Agromyza parvicornis, corn blotch leafminer; Anaphothrips obscrurus, grass thrips; Solenopsis milesta, thief ant; Tetranychus urticae, twospotted spider mite; Busseola fusca; African Maize Stem Borer (AMB);
Sesamia calamistis, African Pink Borer (APB); Eldana sacchharina, African Sugarcane Borer (ASB); Chilo partellus, Sorghum Stem Borer (SSB); Ostrinia furnacalis, Oriental Corn Borer (OCB); Sesamia nonagrioides, Corn borer in Europe/N. Africa; Sor~um: Chilo partellus, sorghum borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus, lesser cornstalk borer; Agrotis subterranea, granulate cutworm;
Phyllophaga crinita, white grub; Eleodes,~ Conoderus, and Aeolus spp., wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis;
corn leaf aphid; Sipha (lava, yellow sugarcane aphid; Blissus leucopterus, chinch bug; Contarinia sorghicola, sorghum midge; Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, twospotted spider mite; Schizaphis graminum, Greenbug (aphid); Wheat: Pseudaletia unipunctata, army worm;

_g_ Spodoptera frugiperda, fall armyworm; Elasmopalpus ~ lignosellus, lesser cornstalk borer; Agrotis orthogonia, plae western cutworm; Oulema melanopus, cereal leaf beetle; Hypera punctata, clover Ieaf weevil; Diabrotica undecimpunctata howardi, spotted cucumber beetle; Russian wheat aphid;
S Schizaphis graminum, greenbug; Sitobion avenae, English grain aphid;
Melanoplus femurrubrum, redlegged grasshopper; Melanoplus dif, f'erentialis, differential grasshopper; Melanoplus sanguinipes, migratory grasshopper;
Mayetiola destructor, Hessian fly; Sitodiplosis mosellana, wheat midge;
Meromyza americana, wheat stem maggot; Hylemya coarctata, wheat bulb fly;
Frankliniella fusca, tobacco thrips; Cephus cinctus, wheat stem sawfly;
Eriophyes tulipae, wheat curl mite; Sunflower: Suleima helianthanu, sunflower bud moth; Homeosoma ellectellum, sunflower head moth;
Zygoramma exclamationis, sunflower beetle; Bothyrus gibbosus, carrot beetle;
Neolasioptera murtfeldtiana, sunflower seed midge; Cochylis hospes, banded sunflower moth; Rachiplusia nu, agentina looper; Smicronyx fulvus, red sunflower seed weevil; Cylindrocopturus adspersus, spotted sunflower stem weevil; Cotton: Heliothis virescens, tobacco budworm; Helicoverpa zea, bollworm; Spodoptera exigua, beet armyworm; Pectinophora gossypiella, pink bollworm; Anthonomus grandis, boll -weevil; Aphis gossypii, cotton aphid;
Pseudatomoscelis seriatus, cotton fleahopper; Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris, tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus dif, ferentialis, differential grasshopper; Thrips tabaci, onion thrips; Franklinkiella fusca, tobacco thrips;
Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, twospotted spider mite; Rice: Diatraea saccharalis, sugarcane borer;
Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm;
Colaspis brunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil; Sitophilus oryzae, rice weevil; Nephotettix nigropictus, rice leafhoper;
Blissus leucopterus, chinch bug; Acrosternum hilare, green stink bug;
Soybean: Pseudoplusia includens, soybean looper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypena scabra, green cloverworm; Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm; Heliothis virescens, cotton boll worm; Helicoverpa zea, cotton bollworm; Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peach aphid; Empoasca , fabae, potato leafhopper; Acrostermrm hilare, green stink bug; Melanoplus femurrubrum, redlegged grasshopper;
Melanoplus d~erentialis, differential grasshopper; Delia platura, seedcorn maggot; Sericothn'ps variabilis, soybean thrips; Thrips tabaci, onion thrips;
Tetrarrychus turkestani, strawberry spider mite; Tetranychus urticae, twospotted spider mite; B, arley: Ostrinia nubilalis, European corn borer;
Agrotis ipsilon, black cutworm; Schizaphis graminum, greenbug; Blissus l0 leucopterus, chinch-bug; Acrosternum hilare, green stink bug; Euschistus servos, brown stink bug; Delia platura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobia laterrs, brown wheat mite; Oil Seed Rape:
Brevicoryne brassicae, cabbage aphid; Flea beetle, Phyllotreta spp.; Bertha Anmyworm; Mamestra configurata; Diamondback Moth; Plutella xylostella;
Alfalfa: alfalfa looper, Autographa californica; alfalfa snout beetle, Otiorhynchus ligusticii; alfalfa caterpillar, Colias eurytheme; alfalfa blotch leafrunner, Agronyza Jrontella; Egyptian alfalfa weevil, Hypera brunneipeonis;
meadow spittlebug, Philaerius spumarius; spotted alfalfa aphid, Theriophis meculata; clover leaf weevil, Hypera punctata; pea aphid, Acyrthosiphon pisum; blue alfalfa aphid, Acyrthosiphor kondoi; green cloverworm, Plathypena scabia; clover root curculio, Sitona hispidulus; alfalfa seed chalcid, Brachophagus roddi; tarnished plantbug, Lygus lineolaris; Say stink bug, Chlorochroa sayi; velvetbean caterpillar, Anlicarsia friegiperda, alfalfa weevil;
Hypera postica; fall armyworm, Spodoptera; potato leafhopper, Empoasca jabae; soybean looper, Psuedolusia includens; Three cornered alfalfa hopper, Spissistilus festinus; See, for example, Manya B. Stoetzel (1989) Common Names of Insects & Related Or anisms. Entomological Society of America.
The nucleotide sequences of the invention can be used to isolate other homologous sequences in other plant species, particularly other Leguminous species. Methods are readily available in the art for the hybridization of nucleic acid sequences. Coding sequences from other plants may be isolated according to well known techniques based on their sequence homology to the coding sequences set forth herein. In these techniques all or part of the known coding sequence is used as a probe which selectively hybridizes to other pesticidal coding sequences present in a population of cloned genomic DNA
fragments or cDNA fragments (i.e. genomic or cDNA libraries) from a chosen organism.
For example, the entire Pentin-1 sequence or portions thereof may be used as probes capable of specifically hybridizing to corresponding coding sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique and are preferably at least about 10 nucleotides in length, and most preferably at least about 20 nucleotides in length. Such probes may be used to amplify Pentin-1 coding sequences from a chosen organism by the well-known process of polymerase chain reaction (PCR). This technique may be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of Pentin-1 coding sequences in an organism.
Such techniques include hybridization screening of plated DNA
libraries (either plaques or colonies; see, e.g. Sambrook et al., Molecular Cloning, eds., Cold Spring Harbor Laboratory Press ( 1989)) and amplification by PCR using oligonucleotide primers corresponding to sequence domains conserved among the amino acid sequences (see, e.g. Innis et al., PCR
Protocols, a Guide to Methods and Applications, eds., Academic Press ( 1990)).
For example, hybridization of such sequences may be carried out under conditions of reduced stringency, medium stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 35-40%
fonnamide with Sx Denhardt's solution, 0.5% SDS and ix SSPE at 37°C;
conditions represented by a wash stringency of 40-45% formamide with Sx Denhardt's solution, 0.5% SDS, and 1 x SSPE at 42 ° C; and conditions represented by a wash stringency of 50% formamide with Sx Denhardt's solution, 0.5% SDS and 1 x SSPE at 42 ° C, respectively), to DNA
encoding the insecticidal genes disclosed herein in a standard hybridization assay. See J.

Sambrook et al., Molecular Cloning, A Laboratory Manual 2d Ed. (1989) Cold Spring Harbor Laboratory.
The terms "stringent conditions" or "stringent hybridization conditions"
includes reference to conditions under which a probe will hybridize to its target sequence, to a delectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing).
Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that tower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1,000 nucleotides in length, preferably less than about 500 nucleotides in length, typically from about SO to about 300 nucleotides in length.
~ Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M
Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 for short probes (e.g., 10 to 50 nucleotides) and at least about 60 ° C for long probes (e.g., greater than SO nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCI, 1% SDS (sodium dodecyl sulphate) at 37°C, and a wash in 1X to 2X SSC (20X SSC= 3.0 M
NaC 1 /0.3 M trisodium citrate) at 50 ° C to 5 5 ° C.
Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M
NaCI, 1% SDS at 37°C, and a wash in O.SX to 1X SSC at 55°C
to 60°C.
Exemplary high stringency conditions include hybridization in 50%
formamide, 1 M NaCI, 1% SDS at 37°C, and a wash in O.1X SSC at 60°C to 65 ° C.
Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem. 138:267-284 (1984): Tm =
81.SC + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent rations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1 ° C for each 1 % of mismatching;
thus, Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. ror example, if sequences with > 90%
identity are sought, the Tm can be decreased 10 ° C. Generally, stringent conditions are selected to be about 5 ° C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength 1 S and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4 ° C
lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8 or 9 or i 0 ° C
lower than the thermal melting point (Tm) ; low stringency conditions can utilize a hybridization and/or wash at 1 I , 12, 13, I 4, 15, or 20 ° C lower than the thermal melting point (Tm) Using the equation, hybridization and wash compositions, and desired Tm those of ordinary skill will understand that variations in the stringency of hybridization andlor wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45 ° C (aqueous solution) or 32 ° C (formamide solution) it is preferred to increase the SSC concentration so that ~a higher temperature can be used.
An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology --Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al.., Eds., Greene Publishing and Wiley-Interscience, New York (1995).
In general, sequences which code for Pentin-1 and other insecticidal proteins of the invention and hybridize to the gene disclosed herein will be at least about 50% homologous, about 70% homologous, up to about 85%
homologous or more up to about 90% to about 95% homologous with the disclosed sequence. That is, the sequence similarity of sequences may range, sharing at least about SO%, about 70%, and about 85% up to about 90% to 95% sequence similarity.
The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence'', (b) "comparison window", (c) "sequence identity", (d) "percentage of sequence identity", and (e) "substantial identity".
(a) As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
(b) As used herein, "comparison window" means includes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100 or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.
Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 ( 1981 ); by the homology alignment algorithm of Needleman and Wunsch, J.

Mol. Biol. 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444 ( 1988); by computerized implementations of these algorithms, including, but not limited to: CLUSTAL
in the PC/Gene program by Intelligenetics, Mountain View, California, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Groups (GCG), 575 Science Drive, Madison, Wisconsin, USA; the CLUSTAL program is well described by Higgins and Sharp, Gene 73:237-244 ( I 988); Higgins and Sharp, CABIDS

5:151-153 (1989); Corpet et al., Nucleic Acids Research 16:10881-90 (1988);
Huang, et al., Computer Applications in the Biosciences 8:155-65 ( 1992), and Pearson, et al., Methods in Molecular Biology 24:307-331 (1994). The BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York ( 1995).
As those of ordinary skill in the an will understand, BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A
number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem. 17:149-163 (1993) and XNU (Claverie and States, Comput.
Chem., 17:191-201 (1993)) low-complexity filters can be employed alone or in combination.
(c) As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by co:iservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of l and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California, USA).
(d) As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions {i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

(e) (i) The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%
sequence identity, preferably at least 80%, more preferably at least 90% and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences fur these purposes normally means sequence identity of at least 60%, more preferably at least 70%, 80%, 90%, and most preferably at least 95%.
Another indication that nucleotide sequences are substantially identical as if two molecules hybridize to each other under stringent conditions.
However, nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the. polypeptide encoded by the second nucleic acid.
(e)(ii) The terms "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, more preferably 85%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Peptides which are "substantially similar" share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes.
It is recognized that the pesticidal proteins may be oligomeric and will vary in molecular weight, number of promoters, component peptides, activity against particular pests, and in other characteristics. However, by the methods set forth herein, proteins active against a variety of pests may be isolated and characterized. Of particular interest are proteins which are active against corn rootworm (CRW). Thus, the purified or partially purified proteins of the invention are tested for insecticidal activity against corn rootworm, including Diabrolica barberi (Northern), D. undecimpunctata howardi (Southern), .and D. virg fera vergifera (Western). In this manner, one protein designated Pentin-1 has been isolated which has insecticidal activity to com rootworm.
Pentin-1 is a glycosylated protein of approximately 45 to about 50 kDal. The amino acid and nucleotide sequence of the Pentin-1 protein is given in Figure 1 and SEQ ID NOS: 1 and 2.
The highest concentration of Pentin-1 in the plant appears to be in mature seeds. The protein is heat stable and has an LC50 of approximately 10 pg/ml of diet against com rootworm.
The Pentin-1 and other proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
For example, amino acid sequence variants of the pesticidal proteins can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, T.
(1985) Proc. Natl. Acad Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra (eds.) Techniques in Molecular Biology, MacMillan Publishing Company, NY
( 1983) and the references cited therein. Thus, the genes and nucleotide sequences of.the invention include both the naturally occurring sequences as well as mutant forms. Likewise, the proteins of the invention encompass both naturally occurring proteins as well as variations and modified forms thereof.

Such variants will continue to possess the desired pesticidal activity.
Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure.
See, EP Patent Application Publication No. E P o o 7 5 4 4 4 ( 19 8 3 ) .
In this manner, the present invention encompasses the pesticidal proteins as well as components and fragments thereof. That is, it is recognized that component promoters, polypeptides or fragments of the proteins may be produced which retain pesticidal activity. These fragments include truncated sequences, as well as N-terminal, C-terminal, internal and internally deleted amino acid sequences of the proteins.
Most deletions, insertions, and substitutions of the protein sequence are not expected to produce radical changes in the characteristics of the protein.
However, when it is difficult to predict the exact effect of the substitution, 1 S deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by insect toxicity assay.
The nucleotide sequences can be used in DNA shuffling protocols.
DNA shuffling is a process for recursive recombination and mutation, performed by random fragmentation of a pool of related genes, followed by reassembly of the fragments by primerless PCR See, for example, Stemmer, W.P.C. (1994) Proc. Natl. Acad Sci. USA 91:10747-10751; Stemmer, W.P.C.
(1994) Nature 370:389-391; Zhang et al. (1997) Proc. Nall. Acad Sci. USA
94:4504-4509; and PCT Publication No. 96119256. An advantage of DNA
shuffling of a rational design is that shuffling can optimize the function of genes without first determining which gene product is rate limiting. The present invention provides methods for sequenced shuffling utilizing polypeptides of the invention, and compositions resulting therefrom.
Generally, sequenced shuffling provides a means for generating libraries of polynucleotides having a desired characteristic which can be selected or screened for. Libraries of recombinant polypeptides are generated from a population of related sequence polypeptides that comprise sequenced regions which have substantial sequence identity and can be homologously recombined in vitro or in vivo.
The population of sequenced-recombined polynucleotides comprises a subpopulation of polynucleotides which' possess desired or advantageous characteristics and can be selected by a suitable selection or screening method.
The characteristics can be any property or attribute capable of being selected for or detected in a screening system, and may include properties of: an encoded protein, a transcriptional element, a sequence controlling transcription RNA processing, RNA stability, chromatin confirmation, translation, or other expression property of a gene or transgene, a replicative element, a protein-binding element, or the like, such as any feature which confers a selectable or detectable property. In some embodiments, the selected characteristic will be an increased Km and/or Kcat over the wild-type protein as provided herein.
In other embodiments, a protein or polynucleotide generated from sequenced shuffling will have a ligand binding affinity greater than the non-shuffled wild-type polynucleotide. The increase in such properties can be at least 110%, 120%, I 30%, 140% or at least 1 SO% of the wild-type value.
Pentin-I is a member of a broader gene family of esterases, and more specifically lipid acyl hydrolases as determined by sequence similarity. Gene shuffling is a method that can improve or alter a biological activity of a given gene product. Gene shuffling, in conjunction with a selection strategy, can be used to improve properties such as substrate specificity, solubility, temperature and pH optima of a protein or enzyme by directed molecular evolution. In the case of Pentin-1 toxicity toward insects as determined by the lethal concentrations is a most relevant parameter.
Gene shuffling can be applied to a single gene which introduces mutations within that gene at a given frequency. Combinations of synergistic mutations can then be selected by subsequent generations of gene shuffling from the primary mutant population. This approach can be applied to Pentin-I .
Alternatively, different members of gene families that are already encoded by divergent but related sequences can be used for gene shuffling.

These could include but not be limited to Pentin-1, from Pentaclethra and an expressed sequence tag from maize identified as SC'9 that encodes a cDNA
that is_about 57% identica~to Pentinrl.at the nucleotide level. See U.S. Patent No. 5,824,864. Concomitantly mutations will also be introduced by gene shuffling further contributing to the genetic diversity. Then synergistic combinations of fusions between the members of the gene family and newly introduced mutations can be selected by directed molecular evolution stategies.
Lipid acyl hydrolases comprise a diverse multigene family that a l0 conserved across many plant species. The enzymes exhibit hydrolyzing activity for many glyco- and phospholipids. Substrates include monogalactosyldiacylglycerol, acylsterylgucoside, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, as well as many other lipid substrates. Similarly membrane composition of various insects as well as plants can vary from species to species and can be affected by diet or growth conditions. Consequently, the activity of a given lipid acyl hydrolase for a given substrate could affect both specificity and potency. Altered substrate specificity could be one parameter for selection of products of gene shuffling.
2 o Solubility and protein stability could also be selected from shuffled gene products. Insecticidal proteins are active iw the harsh environment of the insect gut lumen. Their proteins are digested by proteases, and affected by reducing or oxidizing conditions that vary according to the insect species tested. The solubility and stability of lipid acyl hydrolases both in the 2 5 transgenic plant and in the insect gut lumen could affect biological activity and could be altered through gene shuffling strategies.
Conditions for the enzyme reaction such as pH and temperature optima may also affect the insecticidal activity of the Pentin-1. The gut pH of corn rootworm is 5_5-6Ø Selection of shuffled Pentm-1 gene products for 3 0 enzymatic activity toward lipid substrates in this pH range is another parameter that could affect toocity_ Thus, the pentin sequence of the present invention can be utilized in gene shuffling experiments with other lipid hydrolases such as patatins, and in particular with SC9.
The proteins or other component poiypeptides described herein may be used alone or in combination with other proteins or agents to control different insect pests. Other insecticidal proteins include those from Bacillus, including 8-endotoxins and vegetative insecticidal proteins, as well as protease inhibitors (both serine and cysteine types), lectins; a-amylases, peroxidases, cholesterol oxidase, and the like.
In one embodiment, expression of the proteins of the invention in a transgenic plant is accompanied ~ by the expression of one or more Bacillus thuringiensis (Bt) 8-endotoxins. This co-expression of more than one insecticidal principle in the same transgenic plant can be achieved by genetically engineering a plant to contain and express all the genes necessary.
Alternatively, a plant, Parent l, can be genetically engineered for the expression of proteins of the invention. A second plant, Parent 2, can be genetically engineered for the expression of other principles, such as a Bt 8-endotoxin. By crossing Parent 1 with Parent 2, progeny plants can be obtained which express all the genes present in both Parents 1 and 2.
The present invention also encompasses nucleotide sequences from organisms other than Pentaclethra, where the proteins cross-react with antibodies raised against the proteins of the invention or where the nucleotide sequences are isolatable by hybridization with the nucleotide sequences of the invention. The proteins isolated or those encoded by such nucleotide sequences can be tested for pesticidal activity. The isolated proteins can be assayed for pesticidal activity by the methods disclosed herein or others well-known in the art.
In another embodiment, the proteins of the invention can be used in combination with seed coatings available in the art. In this manner, transformed seed are coated with applications of available insecticide sprays or powders. Such insecticides are known in the art. See, for example, U.S.
Patent Nos. 5,696,144; 5,695,763; 5,420,318; 5,405,612; 4,596,206; 4,356,934;

-22_ 4,886,541; etc.
Once the nucleotide sequences encoding the pesticidal proteins of the invention have been isolated, they can be manipulated and used to express the protein in a variety of hosts including other organisms, including microorganisms and plants.
The proteins of the invention may be used for protecting agricultural crops and products from pests by introduction via a suitable vector into a microbial host, and said host applied to the environment or plants.
Microorganism hosts may be selected which are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops of interest. These microorganisms are selected so as to be capable of successfully competing in the particular environment with the wild-type microorganisms, provide for stable maintenance and expression of -the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.
The proteins of the invention can be used in expression cassettes for expression in any host of interest. Such expression cassettes will comprise a transcriptional initiation region linked to the gene encoding the pesticidal gene of interest. Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes suitable for the particular host organism.
The tr~anscriptional initiation region, the promoter, may be native or analogous or foreign or heterologous to the host. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. By foreign is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. As used herein a chimeric gene comprises a coding sequence operably linked to transcription initiation region which is heterologous to the coding sequence.
While any promoter or promoter element capable of driving expression of a coding sequence can be utilized, of particular interest for expression in plants are root promoters (Bevan et al. (1993) in Gene Conservation and Exploitation. Proceedings of The 20th Stadler Genetics Symposium Gustafson et al. (eds.), Plenum Press, New York pp. 109-129; Brears et al.
(1991) Plant J. 1:235-244; Lorenz et al. (1993) Pant J. 4:545-554; U.S.
Patent Nos. 5,459,252; 5,608,149; 5,599,670);; pith (IJ.S. Patent Nos.
5,466,785; 5,451,514; 5,391,725); or other tissue specific and constitutive promoters (See, for example, U.S. Patent Nos. 5,608,149; 5,608,144;
5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142).
The transcriptional cassette will include in 5'-3' direction of transcription, a transcriptional and translational initiation region, a DNA
sequence of interest, and a transcriptional and translational termination region functional in plants. The termination region may be native with the 1 S transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al., (1991) Mol. Gen_ Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al.
(1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; Joshi et al. (1987) Nucleic Acid Res 15:9627-9639.
The nucleotide sequences encoding the proteins or polypeptides of the invention are particularly useful in the genetic manipulation of plants. In this manner, the genes of the invention are provided in expression cassettes for expression in the plant of interest. The cassette will include 5' and 3' regulatory sequences operably linked to the gene of interest_ The cassette may additionally contain at least one additional gene to be cotransformed or.linked and transformed into the organism. Alternatively, the genes) of interest can be provided on another expression cassette. Where appropriate, the genes) may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant preferred colons for improved expression. Methods are available in the art for synthesizing plant preferred genes. See, for example, U.S. Patent Nos. 5,380,831, 5,436, 391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498.
S Depending upon where the DNA sequence of interest is to be expressed, it may be desirable to synthesize the sequence with plant preferred colons, or alternatively with chloroplast preferred colons. The plant preferred colons may be determined from the colons of highest frequency in the proteins expressed in the largest amount in the particular plant species ~f interest. See; EPA 0359472; EPA 0385962; WO 91/16432; Perlak et al.
(1991) Proc. Natl. Aced Sci. USA 88:3324-3328; and Murray et al. (1989) Nucleic Acids Research 17:477-498. In this manner, the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used.
Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences which may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence may be modified to avoid predicted hairpin secondary mRNA structures.
The expression cassettes may additionally contain 5' leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include:
picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, O., Fuerst. T.R., and Moss, B. (1989) PNAS USA, 86:6126-6130); potyvirus leaders, for example, TEV leader {Tobacco Etch Virus) (Allison et al. (1986)); MDMV leader (Maize Dwarf Mosaic Virus) Virology, 154:9-20); and human immunoglobulin heavy-chain binding protein (BiP), (Macejak, D.G., and Sarnow, P. {1991) Nature, 353:94-94; untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S.A., and Gehrke, L., (1987) Nature, 325:622-625); tobacco mosaic virus leader (TMV), (Gallie, D.R. et al. (1989) Molecular Biology of RNA, pages 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel, S S.A. et al. (1991) Virology, 81:382-385). See also, Dells-Cioppa et al.
(1987) Plant Physiology, 84:965-968. Other methods known to enhance translation can also be utilized, for example, introns, and the like.
The genes of the present invention can be targeted to the chloroplast or amyloplast for expression. In this manner, where the gene of interest is not directly inserted into the chloroplast or amyloplast, the expression cassette will additionally contain a gene encoding a transit peptide to direct the gene of interest to the chloroplasts. Such transit peptides are known in the art. See, for example, Von Heijne et al. ( 1991 ) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; dells-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res Commun.
196:1414-1421; and Shah et al. (1986) Science 233:478-481.
The construct may also include any other necessary regulators such as nuclear localization signals (Kalderon et al. (1984) Cell 39:499-509; and Lassner et al. (1991) Plant Molecular Biology 17:229-234); plant translational consensus sequences (Joshi, C.P. (1987) Nucleic Acids Research 15:6643-6653), introns (Luehrsen and Walbot (1991) Mol. Gen. Genet. 225:81-93) and the like, operably linked to the nucleotide sequence of interest:
It is recognized that the protein can be expressed comprising the native signal sequence. See Figure 3. Alternatively, other signal sequences in the art, for example the barley alpha amylase signal sequence, may be utilized.
In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Towards this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resection, ligation, PCR, or the like may be employed, where insertions, deletions or substitutions, e.g. transitions and transversions, may be involved.
The compositions of the present invention can be used to transform any plant. In this manner, genetically modified plants, plant cells, plant tissue, S seed, and the like can be obtained. Transformation protocols may vary depending on the type of plant or plant cell, i.e. monocot or dicot, targeted for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al. ( 1986) Biotechrriques 4:320-334), electroporadon (Riggs et al. (1986) Proc. Natl. Acad Sci. USA, 83:5602-5606, Agrobacterium mediated transformation (Hinchee et al. (1988) Biotechnology, 6:915-921 ), direct gene transfer (Paszkowski et al. ( i 984) EMBO J., 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al., U.S. Patent No. 4,945,050; and, McCabe et al. (1988) Biotechnology, 6:923-926). Also see, Weissinger et al. (1988) Annual Rev. Genet., 22:421-477; Sanford et al. (1987) Particulate Science and Technology, 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean);
McCabe et al. (1988) BiolTechnology, 6:923-926 (soybean); Dana et al.
(1990) Biotechnology, 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad.
Sci. USA, 85:4305-4309 (maize); Klein et al. (1988) Biotechnology, 6:559-563 (maize); Klein et al. (1988) Plant Physiol., 91:440-444 (maize); Fromm et al.
(1990) Biotechnology, 8:833-839; Tomes et al. "Direct DNA transfer into intact plant cells via microprojectile bombardment, In: Gamborg and Phillips (eds) Plant Cell, Tissue and Organ Culture: Fundamental Methods, Springer-Verlag, Berlin, 1995 (maize); Hooydaas-Van Slogteren & Hooykaas (1984) Nature (London), 311:763-764; Bytebier et al. ( 1987) Proc. Natl. Acad Sci.
USA, 84:5345-5349 (Liliaceae); De Wet et al. (1985) In The Experimental Manipulation of Ovule Tissues, ed. G.P. Chapman et al., pp. 197-209.
Longman, NY (pollen); Kaeppler et al. ( 1990) Plant Cell Reports, 9:41 S-418;
and Kaeppler et al. ( 1992) Theor. Appl. Genet., 84:560-566 (whisker-mediated transformation); D=Halluin et al. (1992) Plant Cell, 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports, 12:250-255 and Christou and Ford (1995) Annals of Botany, 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology, 14:745-750 (maize via Agrobacterium tumeJ'acierrs) .
Where desirable, the plant plastid can be transformed directly. Stable transformation of plastids have been reported in higher plants, see, for example, SVAB et al. (1990) Proc. Natl. Acad Sci. USA 87:8526-8530;
SVAB & Maliga (1993) Proc. Natl. Acad Sci. USA 90:913-917; Staub &
Maliga (1993) Embo J. 12:601-b06. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent ,plastid-borne transgene by tissue-specif c expression of a nuclear-encoded and plastid-directed RNA pol5~nerase. Such a system has been reported in McBride et al.
(1994) Proc. Natl. Acad Sci. USA 91:7301-7305.
The cells which have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al.
(1986) Plant Cell Reports, 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting offspring having the desired phenotypic characteristic identified.
Two or more generations may be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved.
The proteins will be expressed in the transformed organisms in amounts to be toxic to the insects of interest or inhibitory to insect growth.
The following examples are offered by way of illustration and not by way of limitation.
Although the foregoing invention has been described in sole detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the described invention.
EXPERIMENTAL
Purification of Pentin-1 P. macroloba seeds were collected from the lowland moist forest of Costa Rica and transported to the inventors' laboratories where they were sliced, lyophilized and stored at -20'C prior to use. Frozen seeds were diced into smaller pieces and homogenized using a Brinkmann homogenizer. In a typical procedure, 10 grams of seed material was homogenized with 1-2 grams of insoluble polyvinylpyrrolidone and 50-100 ml of 10 mM sodium phosphate buffer, pH 7.5. The homogenate was then stirred at 4'C for 8-10 hours and centrifuged at 5,000 rpm for 15 minutes. The supernatant fluid was carefully decanted and poured through a single layer of Nfiracloth,Mand collected so as to avoid the transfer of lipid-like materials in the extract which have separated and solidified on the surface during centrifugation. The pellet was discarded and the collected liquid, which was still somewhat cloudy, was centrifuged a TM
second time at 18,000 rpm in a Sorvall SS-34 rotor, or its equivalent, for 30 minutes. The slightly turbid supernatant liquid, hereafter called the crude i5 extract, was collected and the pellet was discarded. A sample of the crude extract was saved for testing and the remainder was dialyzed using a 3,500 molecular weight cutoff (MWCO) membrane against five changes of 10 mM
sodium phosphate buffer, pH 7.5, at 3 ° C to 4 ° C. The ratio of dialysis fluid to extract was at least 20:1. Dialysis was continued for 8-16 hours per buffer exchange. The extract became quite turbid during dialysis as a result of protein precipitation. Therefore, the dialyzed extract was clarified by centrifugation at 18,000 rpm for 30 minutes to remove denatured proteins. The resulting material, after centrifugation, is hereafter called crude dialyzed extract. The crude and crude dialyzed extracts were analyzed for protein composition or content, and were found to contain a substance which was an irisecticidaIly active against corn rootworm (CRVi~ in biologicaH assays. The insecticide was found to be a protein or proteinaceous substance.
A 100-ml sample of the dialyzed crude extract was heated to about 80'C using a water bath and held at this temperature for about 5 minutes. The heated extract was then cooled below 25 ° C using an ice bath and, after cooling, centrifuged for 15-30 minutes at 18,000 rprn using a Sorval SS-34 rotor. The clear supernatant liquid was removed, saved and designated hereafter as heat-treated extract. The pelleted material was discarded. It was noted that the heat-treated extract sometimes exhibited a tendency to gel. The heat-treated extract was assayed protein for using the Bradford method with BSA as the standard and was found to possess insecticidal activity against CRW in biological assays.
A sample of the heated extract was fractionated and concentrated using ammonium sulfate. The sample was cooled using an ice bath and powdered ammonium sulfate, 0.6 g/ml sample, was slowly added with stirring. Once the ammonium sulfate addition was completed, the sample was maintained at ice bath temperatures for about 30 minutes. The sample was then centrifuged at 4 °C for 20 minutes at 18,000 rpm using a Sorval SS-34 rotor. The supernatant liquid and the pelleted material were separated, and the pelleted material was resolubilized in a minimum amount of 10 mM sodium phosphate buffer, pH 7.5, and dialyzed extensively against 10 mM sodium phosphate buffer, pH 7.5. The supernatant liquid and the resolubilized pelleted material were assayed for protein content by the Bradford method using BSA as the standard and tested for biological activity against CRW. The majority-of the Pentin-1 was found in the pelleted material and it was insecticidal against CRW. Alternatively, the volume of the heated extract was reduced by centrifugal concentration using Centricon'" or similar concentrating devices according to the manufacturer's directions.
The proteins were also fractionated by size-exclusion chromatography na on either a Pharmacia Sephacryl S-204 column or a Pharmacia Superase 12TM
column. Different column sizes were used depending on the amount of protein in the sample which was to be chromatographed. Generally, the volume of sample was no more than 0.5-~1% of the column volume. The column were equilibrated with at least two to three column volumes of 10 mM
sodium phosphate buffer, pH 7.5, before the sample was applied to the column. T'he proteins were eluted from the column with 10 mM sodium phosphate buffer, pH 7.5. Tlie fractions were assayed for protein content by the Bradford method using BSA as the standard and were bioassayed using com rootworm larvae. Crude or dialyzed extracts, heated extracts, fractions resolubilized after ammonium sulfate precipitation, and extracts or fractions concentrated by other methods can be chromatographed by this method. The biologically-active material was eluted just after the void volume, suggesting that the active material is of moderately high molecular weight. This result is consistent with estimates of size obtained using Centricon"' filtration devices with different molecular weight ranges. The latter indicated that the active material has a native molecular weight greater than 100 kDa, the probable result of combining a plurality of subunits of 40-55 ~ S kDa molecular weight.
The purity of the fractions was estimated after molecular weight determination using SDS-PAGE as described below. These fractions were essentially pure with one primary band detected with an estimated subunit molecular weight in the range of 4t)-55 ~ 5 kDa.
Heat-treated samples or samples which had been subjected to size-exclusion chromatography were fi-actionated by anion exchange chromatography using either a Pharrnacia Q Sepharos column or a Pharmacia Resowce Qmcolumn. Prior to placement of the sample on the column, the column was first washed with 25 mM Tris-HCI or suitable buffer containing 1 M NaCI, and then equilibrated with the same buffer without NaCI. The pH of the buffers used in the chromatography ranged between pH 4 and pH 10. For the purpose of illustrating the methods, chromatography using 25 mM Tris-HCI buffer, pH 9.0, is described herein. Prior to injecting the sample onto the column, the sample was dialyzed using a 3,500 MWCO membrane through 2-3 exchanges of 25 mM Tris-HCl buffer without 1 M NaCI. After placement on the column, the flow-through was collected and the column was washed with 25 mM Tris-HCI, pH 9Ø The wash was also collected- Tire column was then eluted with a gradient ranging from 25 mM Tris-HCI, pH 9.0, no NaCI to 25 mM Tris-HCI, pH 9.0, 1 M NaCI. All fractions collected were dialyzed with a minimum of two buffer exchanges against 10 mM sodium phosphate, pH 7.5. The flow-through, wash and the salt-eluted fractions were assayed for protein by the Bradford method using BSA as the standard and bioassayed using CRW. Active material was found in the flow-through and in fractions which were eluted between 0.2 and 0.5 M NaCI. To determine whether the capacity of the column was exceeded, resulting in additional materials passing through the column without binding. the active material in the flow-through was reapplied to the column after re-equilibration. Most of the W 280 nm absorbing material passed through the column. These S observations suggested this active material has different properties than the material which bound to the column and was eluted with the increasing increments of NaCI. The other buffers used were also suitable for anion exchange chromatography as known to those familiar with the art. Active material could also be purified by ration-exchange chromatography.
The Pentin-1 material was purified to near homogeneity by size-exclusion chromatography or anion-exchange chromatography. Minor protein bands were removed by high pressure liquid chromatography (HI'LC) using a reverse phase column prior to amino acid analysis and determination of the amino acid sequence.
The purity of the samples and the subunit molecular weight were determined by SDS-PAGE using 12% polyacrylamide gels and generally following the method of LaemmLi, Nature 227:684-685 (1970). Gels were stained with either Coomassie Blue 8250 using standard protocols or silver stained (Hammer et al., Phytochemistry 28:3019-3026 (1989)). By SDS-PAGE, the subunit molecular weight of the active substance was found to be in the range 40-55,000 + 5,000 Daltons.
In addition to the procedures described above, other procedures can be used for separating the active substance from a crude seed extract. For example, an extract can be subjected to isoelectric focusing (IEF) using the Rotofor~system (Bio-Rad). The Rotofor separates molecules on the basis of their pI or isoelectric point. Every molecule will have a specific charge, either positive or negative, at a specific pH. The Rotofor, using an electrical current, moves molecules through a pH gradient until they reach their pI; i.e., the pH
at which they have zero net charge. The molecule stops migrating at its pI
because it is no longer affected by the electrical current. The focusing chamber of the Rotofor is separated into twenty smaller chambers by permeable membranes. These twenty samples are removed simultaneously to ensure as little mixing as possible.
Typically, a sample is placed in the focusing medium, a buffered solution (see manufacturer's instructions) which includes 12.5°/ (w/v) glycerol and 2.5% of pH 3-10 Ampholytes (Bio-Rad). After focusing, the fractions are collected, the pH of each determined and each fraction dialyzed against I
M NaCI using a 3,500 MWCO membrane to remove the Ampholytes. The samples are then dialyzed against deionized water to remove the NaCI. Each fraction is lyophilized and resuspended in 0.4 ml of 10 mM NaCI. The Rotofor fractions containing active material can be determined by protein assay and bioassay with insect. larvae. The Rotofor fractions can then be subjected to further treatment or separation as described above.
Biolo~~ical Assays Bioassays were conducted using CRW neonate larvae reared on artificial diets containing Pentin-1 obtained from P. macroloba as described herein. The Pentin-1 may be from crude extract or purified as taught herein.
Pentin-1 was either topically applied to the diet surface or incorporated into the diet as taught by Czapla and Lang, J. Econo. Errto. 83(6):2480-2485 {1990). The culture tray used in the bioassays were divided into treatment groups. One or a plurality of Pentin-I preparations or fractions from the various separations were screened in each tray; each preparation or fraction being applied to a pluraliiy of cells. Each cell was infested with one or two neonate larvae. A MylaTMfilm with ventilation holes was affixed to the top of each tray to prevent escape and allow air exchange.
For the topical (overlay) assays, a 2% Pentin-1 containing solution was prepared in 0.1 M phosphate buffered saline (PBS), pH = 7.8. Seventy-five microliters of Pentin-1 buffer solution were pipetted onto the Stoneville diet medium in each cell. The culture tray was rotated to ensure equal distribution of the Pentin-I solution on the diet medium. The cells were infected and sealed as described above. The control was 75 pl of 0.1 M PBS (only) per cell.
For the diet incorporation assays, StoneviIle medium was prepared in standard fashion, but with only 90% of the prescribed water. Pentin-1 was added such that the amount in the diet was in the range of 1-5 pg/g. The control treatment consisted of 0.9 ml PBS buffer added to 8.1 g of medium.
The medium was poured into the cells and the cells were then infested and covered as described above. Insect weights (Weight or Avg. Wt.) were determined at Day 7 and are given in the tables.
IO Table A. Effect of Pentin-1 on Southern Corn Rootworm.
Sample Concentration (,Ly~/ml diet) % Mortality Control 0 Crude 1,000 100 Crude 400 15 Size Fraction 17 8 14 Size Fraction 18 8 45 Size Fraction 19 8 29 Notes: AS75 = Ammonium sulfate 75%
population Once Pentin-1 was purified and its insecticidal activity was established, cloning efforts were undertaken. The first step of the process was to determine by western blot analysis the temporal and spatial distribution of Pentin-1 in order to identify the plant or seed tissue or tissues most likely expressing this protein. Since Pentin-1 was not isolated in quantities which allowed for the production of antibodies, the protein was sequenced in order to permit the design of peptides for synthesis. The amino acid sequence data for Pentin-1 is shown below in Figure 1. Carboxy-terminus and the internal sequence of approximately forty percent of the Pentin-1 peptides was compiled from fifteen peptides purified from LysC and CNBr digestion of purified Pentin-1 protein. The NH2-terminal sequence was not identified during this process.

Antibodies were raised against five of the peptides. The synthetic peptides used to produce antibodies are listed below.
Synthetic Peptide No. 1 (SEO ID NO:S~
Met Ser Thr Ser Ala Ala Pro Ile Val Phe Pro Pro Tyr Tyr Phe Lys Note: Corresponds to amino acid numbers 213-228 of Figure i.
Synthetic Peptide No. 2 ySEO ID N0:61 Ala Leu Gln Pro Gln Asn Asn Tyr Leu Arg Gln Glu Try Asp Leu Asp Note: Corresponds to amino acid numbers 344-360 of Figure 1.
Synthetic Peptide No. 3 (SEO ID N0:7) Pro Asp Trp Val Val Ile Arg Ser Glu Ser Val Gly Lys Note: No correspondence to amino acids of Figure 1.
Synthetic Peptide No. 4 (SEO ID N0:8) Lys Ala Phe Val Asn Gly Val Tyr Phe Ile Asn Thr Tyr Asp Ser Ala Note: No correspondence to amino acids of Figure 1.
Synthetic Peptide KS (SEO ID N0:9) Asn Asn Tyr Leu Arg Ile Gln Glu Tyr Asp Leu Pro Pro Ala Leu Note: Corresponds to amino acid numbers 349-363 of Figure 1.
Western dot blots of Pentin-1, each of the synthetic peptides and an experimental protein designated SC9 were incubated with each of the antibodies. The incubation results indicated that the antibody raised against synthetic peptide KS (antibody anti-KS) and the antibody raised against synthetic peptide 2 (antibody anti-2) recognized Pentin-1. Western blots of Pentaclethra macroloba tissue extracts treated with anti-KS antibodies indicated that the greatest recognition was with mature seeds 30-40 mm in diameter or larger. The total RNA was isolated from these seeds.
Genomic DNA was isolated, codon-degenerate oligonucleotides based on peptides were used to PCR amplify genomic fragments. Exon sequence of the resulting clones was used to do RT-PCR with specific oligos, then RT-PCR experiments were performed to obtain at least a partial Pentin-1 cDNA
for probing the expression library. Information obtained from the sequencing of random cDNA clones from a P. macroloba immature seed library was used to generate a nascent codon usage table. The data obtained indicated the P.
macroloba tree has no strong codon usage bias and that the GC content is moderate. A matrix of degenerate forward and reverse primers corresponding to Pentin-1 peptides were selected for use. The forward primer sequence was WKR.LAGYFDV (Pentin-1 amino acid Nos. 76-86: Val Val Lys Arg Leu Ala Gly Tyr Phe Asp Val) (SEQ ID NO:10) and the reverse primer sequence was ENMENLEK, (Pentin-1 amino acid Nos. 372-379: Glu Asn Met Glu Asn Leu Glu Lys) (SEQ ID NO:11). Due to the small amount of tissue available, the initial primer testing was conducted using genomic DNA derived from P.
macroloba leaves. One of the sixty-four possible primer combinations yielded a 3.0 kb fragment which encoded the Pentin-1 peptide sequences. The forward and reverse primer pair were then used to amplify a 0.8 kb cDNA
fragment from the total RNA isolated from mature (30-40 mm) seeds.
Subsequent screening of the mature seed expression library with this 0.8 kb cDNA probe produced several related clones, one of which is a 1.4 kb clone that encodes twelve of the fifteen peptide sequences from Pentin-1 (SEQ ID
NO:1).
Western blots were performed with Pentiri-1, the Pentin-1 synthetic peptides, SC9 and BSA proteins after exposure to selected antibodies. The blots were treated with a 1/10,000 dilution of antibodies raised against each of the peptides and SC9. Each antibody recognized its antigen with no detectable cross-reactivity to BSA, the negative control. All the antibodies, except those raised against synthetic peptide number 1, recognized 1.0 micrograms of SC9.
Although the synthetic peptides KS and number 2 were 74% identical, anti-2 antibody did not recognize KS, but did detect Pentin-1 and SC9.
The nucleic acid sequence of the Pentin-1 clone was determined by standard procedures known to those skilled in the art. The cDNA sequence and the predicted Pentin-1 protein sequence is provided in Figure l and SEQ
ID NO:1.
Bioass~s of Cloned Material .
Bioassays were conducted against Western corn rootworm (WCR) utilizing sonicated E coli that had been transformed with one of several plasmids listed (Table 1 ). Transformed cells were grown in approximately 25-3 5 ml of TB broth. The cells were harvested after 24 h by centrifugation.
The pellet was resuspended in approximately 1 ml of PBS buffer and sonicated. The resulting mixture was then top loaded onto the surface of diet and then infested with neonate WCR larvae. Mortality was recorded after four days. A positive result indicated 100% mortality. A negative result indicated mortality less than 10%. A similar experiment involved the use of transformed cells grown on an agar plate. The cells were scraped off after sufficient growth, suspended in a small amount of PBS buffer and then the solution was incorporated into the insect diet. A 4-day bioassay was also conducted with mortality recorded.
Table 1 displays the results of two replicated bioassays. All cells transformed with putative negative (non-lethal WCR genes) plasmids did not cause any larval mortality in either test. These plasmids are P7725, P88126, and P11426. The two plasmids that contain the coding sequence for Pentin-1, but not any promoters to produce the actual protein, PGEM and P11394 did not display any WCR activity. However, all plasmids containing the coding region for Pentin 1 (SEQ ID NO:1) and a functional expression cassette displayed excellent activity against WCR larvae. All such treatments had 100% mortality. Preliminary western blot analysis indicated that a protein similar in size to Pentin-1 was present in these cell extracts, but not in negative control samples. Activity was seen in both types of cell preparation and bioassays.

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Transformation of Protoplasts Isolated from Corn Suspension Cells with Three Pentin-1 Gene Constructs for Gene Expression and for CRW
Bioassay I. Protoplast Transformation Protocol Established HiII (GS3) suspension cells were used to make protoplasts.
Cells were collected 3-4 days after subculture.
Cell digestion: Cells were digested in enzyme solution at 27 ° C
for 3-S
hours with SO-60 RPM shaking speed. The cell wall was digested with cellulase and pectolyase to release the protoplasts.
Io Harvesting of protoplasts: The digested material was passed through 30 mm filter and the protoplasts were recovered by centrifuging the filtrate at 1,000 RPM for 10 min.
The protoplast pellet was resuspended in 20m1 or 40 ml ICMC solution.
The protoplast density and total protoplast yield was determined by counting the number of protoplasts with a hemacytometer. The suspension was centrifuged to pellet the protoplasts. The protoplast pellet was suspended in MaMg transformation solution in a concentration of 2 million protoplasts per ml.
The solution in 2 ml quantities (about 4 million protoplasts) was 2o dispensed into 1 S-ml round-bottom tubes. Each tube was a replication. At least three replications were used for each Pentin-1 gene construct. The constructs included both native Pentin-1 and the optimized Pentin-1 sequence. See SEQ
ID NOS:I and 3, respectively. Plasmid DNA was added to the protoplast suspension in the tubes (15 mg plasmid DNA/million protoplasts) and mixed.
After a five minute incubation, 2 ml 40% polyethylene glycol (PEG-8000, Sigma) was added to the protoplast/DNA mixture (the final PEG
concentration is about 20%) and mixed by inverting tubes several times and incubated at room temperature for 20-30 min.
About 3 ml of WS salt solution was added to each tube. The tubes 3o were covered and gently inverted. This was repeated two times until the final volume was 13-14 ml. The suspension was centrifuged 8 min at 1,000 RPM.

3 ml FW medium was used to resuspend protoplasts. (See below}. Using plastic squeeze Pasteur pipette, one treatment (3 ml) was dispensed into two wells of a 6-well culture plate, sealed with parafilm, and incubated 24-48 hours in the dark, at 28 ° C.
After culture, the protoplasts were transferred into a I S ml tube using plastic squeeze Pasteur pipette and centrifuged 8 min at 500 RPM to pellet the protoplasts.
Protein analysis and Bioassay: One fifth to one quarter of the protoplast pellet in each replication for each transformation treatment was sampled for 1o analysis of Pentin-1 expression by Western blot. The remainder of the' protoplasrt pellet was used for bioassay. All replication samples from the same transformation treatment, that is transformed with the same Pentin-1 construct, were pooled and incorporated into diet for CRW bioassay. The bioassay results are provided in Table 2.

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H o G. 0. G. p. 0~.. pM.. a' ~2_ II. Pentin-1 Gene Constructs for Transformation P11184 - Ubi promoter::Pentin-1 (original full-length clone) P11335 - U6i promoter::Pentin-1 (partially modified gene) P11443 - Ubi promoter::moPentin-1 (optimized gene) S Solutions and Media Used for Transformation KMC solution - 1,000 ml KCl 8.65 g MgCl2-6H20 16.47 g CaCl2-2H20 12.50 g MES 0.5% S.0 g PH 5.8 with KOH

Filter sterilize MaMg transformation solution - 1,000 ml M mannitol 108.1 g 15 mM MgCl2-6H20 3.05 g 10 mM MES 1.95 g PH 5.7 Filter sterilize 40% PEG - 100 ml Add 40 g PEG to 60 ml MaMg transformation solution. Briefly microwave to dissolve PEG. Add more MaMg solution to final volume of 100 ml. Adjust to pH 7Ø Filter sterilize.
Enzyme solution for digesting suspension cell (Enzyme solution) Enzyme solution contains 3% cellulase RS and 0.3% pectolyase Y23 in protoplast solution.
Protoplast solution - 1,000 ml M mannitol 108.1 g 10 mM MES 1.95 g 1 mM CaCI2-2H20 147 mg 1 mM MgCl2-6H20 203 mg 1 % BSA (optional) 1 g PH 5.7 Filter sterilize WS salt solution - 1,000 ml 154 mM NaC! 9.0 g 125 mM CaCl2-2H20 18_56 g mM KCl 0.373 g S 5 mM Glucose 0.901 g PH 5.5 with KOH
Filter sterilize FW medium - 1,000 ml MS salts (Sigma M5519) 4.3 g sucrose 30.0 g ~~l 54.0 g Proline I.5 g 2,4-D 3.0 mg 1,OOOx BS Vitamins I ~

PH 5.8 Filter sterilize Transformation and Regeneration of Maize Callus Immature maize embryos from green house donor plants are bombarded with a plasmid containing the three Pentin-1 constructs plus a plasmid containing the selectable marker gene, PAT, (Wohlleben, W., Arnold, W., Broer, L, Hillemann, D., Strauch, E. and Puehler, A. "Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tue494 and its expression in Nicotiana tabacum" Gene 70:25-37 (1988) that confers resistance to the herbicide Bialophos by the following method:
Please note: All media recipes are in the Appendix.
Preparation of target tissue: The ears are surface sterilized in 30%
Chlorox bleach plus 0.5% Micro detergent for 20 minutes, and iinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate. These are cultured on 560L medium 4 days prior to bombardment, in the dark. The day of bombardment, the embryos are transferred to 560Y medium for 4 hours, arranged within the 2.5 cm target zone.
Preparation of DNA:

_ø4_ 100 pl prepared tungsten particles in water l 0 ~l ( 1 pg) DNA in TrisEDTA buffer ( 1 pg total) 100 pl 2.5 M CaCl2 ~l 0.1 M spermidine 5 Each reagent is added sequentially to the tungsten particle suspension, while maintained on the mufti-tube vortexer. The plasmids are adjusted for a final l : i ratio by size. The final mixture is sonicated briefly, and allowed to incubate under constant vortexing for ten minutes. After the precipitation period, the tubes are centrifuged briefly, liquid removed, washed with 5 ~0 ml 10 100% ethanol, and centrifuged 30 seconds. Again the liquid is removed, and 105 pl 100% ethanol added to the final tungsten particle pellet. For particle gun bombardment, the tungsten/DNA particles are briefly sonicated and 10 pl spotted onto the center of each macro-carrier and allowed to dry about 2 minutes before bombardment.
Particle Gun Treatment: The sample plates are bombarded at level #4 in particle gun #HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.
Subsequent treatment: Following bombardment; the embryos are kept on 560Y medium for 2 days then transferred to 5608 selection medium containing 3 mg/liter Bialophos, and sub-cultured every 2 weeks. After approximately IO weeks of selection, selection-resistant callus clones are sampled for PCR and fumonisin esterase TLC activity analysis. Positive lines are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to 2.5" pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots {1.6 gallon) and grown to rnaturity.

APPENDIX
Ingredient Unit Amount D-I H20 900.000 ml CHLJ (N6) Basal Salts (SIGMA C-1416) 1.600 g N6 Macronutrients lOX Stock ## 60.000 ml Potassium Nitrate 1.680 g BSH Minor Salts IOOOX ### 0.600 ml BSH Fe Na EDTA 100X 6.000 ml Eriksson's Vitamin Mix (IOOOX SIGMA-1511) 0.400 ml S & H Vitamin Mixture 100X Stock (53766) 6.000 Thiamine .HCL 0.4mg/ml 0.500 ml L-Proline 1.980 g Casein Hydrolystate (acid) 0.300 g Sucrose 20.000 g Glucose p,(pp 2,4-D 0.5 mglml 1.600 ml Geli-ite @ 2.~0. g Dicamba I mglml # 1.200 ml Silver Nitrate 2 mg/ml # 1.700 ml Directions:
@= Add after bringing up to volume ~ Add after sterilizing and cooling to temp.

Dissolve ingredients in polished D-I HZO in sequence Adjust to pH 5.8 Bring up to volume with polished D-I HZO after adjusting pH
Sterilize and cool to 60 ° C.
##~ Dissolve 1.660 g of Calcium Chloride Dihydrate in 950.000 ml of polished D-I HzO. Then dissolve 4.629 of Ammonium Sulfate; 4.000 g of Potassium Phosphate Monobasic KH2P04; 1.850 g of Magnesium Sulfate 7-HZO, MgS04, 7H20; and 28.300 g of Potassium Nitrate into sequence. Bring up to volume with polished D-I H20.
### = Dissolve 3.000 g of Boric Acid; 10.000 g of Manganous Sulfate Monohydrate; 0.250 g of Sodium Molybdate Dihydrate; and 0.750 g of Potassium Iodide in polished D-I HBO in sequence. Bring up to volume with polished D-I H20.
~ = Dissolve 3.700 g of Disodium EDTA Dihydrate and 2.790 g of Ferrous 1 S Sulfate 7-Hydrate into D-I H20. Bring up to volume with D-I H,O.
Total Volume (L) = 1.00 - ---Ingredient Amount Unit D-I H20 900.000 ml CHU (N6) Basal Salts (SIGMA C-1416) 1.600 g N6 Macronutrients lOX Stock ## 60.000 ml Potassium Nitrate 1.680 g BSH Minor Salts 1000X ### 0.600 ml BSH Fe Na EDTA 100X 6.000 ml Eriksson's Vitamin Mix (1000X SIGMA-1511) 0.400 ml S & H Vitamin Mixture IOOX Stock {53766) 6.000 ml Thiamine .HCL 0.4mg/rnl 0.500 ml L-Proline 1.980 g Casein Hydrolysate (acid) 0.300 g Sucrose 20.000 g Glucose 0.600 g 2, 4-D O.Smg/ml 1:600 ml Gelrite @ 2.000 g Dicamba I mg/ml # 1.200 ml Silver Nitrate 2mg/ml # 1.700 ml Bialaphos 1 mg/ml # 3.000 ml Directions:
@ = Add after bringing up to volume # = Add after sterilizing and cooling to temp.

Dissolve ingredients in polished D-I H20 in sequence Adjust to pH 5.8 Bring up to volume with polished D-I HZO after adjusting pH
Sterilize and cool to 60 ° C.
### = Dissolve 1.660 g of Calcium Chloride Dihydrate in 950.000 ml of polished D-I H20. Then dissolve 4.629 of Ammonium Sulfate; 4.000 g of Potassium Phosphate Monobasic KH2P04; 1.850 g of Magnesium Sulfate 7-H,O, MgSO,, 7H20; and 28.300 g of Potassium Nitrate into sequence. Bring up to volume with polished D-I H20.
### = Dissolve 3.000 g of Boric Acid; 10.000 g of Manganous Sulfate Monohydrate; 0.254 g of Sodium Molybdate Dihydrate; and 0.750 g of Potassium Iodide in polished D-I Hz0 in sequence. Bring up to volume with polished D-I H20.
= Dissolve 3.700 g of Disodium EDTA Dihydrate and 2.790 g of Ferrous Sulfate 7-Hydrate into D-I H,O. Bring up to volume with D-I H20.
Total Volume (L} = 1.00 Ingredient Amount Unit D-I H20 900.000 ml CHU (N6) Basal Salts (SIGMA C-1416) 1.600 g N6 Macronutrients lOX Stock ## 60.000 ml Potassium Nitrate 1.680 g BSH Minor Salts 1000X ### 0.600 ml BSH Fe Na EDTA IOOX 6.000 ml Eriksson's Vitamin Mix (IOOOX SIGMA-1511) 0.400 ml S & H Vitamin Mixture 100X Stock (S3766) 6.000 ml Thiamine .HCL 0.4mg1m1 0.500 ml Sucrose 20.000 g Glucose 0.600 g 2, 4-D O.Smg/ml 1.600 ml Gelrite @ 2.000 g Dicamba 1 mg/ml # I .200 ml Silver Nitrate 2mg/ml # 0.425 ml Bialaphos I mg/ml # 3.000 ml Directions:
@ = Add after bringing up to volume # = Add after sterilizing and cooling to temp.
Dissolve ingredients in polished D-I H20 in sequence Adj ust to pH S . 8 Bring up to volume with polished D-I HZO after adjusting pH

Sterilize and cool to 60 ° C.
## = Dissolve 1.660 g of Calcium Chloride Dihydrate in 950.000 ml of polished D-I HzO. Then dissolve 4.629 of Ammonium Sulfate; 4.000 g of Potassium Phosphate Monobasic KH2P04; 1.850 g of Magnesium Sulfate 7-H20, MgSO,,, 7H20; and 28.300 g of Potassium Nitrate into sequence. Bring up to volume with polished D-I H20.
### = Dissolve 3.000 g of Boric Acid; 10.000 g of Manganous Sulfate Monohydrate; 0.250 g of Sodium Molybdate Dihydrate; and 0.750 g.of Potassium Iodide in polished D-I H20 in sequence. Bring up to volume with polished D-I H20.
= Dissolve 3.700 g of Disodium EDTA Dihydrate and 2.790 g of Ferrous Sulfate 7-Hydrate into D-I H20. Bring up to volume with D-I HBO.
Total Volume (L) = 1.00 Ingredient Amount Unit D-I H20 800.000 ml CHU (N6) Basal Salts (SIGMA C-1416) 1.600 g N6 Macronutrients lOX Stock ## 60.000 ml Potassium Nitrate 1.680 g BSH Minor Salts 1000X ### 0.600 ml BSH Fe Na EDTA 100X- 6.000 ml Eriksson's Vitamin Mix (1000X SIGMA-1511) 0.400 ml S & H Vitamin Mixture 100X Stock (S3766) 6.000 ml Thiamine .HCL 0.4mg/ml 0.500 ml L-Proline 1.980 g Casein Hydrolysate (acid) 0.300 g Sucrose 120.000 g Glucose 0.600 g 2, 4-D O.Smg/ml 1.600 ml Gelrite @ 2.000 g Dicamba lmg/ml # 1.200 ml Silver Nitrate 2rng/ml # 1.700 ml Directions:
@ = Add after bringing up to volume # = Add after sterilizing and cooling to temp.
Dissolve ingredients in polished D-I H20 in sequence Adjust to pH 5.8 Bring up to volume with polished D-I HZO after adjusting pH
Sterilize and cool to 60 ° C.
### = Dissolve 1.660 g of Calcium Chloride Dihydrate in 950.000 ml of polished D-I HZO. Then dissolve 4.629 of Ammonium Sulfate; 4.000 g of S Potassium Phosphate Monobasic KH2P04; 1.850 g of Magnesium Sulfate 7-H20, MgSO,, 7H20; and 28.300 g of Potassium Nitrate into sequence. Bring up to volume with polished D-I H20.
## = Dissolve 3.000 g of Boric Acid; 10.000 g of Manganous Sulfate wMonohydrate; 0.250 g of Sodium Molybdate Dihydrate; and 0.750 g of Potassium Iodide in 950.000 ml of polished D-I HzO. in sequence. Bring up to volume with polished D-I H20.
= Dissolve 3.700 g of Disodium EDTA Dihydrate and 2.790 g of Ferrous Sulfate 7-Hydrate into 950.000 mI of D-I H,O. Bring up to volume with D-I
HzO.
1 S Total Volume (L) = I .00 Ingredient Amount Unit D-I HZO 950.000 ml MS Salts (GIBCO 11117-074) 4.300 g S Myo-Inositol 0.100 g MS Vitamins Stock Solution ## 5.000 ml Sucrose 40.000 g Bacto-Agar @ 6.000 g Directions:
@ = Add after bringing up to volume Dissolve ingredients in polished D-I H20 in sequence Adjust to pH 5.6 Bring up to volume with polished D-I H20 after adjusting pH
Sterilize and cool to 60 o C.
## = Dissolve 0.100 g of Nicotinic Acid; 0.020 g of Thiamine.HCL; 0.100 g of Pyridoxine.HCL; and 0.400 g of Glycine in 875.00 ml of polished D-I HZO in sequence. Bring up to volume with polished D-I HzO. Make in 400 ml portions. Thiamine.HCL & Pyridoxine.HCL are in Dark Descicator. Store for one month, unless contamination or precipitation occur, then make fresh stock.
Total Volume (L) = 1.00 Ingredient Amount Unit D-I HBO 950.000 ml MS Salts 4.300 g S Myo-Inositol 0.100 g MS Vitamins Stack Solution ## 5.000 ml Zeatin .Smg/ml 1.000 ml Sucrose 60.000 g Gelrite @ 3.000 g Indole Acetic Acid 0.5 mg/ml # 2.000 ml .1 mM Absissic Acid 1.000 ml Bialaphos lmglml # 3.000 ml Directions:
@ = Add after bringing up to volume 1 S Dissolve ingredients in polished D-I H20 in sequence Adjust to pH 5.6 Bring up to volume with polished D-I H20 after adjusting pH
Sterilize and cool to 60 o C.
Add 3.Sg/L of Gelrite for cell biology.
## = Dissolve 0.100 g of Nicotinic Acid; 0.020 g of Thiamine.HCL; 0.100 g of Pyridoxine.HCL; and 0.400 g of Glycine in 875.00 ml of polished D-I H,O in sequence. Bring up to volume with polished D-I H.,O. Make in 400 ml portions. Thiamine.HCL & Pyridoxine.HCL are in Dark Descicator. Store for one month, unless contamination or precipitation occur, then make fresh stock.
Total Volume (L) = 1.00 -SS-Ingredient Amount Unit D-I Water, Filtered 950:000 ml CHU (N6) Basal Salts (SIGMA C-1416) 4.000 g I

Eriksson's Vitamin Mix (1000X SIGMA-1511 0.400 ml Thiamine .HCL 0.4mg/ml 1.250 ml Sucrose 20.000 g .

2, 4-D O.Smg/ml 2.000 ml -L-Proline 2.880 g Gelrite @ 2.000 g Silver Nitrate 2mg/ml # 4.250 ml Directions:
@ = Add after bringing up to volume # = Add after sterilizing and cooling to temp.
1 S Dissolve ingredients in D-I HZO in sequence Adjust to pH 5.8 with KOH
Bring up to volume with D-I HBO
Sterilize and cool to room temp.
Total Volume (L) = 1.00 Ingredient Amount Unit D-I Water, Filtered 950.000 ml CHU (N6) Basal Salts (SIGMA C-1416) 4.000 g S Eriksson's Vitamin Mix (1000X SIGMA-1511 1.000 m1 Thiamine .HCL 0.4mg/ml 1.250 m1 Sucrose 30.000 g 2, 4-D O.Smg/Iril 4.000 ml Gelrite @ 3.000 g Silver Nitrate 2mg/ml # 0.425 ml Bialaphos lmg/ml # 3.000 ml Directions:
@ = Add after bringing up to volume # = Add after sterilizing and cooling to temp.
Dissolve ingredients in D-I H20 in sequence Adjust to pH 5.8 with KOH
Bring up to volume with D-I H20 Sterilize and cool to room temp.
Total Volume (L) = 1.00 Ingredient Amount Unit i D-I Water, Filtered 950.000 ml CHU (N6) Basal Salts (SIGMA C-1416) 4.000 g Eriksson's Vitamin Mix (1000X SIGMA-1511 1.000 ml Thiamine .HCL 0.4mg/ml 1.250 ml Sucrose 120.000 g 2,4-D O.Smg/rml 2.000 ml I

L-Proline 2.880 g Gelrite @ 2.000 g Silver Nitrate 2mg/ml # 4.250 ml Directions:
@ = Add after bringing up to volume # = Add after sterilizing and cooling to temp.
Dissolve ingredients in D-I H,O in sequence Adjust to pH 5.8 with KOH
Bring up to volume with D-I HZO
Sterilize and cool to room temp.
* * Autoclave less time because of increased sucrose*
Total Volume (L) = 1.00 Plasmids PHP11361 and PHP11511 were deposited with the American Type Culture Collection, Bethesda, Maryland, and given Accession Nos.
209026 and 209025, respectively. PHP11361 comprises the nucleotide sequence of the native Pentin-1 sequence. PHP 11 S 11 comprises the optimized Pentin-1 sequence.
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: PIONEER HI-BRED INTERNATIONAL INC. -AND- THE BOARD OF
REGENTS FOR THE UNIVERSITY OF OKLAHOMA
{ii) TITLE OF INVENTION: PROTEINS HAVING INSECTICIDAL ACTIVITIES AND
METHOD OF USE
{iii) NUMBER OF SEQUENCES: 11 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SMART & BIGGAR
(B) STREET: P.O_ BOX 2999, STATION D
(C) CITY: OTTAWA
(D) STATE: ONT
(E) COUNTRY: CANADA
(F) ZIP: K1P SY6 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
2 O {D) SOFTWARE: ASCII {text) (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:. CA 2,290,77_6 {B) FILING DATE: 23-NOV-1999 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/047,869 {B) FILING DATE: 29-MAY-1997 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 09/079,912 30 (B) FILING DATE: 08-MAY-1998 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SMART & BIGGAR
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: 62451-843 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613)-232-2486 (B) TELEFAX: (613)-232-8940 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1469 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Pentaclethra macroloba 2 O (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 31..1257 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:

Met Lys Ser Lys Met Ala Met Leu Leu Leu Leu Phe Cys Val Leu Ser Asn Gln Leu Val Ala Ala Phe Ser 30 l0 15 20 Thr Gln Ala Lys Ala Ser Lys Asp Gly.Asn Leu Val Thr Val Leu Ala Ile Asp Gly Gly Gly Ile Arg Gly Ile Ile Pro Gly Val Ile Leu Lys Gln Leu Glu Ala Thr Leu Gln Arg Trp Asp Ser Ser Ala Arg Leu Ala Glu Tyr Phe Asp Val Val Ala Gly Thr Ser Thr Gly Gly Ile Ile Thr Ala Ile Leu Thr Ala Pro Asp Pro Gln Asn Lys Asp Arg Pro Leu Tyr Ala Ala Glu Glu Ile Ile Asp Phe Tyr Ile Glu His Gly Pro Ser Ile Phe Asn Lys Ser Thr Ala Cys Ser Leu Pro Gly Ile Phe Cys Pro Lys Tyr Asp Gly Lys Tyr Leu Gln Glu Ile Ile Ser Gln Lys Leu Asn Glu Thr Leu Leu Asp Gln Thr Thr Thr Asn Val Val Ile Pro Ser Phe Asp Ile Lys Leu Leu Arg Pro Thr Ile Phe Ser Thr Phe Lys Leu Glu Glu Val Pro Glu Leu Asn Val Lys Leu Ser Asp Val Cys Met Gly Thr Ser GCA GCA CCA ATC GTA TTT CCT CCC,TAT TAT TTC AAG CAT GGA GAT ACT 678 Ala Ala Pro Ile Val Phe Pro Pro Tyr Tyr Phe Lys His Gly Asp Thr Glu Phe Asn Leu Val Asp Gly Ala Ile Ile Ala Asp Ile Pro Ala Pro Val Ala Leu Ser Glu Val Leu Gln Gln Glu Lys Tyr Lys Asn Lys Glu Ile Leu Leu Leu Ser Ile Gly Thr Gly Val Val Lys Pro Gly Glu Gly 250 255 .260 Tyr Ser Ala Asn Arg Thr Trp Thr Ile Phe Asp Trp Ser Ser Glu Thr Leu Ile Gly Leu Met Gly His Gly Thr Arg Ala Met Ser Asp Tyr Tyr GTT GGC TCACAT TTCAAA CTTCAA CCCCAG AACTACCTC CGA
GCC AAT

Val Gly SerHis PheLys AlaLeuGln ProGlnAsn AsnTyrLeu Arg Ile Gln GluTyr AspLeu AspProAla LeuGluSer IleAspAsp Ala Ser Thr GluAsn MetGlu AsnLeuGlu LysValGly GlnSerLeu Leu Asn Glu ProVal LysArg MetAsnLeu AsnThrPhe ValValGlu Glu Thr Gly GluGly ThrAsn AlaGluAla LeuAspArg LeuAlaGln Ile Leu Tyr GluGlu LysIle ThrArgGly LeuGlyLys IleSerLeu Glu Val Asp AsnIle AspPro TyrThrGlu ArgValArg LysLeuLeu Phe TGA AGTTGTTTC TCCTTG

AATAAATAAA AAATG TTTGGTT TGATGTCCTTGACTT TG CATATATGC

TAAAT TAATCG T

TGGC TCTATA CACCA AGATAAAT AAAGGTTAAT GTTTGAGGTA

AGAAG GC TWAARWAAAA

P~ ~~~AAAAAA AAAAAAAAAAAAAAACTC GA 14 69 p,~~

(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 408 amino acids 40 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Lys Ser Lys Met Ala Met Leu Leu Leu Leu Phe Cys Val Leu Ser Asn Gln Leu Val Ala Ala Phe Ser Thr Gln Ala Lys Ala Ser Lys Asp Gly Asn Leu Val Thr Val Leu Ala Ile Asp Gly Gly Gly Ile Arg Gly Ile Ile Pro Gly Val Ile Leu Lys Gln Leu Glu Ala Thr Leu Gln Arg Trp Asp Ser Ser Ala Arg Leu Ala Glu Tyr Phe Asp Val Val Aia Gly Thr Ser Thr Gly Gly Ile Ile Thr Ala Ile Leu Thr Ala Pro Asp Pro Gln Asn Lys Asp Arg Pro Leu Tyr Ala Ala Glu Glu Ile Ile Asp Phe Tyr Ile Glu His Gly Pro Ser Ile Phe Asn Lys Ser Thr Ala Cys Ser 2 0 Leu Pro Gly Ile Phe Cys Pro Lys Tyr Asp Gly Lys Tyr Leu Gln Glu_ 130 - 135 ' 140 Ile ile Ser Gln Lys Leu Asn Glu Thr Leu Leu Asp Gln Thr Thr Thr Asn Val Val Ile Pro Ser Phe Asp Ile Lys Leu Leu Arg Pro Thr Ile Phe Ser Thr Phe Lys Leu Glu Glu Val Pro Glu Leu Asn Val Lys Leu Ser Asp Val Cys Met Gly Thr Ser Ala Ala Pro Ile Val Phe Pro Pro Tyr Tyr Phe Lys His Gly Asp Thr Glu Phe Asn Leu Val Asp Gly Ala Ile Ile Ala Asp Ile Pro Ala Pro Val Ala Leu Ser Glu Val Leu Gln Gln Glu Lys Tyr Lys Asn Lys Glu Ile Leu Leu Leu Ser Ile Gly Thr Gly Val Val Lys Pro Gly Glu Gly Tyr Ser Ala Asn Arg Thr Trp Thr Ile Phe Asp Trp Ser Ser Glu Thr Leu Ile Gly Leu Met Gly His Gly 50 Thr Arg Ala Met Ser Asp Tyr Tyr Val Gly Ser His Phe Lys Ala Leu Gln Pro Gln Asn Asn Tyr Leu Arg Ile Gln Glu Tyr Asp Leu Asp Pro Ala Leu Glu Ser Ile Asp Asp Ala Ser Thr Glu Asn Met Glu Asn Leu Glu Lys Val Gly Gln Ser Leu Leu Asn Glu Pro Val Lys Arg Met Asn Leu AsnThr PheValVal GluGluThr Glu GlyThrAsn Ala 355 360Gly 365Glu Ala LeuAsp ArgLeuAla GlnIleLeu Glu GluLysIle Thr 370 375 Tyr 380 Arg Gly LeuGly LysIleSer LeuGluVal Asn IleAspPro Tyr 385 390 Asp 395 Thr Glu ArgVal ArgLysLeu LeuPhe (2) INFORMATION FOR SEQ ID N0:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1227 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "cDNA Pentin- 1 of optimized f or enhanced expression"

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Pentaclethr a croloba ma (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1227 (xi) SEQUENCE DESCRIPTION: ID
SEQ N0:3:

CTC CTC

Met Lys Ser Lys Met Ala Met LeuLeu PheCys ValLeuSer Leu Leu TCC ACC

Asn Gln Leu Val Ala Ala Phe GlnAla LysAla SerLysAsp Ser Thr GCC ATC

Gly Asn Leu Val Thr Val Leu AspGly GlyGly IleArgGly Ala Ile AAG CAG

Ile Ile Pro Gly Val Ile Leu LeuGlu AlaThr LeuGlnArg Lys Gln Trp Asp Ser Ser Ala Arg Leu Ala Glu Tyr Phe Asp Val Val Ala Gly Thr Ser Thr Gly Gly Ile Ile Thr Ala Ile Leu Thr Ala Pro Asp Pro Gln Asn Lys Asp Arg Pro Leu Tyr Ala Ala Glu Glu Ile Ile Asp Phe Tyr Ile Glu His Gly Pro Ser Ile Phe Asn Lys Ser Thr Ala Cys Ser Leu Pro Gly Ile Phe Cys Pro Lys Tyr Asp Gly Lys Tyr Leu Gln Glu Ile Ile Ser Gln Lys Leu Asn Glu Thr Leu Leu Asp Gln Thr Thr Thr Asn Val Val Ile Pro Ser Phe Asp Ile Lys Leu Leu Arg Pro Thr Ile Phe Ser Thr Phe Lys Leu Glu Glu Val Pro Glu Leu Asn Val Lys Leu Ser Asp Val Cys Met Gly Thr Ser Ala Ala Pro Ile Val Phe Pro Pro Tyr Tyr Phe Lys His Gly Asp Thr Glu Phe Asn Leu Val Asp Gly Ala Ile Ile Ala Asp Ile Pro Ala Pro Val Ala Leu Ser Glu Val Leu Gln Gln Glu Lys Tyr Lys Asn Lys Glu Ile Leu Leu Leu Ser Ile Gly Thr Gly Val Val Lys Pro Gly Glu Gly Tyr Ser Ala Asn Arg Thr Trp Thr Ile Phe Asp Trp Ser Ser Glu Thr Leu Ile Gly Leu Met Gly His Gly Thr Arg Ala Met Ser Asp Tyr Tyr Val Gly Ser His Phe Lys Ala Leu AAC

Gln ProGln AsnAsnTyr LeuArg IleGlnGlu TyrAsp LeuAspPro Ala LeuGlu SerIleAsp AspAla SerThrGlu AsnMet GluAsnLeu Glu LysVal GlyGlnSer LeuLeu AsnGluPro ValLys ArgMetAsn Leu AsnThr PheValVal GluGlu ThrGlyGlu GlyThr AsnA1aGlu Ala LeuAsp ArgLeuAla GlnIle LeuTyrG1u GluLys IleThrArg Gly LeuGly LysIleSer LeuGlu ValAspAsn IleAsp ProTyrThr Glu ArgVal ArgLysLeu LeuPhe (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 408 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Lys Ser Lys Met Ala Met Leu Leu Leu Leu Phe Cys Val Leu Ser Asn Gln Leu Val Ala Ala Phe Ser Thr Gln Ala Lys Ala Ser Lys Asp Gly Asn Leu Val Thr Val Leu Ala Ile Asp Gly Gly Gly Ile Arg Gly Ile Ile Pro Gly Val Ile Leu Lys Gln Leu Glu Ala Thr Leu Gln Arg 50 Trp Asp Ser Ser Ala Arg Leu Ala Glu Tyr Phe Asp Val Val Ala Gly Thr Ser Thr Gly Gly Ile Ile Thr Ala Ile Leu Thr Ala Pro Asp Pro Gln Asn Lys Asp Arg Pro Leu Tyr Ala A1a Glu Glu Ile Ile Asp Phe Tyr Ile Glu His Gly Pro Ser Ile Phe Asn Lys Ser Thr Ala Cys Ser Leu Pro Gly Ile Phe Cys Pro Lys Tyr Asp Gly Lys Tyr Leu Gln Glu Ile Ile Ser Gln Lys Leu Asn Glu Thr Leu Leu Asp Gln Thr Thr Thr Asn Val Val Ile Pro Ser Phe Asp Ile Lys Leu Leu Arg Pro Thr Ile Phe Ser Thr Phe Lys Leu Glu Glu Val Pro Glu Leu Asn Val Lys Leu.

Ser Asp Val Cys Met Gly Thr Ser Ala Ala Pro Ile Val Phe Pro Pro Tyr Tyr Phe Lys His Gly Asp Thr Glu Phe Asn Leu Val Asp Gly Ala Ile Ile Ala Asp Ile Pro Ala Pro Val AIa Leu Ser Glu Val Leu Gln Gln Glu Lys Tyr Lys Asn Lys Glu Ile Leu Leu Leu Ser Ile Gly Thr Gly Val Val Lys Pro Gly Glu Gly Tyr Ser Ala Asn Arg Thr Trp Thr Ile Phe Asp Trp Ser Ser Glu Thr Leu Ile Gly Leu Met Gly His Gly Thr Arg Ala Met Ser Asp Tyr Tyr Val Gly Ser His Phe Lys Ala Leu Gln Pro Gln Asn Asn Tyr Leu Arg Ile Gln Glu Tyr Asp Leu Asp Pro Ala Leu Glu Ser Ile Asp Asp Ala Ser Thr Glu Asn Met Glu Asn Leu Glu Lys Val Gly Gln Ser Leu Leu Asn Glu Pro Val Lys Arg Met Asn Leu Asn Thr Phe Val Val Glu Glu Thr Gly Glu Gly Thr Asn Ala Glu Ala Leu Asp Arg Leu Ala Gln Ile Leu Tyr Glu Glu Lys Ile Thr Arg Gly Leu Gly Lys Ile Ser Leu Glu Val Asp Asn Ile Asp Pro Tyr Thr Glu Arg Val Arg Lys Leu Leu Phe (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single-(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE:
(A) ORGANISM: Pentaclethra macroloba (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Met Ser Thr Ser Ala Ala Pro Ile Val Phe Pro Pro Tyr Tyr Phe Lys (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE:
(A) ORGANISM: Pentaclethra macroloba 3 O (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Ala Leu Gln Pro Gln Asn Asn Tyr Leu Arg Gln Glu Tyr Asp Leu Asp (2) INFORMATION FOR SEQ ID N0:7:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE:
(A) ORGANISM: Pentaclethra macroloba (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Pro-Asp Trp Val Val Ile Arg Ser Gln Ser Val Gly Lys (2) INFORMATION FOR SEQ ID NO: B:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE:
(A) ORGANISM: Pentaclethra macroloba (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Lys Ala Phe Vai Asn Gly Val Tyr Phe Ile Asn Thr Tyr Asp Ser Ala (2) INFORMATION FOR SEQ ID N0:9:
30 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE:
(A) ORGANISM: Pentaclethra macroloba (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Asn Asn Tyr Leu Arg Ile Gln Glu Tyr Asp Leu Pro Pro Ala Leu (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
10 (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE:
(A) ORGANISM: Pentaclethra macroloba (xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
Val Val Lys Arg Leu Ala Gly Tyr Phe Asp Val 2 0 (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE:
(A) ORGANISM: Pentaclethra macroloba (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Glu Asn Met Glu Asn Leu Glu Lys

Claims (17)

CLAIMS:

1. An organism which has been transformed with a vector comprising a DNA sequence encoding a substantially purified protein having insecticidal activity, wherein said protein comprises the amino acid sequence set forth in Figure 1 and SEQ
ID NO:2.

2. The organism of claim 1, wherein the organism is a plant.

3. A plant that has been stably transformed with an expression cassette comprising a promoter that drives expression in a plant cell operably linked to a nucleotide sequence that encodes an insecticidal protein wherein said protein is selected from the group consisting of:

a) the amino acid sequence set forth in SEQ ID NO:2;

b) residues 22-409 of the sequence set forth in SEQ
ID NO:2;

c) residues 29-409 of the amino acid sequence set forth in SEQ ID NO:2;

d) a polypeptide comprising a variation of (a), (b) or (c), wherein said variation comprises at least one amino acid substitution, truncation, internal deletion or insertion;
and e) a polypeptide comprising at least 60% amino acid sequence identity to the sequence of (a), (b), or (c).

4. The plant of claim 3, wherein said promoter is a root preferential promoter.

5. The plant of claim 3 or 4, wherein said nucleotide sequence has a sequence selected from the group consisting of:

(a) the sequence set forth in Figure 1 and SEQ ID
NO: 1;

(b) nucleotide sequences which encode a polypeptide having insecticidal activity and which hybridize to sequences of (a) above under stringent conditions defined by a wash stringency of 0.3M NaCl, 0.03M sodium citrate, 0.1% SDS at 70°C;

(c) nucleotide sequences which encode a polypeptide having insecticidal activity and which differ from the sequence of (a) due to the degeneracy of the genetic code; and (d) nucleotide sequences comprising at least 70%
sequence identity to the sequence of (a).

6. A plant which has been stably transformed with an expression cassette comprising a promoter that drives expression in a plant cell operably linked to a nucleotide sequence that encodes a protein having insecticidal activity, wherein said protein is selected from the group consisting of:

a) the amino acid sequence set forth in SEQ ID NO:2;

b) residues 22-409 of the sequence set forth in SEQ
ID No:2;

c) residues 29-409 of the amino acid sequence set forth in SEQ ID NO:2;

d) a polypeptide comprising at least 60% amino acid sequence identity to the sequence of (a), (b), or (c).

7. The plant of claim 6, wherein said nucleotide sequence encodes the amino acid sequence set forth in Figure 1 and SEQ ID NO:2.

8. The plant of claim 6, wherein said nucleotide sequence comprises a sequence selected from the group consisting of:

(a) the sequence set forth in Figure 1 and SEQ ID
NO:1;

(b) nucleotide sequences which encode a polypeptide having insecticidal activity and which hybridize to sequences of (a) above under stringent conditions defined by a wash stringency of 0.3M NaCl, 0.03M sodium citrate, 0.1% SDS at 70°C; and (c) nucleotide sequences comprising at least 70%
sequence identity to the sequence of (a).

9. The plant of any one of claims 6 to 8, wherein said nucleotide sequence is operably linked to a root preferential promoter.

10. The plant of any one of claims 2 to 9, wherein said plant is from a monocot.

11. The plant of claim 10, wherein said monocot is maize.

12. A seed of the plant of any one of claims 2 to 11.

13. A seed which has been stably transformed with an expression cassette comprising a promoter that drives expression in a plant operably linked to a nucleotide sequence that encodes a protein having insecticidal activity, wherein said protein is selected from the group consisting of:

a) the amino acid sequence set forth in SEQ ID NO:2;

b) residues 22-409 of the sequence set forth in SEQ
ID NO:2;

c) residues 29-409 of the amino acid sequence set forth in SEQ ID NO:2;

d) a polypeptide comprising at least 60% amino acid sequence identity to the sequence of (a), (b), or (c).

14. The seed of claim 13, wherein said nucleotide sequence encodes the amino acid sequence set forth in Figure 1 and SEQ ID NO:2.

15. The seed of claim 13, wherein said nucleotide sequence comprises a sequence selected from the group consisting of:

(a) the sequence set forth in Figure 1 and SEQ ID
NO:1;

(b) nucleotide sequences which encode a polypeptide having insecticidal activity and which hybridize to sequences of (a) above under stringent conditions defined by a wash stringency of 0.3M NaCl, 0.03M sodium citrate, 0.1% SDS at 70°C;
and (c) nucleotide sequences comprising at least 70%
sequence identity to the sequence of (a).

16. The seed of any one of claims 13 to 15, wherein said nucleotide sequence is operably linked to a root preferential promoter.

17. The seed of any one of claims 12 to 16, wherein said seed is a maize seed.