CA2247798A1 - Soybean cysteine proteinase inhibitors, nucleotides encoding the same, and methods of use thereof - Google Patents

Abstract

The invention relates to the isolation, purification and use of three soybean cysteine proteinase inhibitors, designated L1, R1 and N2, which have strong inhibitory activity with respect to cysteine proteinases found in the digestive tracts of certain herbivorous insects. The invention further relates to three nucleotide sequences which have been cloned, isolated and sequenced, these nucleotide sequences coding for the three above-named proteins, as well as a vector having one or more of these sequences provided therein. The invention also relates to plants transformed by inventive vectors, transformed plants having an enhanced ability to resist predation by said insects, as well as microorganisms transformed using inventive vectors or plasmids.

Description

CA 02247798 19s8-08-27 W097/32007 - 1 - PCTrUS97/03234 SOYBEAN CYSTEINE PROTEINASE INHIBITORS, NUCLEOTIDES
ENCODING THE SAME, AND METHODS OF USE ~ K~Ob This invention was made with government support under the following USDA grants: grant number 91-37307-6443 and grant number 89-37153-4353. The government has certain rights in the invention.

CROSS-k~r~:n~ TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 60/012,423, filed February 28, 1996, which is hereby incorporated by reference herein in its- entirety.

BACKGROUND OF THE lNv~ ON

Field of the Invention This invention relates to methods and materials for the protection of plants against pests through plant genetic engineering. More specifically, it relates to proteins which inhibit the activity of cysteine proteinases found in the digestive tracts of certain pests, isolated DNA sequences which encode these proteins, and methods for using inventive DNA
sequences to transform cells such that the cells are capable of expressing the DNA sequence.

Description of Related Art Damage to crops by pests (i.e. insects, herbivores, and pathogens, including fungi, bacteria, and viruses), results in substantial annual losses in agricultural production. This W097/32007 - 2 - PCT~S97/03234 problem has been handled in the past by employing a wide variety of chemicals to reduce pest damage to plant crops. This approach, however, has been associated with many environmental problems created by the widespread use of pesticidal chemicals, S and the chemicals often only provide a transient level of protection for crops. Chemicals also suffer from the disadvantage that all organisms in an area may be indiscriminately treated, causing needless damage to many beneficial organisms. Perhaps more importantly, many chemicals are also potentially toxic to man and animals and often become concentrated in, for example, lakes and ponds and/or other water supplies.
As a result, alternate methods have been explored to reduce crop damage, one example being selective breeding of plants based upon pest resistance characteristics. However, resistance traits are commonly controlled by many genes, making it difficult (or impossible) to genetically select a desired attribute. Decreased crop yields are also commonly encountered in resistance strains developed by selective breeding. Accordingly, there exists a strong need for compositions and processes to improve the resistance of plants from attack by herbivorous pests. Such are provided by the present invention, which provides compositions and methods useful for transforming plants and thereby providing to plants advantageous ability to resist, for example, insect predation.
Commenting now upon plant resistance to insect predation, plants have evolved inducible defensive mechanisms that respond to attacks by pests. One mechanism involves plant synthesis of proteins which function to inhibit activity of various enzymes found in the digestive tracts of various insects. One example of this response mechanism is the systemic synthesis of serine proteinase inhibitors (SPIs) that are accumulated at distal tissue sites in some plants. The SPIs can inhibit various insect - and microorganism digestive enzymes, such as extracellular proteases in the lumen of insect guts which hydrolyze dietary W097/32007 _ 3 PCTrUS97/03234 protein for amino acid assimilation. SPIs can thus be detrimental to the growth and development of insects from a variety of genera including Heliothis, Spodoptera, Diabrotica and Tribolium. Several families of proteins have been described that inhibit serine proteinases. It has additionally been found that some such proteinaceous inhibitors have widely varying activity against differing insect gut enzymes such as those found in different insect species.
Insects in several coleopteran and hemipteran families are unique in the utilization of cysteine proteinases such as cysteine endopeptidases (rather than serine pro'einases) for protein digestion. The use of cysteine proteinases for protein digestion has been hypothesized to be an evolutionary adaptation that enables these insects to consume legume seeds and other plant materials that contain high levels of serine proteinase inhibitors. Proteinaceous inhibitors of cysteine proteinases are widely distributed in plants and have been isolated from a number of plant sources, including rice, cowpea and maize. While the natural function of these cysteine proteinase inhibitors has not previously been established, it has been proposed that these proteins also may be involved in plant defense against insects, particularly those in the coleopteran and hemipteran orders.
However, no mechanism by which this occurs has been shown.
The larvae of Western corn rootworm (WCR) (Diabrotica virgifera), which feed almost exclusively on roots, are serious pests of corn that substantially reduce potential yields. Larval damage to roots has a seriously detrimental impact on plant productivity, and third instar larval feeding inflicts the majority of damage. In addition to the economic impact of crop yield reduction, more than l billion dollars per year is used to control WCR and northern corn rootworm in the U.S. Therefore, there is a great need for compositions and processes which may be used to transform plants, specifically corn plants, to make the plants advantageously more resistant to, for example, WCR

W097/32007 _ 4 _ PCT~S97/03234 predation. The present invention provides methods for making transgenic p~ants having improved resistance to such pests.

W097132007 _ 5 _ PCT~S97/03234 SU~ RY OF THE lNV~ ION

The invention relates to the isolation, purification and use of three soybean cysteine proteinase inhibitors, designated L1, R1 and N2. These proteins are shown to efficaciously inhibit the activity of cysteine proteinases found in the digestive tracts of certain herbivorous insects. The invention therefore involves the amino acid sequences of the L1, R1 and N2 proteins, as set forth herein, as well as proteins having substantial identity thereto and having similar levels of inhibitory activity with respect to cysteine proteinases.
The invention further relates to the cloning of DNA
sequences which encode these proteins. As such, the present disclosure sets forth three nucleotide sequences which have been cloned, isolated and sequenced, these nucleotide sequences coding for the three above-named proteins. These nucleotide sequences, or nucleotide sequences having substantial similarity thereto, e.g. encoding an amino acid sequence having substantial identity to those disclosed herein, may, for example, be advantageously incorporated into a vector and used to transform a plant. Plants transformed with inventive nucleotide sequences thereby have an enhanced ability to resist predation by insects which utilize one or more cysteine proteinases for digestion.
Inventive nucleotide sequences may also be used to transform microorganisms. Methods for transforming microorganisms find advantageous use in producing relatively large amounts of inventive proteins which may then be purified for use, for example, in biological assays. Alternatively, purified inventive proteins may advantageously be applied to plants or other tissues where the inhibition of cysteine proteinase activity is desired.
Purified inventive proteins may also be advantageously used together with, for example, carrier compositions and/or additional active agents.

W097/32007 - 6 - PCT~S97/03234 It is an object of the present invention to provide isolated, sequenced and purified soybean cysteine proteinase inhibitors which have widely applicable inhibitory activity.
Another object of the invention is to provide isolated nucleotide sequences which encode soybean cysteine proteinase inhibitors and thereby find advantageous use when incorporated into a vector or plasmid as a transformant for a plant or microorganism.
Additionally, it is an object of the present invention to provide transformed plants which have an enhanced ability to resist predation by herbivorous insects.
It is also an object of the invention to provide transformed microorganisms capable of expressing an inventive nucleotide sequence and thereby producing relatively large amounts of inventive proteins.
Further objects, advantages and features of the present invention will be apparent from the detailed description herein.

W097/32007 _ 7 _ PCT~S97/03234 BRIEF DESCRIPTION OF THE FIGURES

Although the characteristic features of this invention will be particularly pointed out in the claims, the invention itself, and the manner in which it may be made and used, may be better understood by referring to the following descriptions taken in connection with the accompanying figures forming a part hereof.
FIG. 1 is a plot of percentage inhibition of digestive enzymes in Western corn rootworm versus molar concentrations of a given proteinase inhibitor. This plot gives data relating to the inhibition of protease activity in Western corn rootworm guts (in vitro) by various protease inhibitors and control, and the data were collected as described in Example 4. E-64 is a low molecular weight, commercially available cysteine proteinase inhibitori SCPI is a native soybean cysteine protease inhibitor that is encoded by pR1; and L1, R1 and N2 are inventive recombinant soybean cysteine proteinase inhibitors.
FIG. 2 is a plot of percentage inhibition of digestive enzymes in Colorado potato beetle versus molar concentrations of a given proteinase inhibitor. This plot gives data relating to the inhibition of protease activity in crude gut extracts from Colorado potato beetle (in vitro) as is described in greater detail in Example 4.
FIG. 3 is a plot of larval weight versus dose of a given proteinase inhibitor. This plot gives data relating to the effect of inventive proteinase inhibitors upon live third instar larvae of Western corn rootworm (in vivo) as is described in greater detail in Example 5.

DESCRIPTION OF THE ~X~b-~ ~K~ EMBODIMENTS

For the purposes of promoting an understanding of the principles of the invention, reference will now be made to particular embodiments of the invention and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the invention, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
The present inventors have isolated, sequenced and characterized three biologically and commercially useful proteins (Ll, Rl and N2), and have isolated, sequenced and cloned three novel nucleotide sequences which encode them (pLl, pRl and pN2, respectively). The Ll, Rl and N2 proteins are shawn to have a surprisingly strong inhibitory effect on cysteine proteinases, specifically, cysteine proteinases found in the digestive tracts of a number of herbivorous insect species. As such, the three novel soybean proteins described herein may be generally referred to as cysteine proteinase inhibitors (CPIs). Preferred embodiments of the invention described and illustrated herein provide proteinaceous CPIs having very strong inhibitory activity with respect to digestive enzymes of herbivorous insects such as, for example, Western corn rootworm (WCR), Colorado potato beetle ~CPB) and cowpea weevil (CW).
A feature of the present invention is the inhibitory effect that inventive proteins, especially the protein N2, have on digestive cysteine proteinases of the larvae of WCR (Diabrotica virgifera ), which feed almost exclusively on corn plant roots and have a serious detrimental impact on the health and productivity of corn plants. In one advantageous aspect, the present invention provides transgenic corn plants expressing foreign DNA
encloding a cysteine proteinase inhibitor and having an increased W097132007 - 9 - PCTrUS97/03234 ability to resist predation by WCR, and methods for making such transgenic corn plants.
The term "protein" is used herein to mean a plurality of amino acids linked in a serial array. The term "nucleotide sequence" is intended to refer to a natural and/or synthetic linear and sequential array of nucleotides and/or nucleosides, and derivatives thereof. Inventive nucleotide sequences were cloned from a soybean cDNA library.
As is described in more detail below in the Examples, the present inventors have tested the L1, R1 and N2 proteins to assess their inhibitory activity against digestive enzymes of the three above-mentioned insects. The amino acid sequences of inventive expressed, processed and cleaved CPIs are set forth below in Table I ~SEQ ID NOS. 1, 2 and 3, respectively).

TABLE I

L1 (SEQ ID NO: 1) GLY ASN ARG ASP VAL THR GLY SER GLN ASN SER VAL GLU ILE ASP ALA
LEU ALA ARG PHE ALA VAL GLU GLU HIS ASN LYS LYS GLN ASN ALA LEU
LEU GLU PHE GLU LYS VAL VAL THR ALA LYS GLN GLN VAL VAL SER GLY
THR LEU TYR THR ILE THR LEU GLU ALA LYS ASP GLY GLY GLN LYS LYS
VAL TYR GLU ALA LYS VAL TRP GLU LYS SER TRP LEU ASN PHE LYS GLU
VAL GLN GLU PHE LYS LEU VAL GLY ASP ALA PRO ALA
R1 (SEQ ID NO: 2) LEU GLY GLY PHE THR ASP ILE THR GLY ALA GLN ASN SER ILE ASP ILE
GLU ASN LEU ALA ARG PHE ALA VAL ASP GLU HIS ASN LYS LYS GLU ASN
ALA VAL LEU GLU PHE VAL ARG VAL ILE SER ALA LYS LYS GLN VAL VAL
SER GLY THR LEU TYR TYR ILE THR LEU GLU ALA ASN ASP GLY VAL THR
LYS LYS VAL TYR GLU THR LYS VAL LEU GLU LYS PRO TRP LEU ASN ILE
LYS GLU VAL GLN GLU PHE LYS PRO ILE THR VAL ALA VAL ASN PRO LEU
SER VAL THR VAL

W097/32007 - 10 - PCT~S97/03234 N2 (SEQ ID N0: 3) ALA ALA LEU GLU LYS VAL GLN GLU LEU GLY GLY ILE THR ASP VAL HIS
GLY ALA ALA ASN SER VAL GLU ILE ASN ASN LEU ALA ARG PHE ALA VAL
G~U GLU GLN ASN LYS ARG GLU ASN SER VAL LEU GL~ PHE VAL ARG VAL
ILE SER ALA LYS GLN GLN VAL VAL ALA GLY VAL ASN TYR TYR ILE THR
LEU GLU ALA LYS ASP GLY LEU ILE LYS ASN GLU TYR GLU ALA LYS VAL

VAL ASN VAL SER SER THR GLN

Inhibitory profiles of crude gut extracts of WCR and CPB with regard to the L1, R1 and N2 proteins indicate the relative susceptibility of these insect species to the recombinant CPIs.
Based upon these data, it is expected that additional species of herbivorous insects which also utilize cysteine proteinases to digest plant tissue are also susceptible to inventive CPIs, and that predation by these species will similarly be advantageously resisted by plants transformed according to the present invention.
For purposes of comparison, inhibitory activity of an inventive protein is compared to the inhibitory activity of the commercially available chemical CPI inhibitor E-64. E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane) is a specific and potent low molecular weight tripeptidyl cysteine proteinase inhibitor which is commonly used as a standard with which to compare the level of inhibitory activity of other compositions, such as the proteins of the present invention. Based upon the ability of inventive proteins to strongly inhibit the cysteine proteinase enzymes tested, as compared to the inhibitory activity of E-64, it is predicted that inventive proteins described and illustrated herein will be found to have good inhibitory effect with regard to additional cysteine proteinases of a wide variety of insect species. As such, advantageous features of the present invention include the transformation of a wide variety of plants from various agriculturally and/or commercially valuable species to provide advantageous resistance to insect predation.
Candidate herbivorous insect species may be identified as W097l32007 - 11 - PCT~S97/03234 susceptible to inventive protein inhibitors by routine biological assays using E-64.
Skilled artisans will recognize that through the process of mutation and/or evolution, proteins of different lengths and having differing constituents, e.g., with amino acid insertions, substitutions, deletions, and the like, may arise that are related to the proteins of the present invention by virtue of (a) amino acid sequence homology; and (b) functionality in terms of inhibiting cysteine proteinases. Many deletions, insertions, and substitutions, are not expected to produce radical changes in the characteristics of the CPI protein. Given the disclosures herein, one skilled in the art will be readily able to make and select suitable CPI proteins based on routine screening assays.
For example, a variant typically may be made by site-specific mutagenesis of a native CPI-encoding nucleotide sequence, expression of the variant nucleotide sequence in recombinant cell culture, and, optionally, purification from the cell culture by any method known in the art. This purified variant may then be tested for inhibiting activity with regard to various cysteine proteinases.
In a preferred aspect of the invention, inhibitory activity is represented by a Cgo with respect to a particular cysteine proteinase of at most about 10-4. The term Cgo, as used herein, represents the molar concentration of a cysteine proteinase

2~ inhibitor necessary to achieve 80% inhibition of a given cysteine proteinase. Preferably, the protein has a Cgo ~f at most about 10-5 with respect to a cysteine proteinase, more preferably about 10-6 and most preferably about 10-7. As such, in a preferred aspect of the present invention, there is provided a protein having substantial identity to the protein sequence of SEQ ID NO
1, SEQ ID NO 2 or SE~ ID NO 3 and having a C80 Of at most about 10-4 with respect to one or more insect gut cysteine proteinases.
Although inventive protein are expected to have good inhibitory activity with respect to cysteine proteinases of a wide variety CA 02247798 19s8-08-27 W097/32007 - 12 - PCT~S97/03234 o~ insect species, particularly preferred embodiments have strong inhibitory activity with respect to the cysteine proteinases of Western corn rootworm, Colorado potato beetle and/or cowpea weevil.
For purposes of describing the present invention, variants having such potential modifications as those mentioned above, which have at least about 60~ similarity to the amino acid sequences set forth in Table I, are considered to have "substantial identity" thereto. More preferred sequences will have at least 80% or 90% or more similarlty to the amino acid seqences set forthin in Table I. In addition, sequences having lesser degrees of similarity but comparable biological activity are considered to be equivalents. It is believed that the identity required to maintain proper functionality is related to maintenance of the tertiary structure of the protein such that specific interactive sequences will be properly located and will have the desired activity. As such, it is believed that there are discreet domains and motifs within the protein sequence which must be present for the protein to retain its advantageous functionality and specificity. It is contemplated that a protein including these discreet domains and motifs in a proper spatial context will retain good inhibitory activity with respect to cysteine proteinases, even where substantial substitutions, insertions and/or deletions have taken place elsewhere in the sequence. However, it is not intended that the present invention be limited by any theory by which it achieves it advantageous result.
Inventive nucleotide sequences of the present invention, i.e. those which encode inventive proteins L1, R1 and N2, are set forth below in Table II (SEQ ID NOS. 4, 5 and 6, respectively).

CA 02247798 l998-08-27 W097/32007 - 13 - PCT~S97/03234 TABLE II
pL1 (SEQ ID NO: 4) 451 TATGTGTTTC TTTCAAAAAA AAP~ AA AP~ AAA AAAAA~5 pR1 (SEQ ID NO:5) pN2 (SEQ ID NO:6) The present invention also contemplates nucleotide sequences having substantial identity to those set forth in Table II, e.g.
mutants or allelic variants. The term "substantial identity" is used herein with respect to a nucleotide sequence to designate that the nucleotide sequence has a sequence sufflciently similar to one of those explicitly set forth above in Table II that it will hybridize therewith under moderately stringent conditions, this method of determining identity being well known in the art to which the invention pertains. Briefly, moderately stringent W097132007 - 14 - PCT~S97/03234 conditions are defined in Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2ed. Vol. 1, pp. 101-104, Cold Spring Harbor Laboratory Press (1989) as including the use of a prewashing solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions of about 55~C, 5 X SSC, overnight. A
further requirement of the term "substantial identity" as it relates to inventive nucleotide sequences is that the protein encoded thereby must be an inventive protein, i.e. must have good inhibitory activity with respect to one or more cysteine proteinase inhibitors. DNA fragments comprising inventive nucleotide sequences may be obtained, for example, by cloning techniques, these techniques being well known in the relevant art, or may be made by chemical synthesis techniques which are also well known in the relevant art.
With regard to one inventive use of nucleotide sequences described herein, embodiments of the invention provide processes for enhancing in vivo synthesis of CPIs in a plant by introducing inventive nucleotide sequences into, for example, a precursor plant cell. Recombinant DNA in accordance with the invention may advantageously be incorporated into the genome of plants by methods well known in the art, thereby making transformed plants having greater ability to resist, for example, insect predation.
In this regard, the term "genome" as used herein is intended to refer to DNA which is present in the plant and which is heritable by progeny during propagation of the plant. As such, inventive transgenic plants may alternatively be produced by breeding a transgenic plant made according to the invention with a second plant or selfing an inventive transgenic plant to form an F1 or higher generation plant. The subject transgenic plants and progeny are all contemplated by the invention and are all intended to fall within the meaning of the term "transformed plant."
According to the invention, plants may advantageously be - transformed by inserting inventive nucleotide sequences into vectors, e.g. viral vectors, and introducing the vectors into W097/32007 - 15 - PCTrUS97/03234 cells of the plant using conventional techniques. As an example of a manner in which to transform a plant, this may be accomplished utilizing Agrobacterium tumefaciens-mediated transformation, although other techniques can also be used and are within the purview of the ordinarily skilled artisan. The technique used for a given plant species or specific type of plant tissue will depend upon the known preferred techniques for that species or tissue. Additional means for introducing recombinant DNA into plant tissue include but are not limited to electroporation, microprojectiles and microinjection, as well as other T-DNA mediated transfer from Agrobacterium tumefaciens.
In one representative example, enhanced CPI production may be achieved by inserting a CPI nucleotide sequence in a vector downstream from and operably linked to a promoter sequence capable of driving constitutive high-level expression in a plant cell. Two DNA sequences (such as a promoter region sequence and a CPI-encoding sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the desired CPI-encoding gene sequence, or (3) interfere with the ability of the desired CPI
sequence to be transcribed by the promoter region sequence.
Expression is provided in transformed plants in the above embodiments by regulatory elements within about 3000 bp of the 5' region of an inventive CPI coding sequence. Promoter, enhancer, and other regulatory elements within the 3000 bp 5' region are expected to be useful for insertion into recombinant vectors for controlling gene expression in plants. As such, in accordance with the present invention, an inventive nucleotide sequence is incorporated in a recombinant DNA molecule under the control of a promoter. In this regard, a recombinant DNA molecule is one which has either been naturally or artificially produced from parts derived from heterologous sources, which parts may be naturally occurring or chemically synthesized molecules, and CA 02247798 l998-08-27 W097l32007 - 16 - PCT~US97/03234 wherein those parts have been joined by ligation or other means known in the art. The introduced coding sequence is under control of the promoter and thus will be downstream from the promoter. Stated alternatively, the promoter sequence will be upstream (i.e., at the 5' end) of the coding sequence. Also, the recombinant DNA will preferably include a termination sequence downstream from the introduced sequence.
As was mentioned above, a constitutive promoter may be used according to one aspect of the invention. However, as is well known in the art, targeting of the DNA product can be obtained using, for example, a constitutive, tissue specific, inducible or developmentally regulated promoter to construct the vectors. As such, in another embodiment, the invention involves processes for transforming plants such that the plant selectively initiates expression of the inserted CPI nucleotide sequence or sequences.
One example of selective expression which may be considered particularly advantageous in one application of the invention is wound-inducible expression, for example in the roots of a corn plant. Alternatively, expression may be tissue specific and, thereby, be selectively initiated in particular tissues of a plant that are susceptible to predation by a specific insect.
Transgenic plants according to the present invention exhibit increased synthesis of CPI proteins and, thus, increased resistance to herbivorous insects.
In one preferred aspect of the invention, transformed corn plants are provided which are transformed by nucleotide sequences of the present invention. Root cells of these transformed corn plants are advantageously capable of expressing the DNA sequences and, as such, transformed corn plants have an increased ability to resist predation by Western corn rootworm. For example, as is seen in FIG. 1 and described in Example 4, the protein N2 is shown to have an extremely strong inhibitory effect on the digestive enzymes of Western corn rootworm. As corn root infestation by Western corn rootworm larvae has a very de_rimental impact on the survival and productivity of a corn W097t32007 - l7 - PCTrUS97/03234 plant, an extremely advantageous aspect of the present invention provides transgenic corn plants capable of expressing N2.
Western corn rootworm larvae feeding upon the roots of such a transformed corn plant will ingest the N2 protein, and the N2 will inhibit the digestive enzymes of the larvae. This inhibition results in resistance of larvae predation by decreasing ability of the larvae to assimilate nutrients, and thus hinders growth and development and hinders survival rates of larval Western corn rootworm which feed on inventive transformed plants. This result not only minimizes immediate damage caused by the feeding larvae, but also prevents the establishment of a strong Western corn rootworm population in, for example, a given corn field.
Once inventive recombinant DNA is introduced into plant tissue, successful transformants can be screened using standard techniques such as the use of marker genes, e.g., genes encoding resistance to antibiotics. Additionally, the level of expression of the inserted CPI coding sequence of transgenic plants may be measured at the transcriptional level, e.g. by the detection in transformed cells of the mRNA products of the same, or as protein synthesized. Transgenic plants in accordance with the present invention can also be identified by detection of a significant increase in the plant's ability to resist predation by herbivorous insects such as, for example, Western corn rootworm, as compared to non-transformed plants.
Those skilled in the art will recognize the agricultural advantages inherent in plants constructed to have increased or selectively increased expression of CPI proteins. For example, such plants have increased resistance to attack by pests which utilize cysteine proteinases for digestion such as, for example, insects, pathogens, microorganisms, herbivores, and the like.
Representative examples of plants in which the invention may find advantageous use include (but are not limited to) corn, potato, cowpea, tomato, tobacco, wheat, rice, cotton, soybean, alfalfa, and the like.

W097/32007 - 18 - PCT~S97/03234 Also provided by the present invention are microorganisms transformed using inventive nucleotide sequences. For example, using methods well known in the relevant art, microorganisms such as, for example, E. coli cells, may be transformed such that they synthesize inventive proteins in relatively large amounts.
Unicellular hosts are selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded on expression by the DNA sequences of this invention to them, their secretion characterlstics, their ability to fold proteins correctly, their stability and culturing requirements, and the ease of purification of the products coded on expression by the DNA sequences of this invention.
The present invention is not intended to be limited by the choice of vector or host cell. It should of course be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences, and hosts without undue experimentation and without departing from the scope of this invention.
Such synthesis by a microorganism finds advantageous use in providing quantities of inventive proteins which may be purified and used, for example, for biological assays or, alternatively, for administration to plants or other products to prevent tissue degeneration or destruction by cysteine proteinases such as those used by various insects for digestion.
Additionally, it is contemplated that inventive purified proteins may be advantageously mixed with additional compositions such as, for example, compatible liquid or solid carrier compositions or other active agents. For example, in one advantageous use, inventive proteins may be combined with another insecticidal chemical, thus providing a composition which prevents crop damage by a particular pest and has reduced detrimental effects, for example, on other organisms or water CA 02247798 l998-08-27 W097/32007 - l9 - PCT~US97/03234 supplies than application of a more concentrated form of the chemical. As another example, inventive proteins may be mixed with a suitable carrier alone or in combination with other agents, to provide a composition which may be applied to plants, e.g. sprayed or dusted onto the plants, to prevent damage by insects which feed, for example, on the leaves of the plants.
It is understood that the present invention contemplates not only proteins and nucleotide sequences which are naturally produced and subsequently isolated, but also proteins and nucleotide sequences which are constructed artificially such as, for example, through chemical synthesis.
The invention will be further described with reference to the following specific Examples. It will be understood that these Examples are illustrative and not restrictive in nature.
The following materials were purchased from Sigma Chemical Company ~St. Louis, MO): E-64, BSA (bovine serum albumin), BBTI
(Bowman Birk trypsin inhibitor), SKTI (Kunitz trypsin inhibitor), IPTG (isopropylthio-2-D-galactoside), Triton, EDTA
~ethylenediaminetetracetic acid), PMSF (phenylmethylsulfonyl fluoride).

EXAMPLE ONE
ISOLATION OF cDNA CLONES ENC0DING INVENTIVE
SOYBEAN CYSTEINE PROTEINASE INHIBITORS
A lambdaZAPII cDNA library (Stratagene) was prepared using poly~A)+ RNA isolated from immature soybean (variety Del Soy) embryos that contained 7.8 x 106 recombinant plaques prior to amplification. This library was screened with two probes obtained by RT-PCR of mRNA from immature embryos. To produce the probes, first strand cDNA was generated using oligo-dT as primer.
Soybean CFI sequences were amplified by PCR using degenerate

3~ oligonucleotide primers (5'-encoded DEHNKKENA and 3' antisense of sequence encoding KELQEF), designed based on conserved motifs in W097/32007 - 20 - PCT~S97/03234 soybean CPI and oryzacystatin I and II- Three different cDNA
clones were isolated (pL1, pR1 and pN2) (GenBank accession nos.
U51853, U51854, and U51855, respectively). The sequences of these three clones are set forth in Table II above.

EXAMPLE TWO
TRANSFORMATION OF A MICROORGANISM TO PROVIDE
EXPRESSION OF RECOMBINANT CPI PROTEINS
PCR products of the open reading frame of pLI, pR1 and pN2 (SEQ ID NOS. 4, 5 and 6, repectively) were inserted into pGEX in frame with the glutathione-S-transferase (GST) gene using methods well known in the art. E. coli strain DH5 alpha was used to express the recombinant proteins.

EXAMPLE THREE
ISOLATION AND PURIFICATION OF INVENTIVE
CLEAVED RECOMBINANT POLYPEPTIDES
Escherichia coli strains DH5 alpha that expressed recombinant glutathione-fusion (GST-fusion) proteins ~1, R1 and N2 were inoculated into Luria broth containing ampicillin. When the growth of the culture indicated an absorption of 1.0 at 600 nm, IPTG was added to a final concentration of 0.2 mM. Cells were incubated for 4 hours, then harvested by centrifuging at 13,800 x g for 10 minutes. The pellet from 1 liter of culture was suspended in 6 ml of phosphate buffered saline with triton (PBST: 150 mM NaCl, 16 mM Na2HPO4, 4 mM NaH2PO4, 1% Triton, 2 mM
EDTA, 0.1% 2-mercaptoethanol, 0.2 mM PMSF, pH 7.3). Cells were lysed at 4~C using a sonicator and the bacterial lysate was centrifuged at 12,000 x g for 20 minutes to separate the insoluble fraction. The supernatant was mixed with 2 milliliters of glutathione-agarose beads and allowed to shake gently for 8 hours at 4~C. Agarose beads bound to the recombinant GST-CPI
fusion protein were packed into a spun column and washed with buffer A ~150 mM NaC1, 16 mM Na2HPO4, 4 mM NaH2PO4, 1% Triton, pH

W097~32007 - 21 - PCT~US97/03234 7.3). The column was equilibrated with buffer B (50 mM TrisCl, 150 mM NaCl, 2.5 mM CaCl2 and 0.1% 2-mercaptoethanol, pH 8.0).
Thrombin (4 micrograms) was added to the column to aid thrombin hydrolysis at 22~C for 3 hours. At completion, the cleaved recombinant protein was eluted with 50 mM Tris-HCL, 150 mM NaCl, pH 8Ø
Homogeneity of the cleaved recombinant proteins were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

EXAMP~E FOUR
MEASUREMENT OF SOYBEAN CPI INHIBITORY ACTIVITIES
AGAINST INSECT DIGESTIVE CYSTEINE PROTEINASES
Preparation of crude gut extracts of Western corn rootworm, Colorado potato beetle and cowpea weevil Digestive tracts of 10, 6 and 20 third and fourth instar neonate Western corn rootworm (WCR), (Diabrotica virgifera), Colorado potato beetle (CPB), (Leptinotarsa decemlineata) and cowpea weevil (CW) (Callosobruchus maculatus) were dissected into 100 microliters of 0.2 M sodium acetate buffer, pH 5Ø Crude gut extracts of these insects were prepared by homogenizing the guts in a microcentrifuge tube using a close fitting ground glass pestle. The homogenate was centrifuged at 12,800 x g for 5 minutes at 22~C to sediment the gut debris. The supernatant was used as the crude gut extract.
Inhibition studies of the cysteine proteinase in crude gut extracts A (3H)-methemoglobin assay was used to monitor the protease activity in crude gut extracts of larval WCR, CPB and CW. Crude gut extracts as prepared above were first diluted to 1 gut equivalent in 10 microliters of 0.2 M sodium acetate buffer (pH
5.0). Proteolytic activity was assayed using (3H)-methemoglobin as the substrate. The reaction mixture included 50 microliters of (3H)-methemoglobin, 10 microliters of 50 mM cysteine, 10 W097l32007 - 22 - PCT~S97103234 microliters of crude gut extract, 10 microliters of an inhibitor or control at different concentrations, and 20 microliters of 0.2 M sodium acetate buffer, pH 5.0 in a final volume of 100 microliters. Controls included BSA and the serine proteinase inhibitors, SKTI and BBTI. After incubation at 37~C, the reaction was stopped with 100 microliters of 10% (w/v) trichloroacetic acid. The mixture was held on ice for 20 minutes and then centrifuged at 12,000 x g for 5 minutes at 22~C. The radioactivity in a 150 microliter aliquot of the supernatant was determined by liquid scintillation spectrometry.

RESULTS
~ It was found that crude gut proteinases of Western corn rootworm and Colorado potato beetle were inhibited by recombinant soybean cysteine proteinase inhibitors L1, R1 and N2. Inhibi~ory profiles of the crude gut extracts of WCR and CPB are given in FIGS. 1 and 2, respectively. These data show that the presence of inventive proteins show a substantial inhibitory effect upon the activity of the three crude gut extracts. Data have not been collected for inhibition of CW by inventive proteins; however, based upon the inhibitory effect of E-64 with respect to CW crude gut extracts, and based upon in vivo results as described below, it is expected that inventive proteins will have good inhibitory effect on CW digestive enzymes.

EXAMPLE FIVE
INHIBITION OF WESTERN CORN ROOTWORM
COLORADO POTATO BEETLE, AND COWPEA WEEVIL
Cowpea weevil assays Artificial seeds containing E-64 were prepared using a procedure by Shade et al. (Shade, R.E., Murdock, L.L., Foard, D.E., and Pomeroy, M.A. 1986. Artificial seed system for bioassay of cowpea weevil (Coleoptera bruchidae) growth and development. Environ. Entomol. 15:1286-1291.). Briefly, CW
susceptible seeds were decorticated and milled into flour. The W097/32007 - 23 - PCT~S97/03234 flour was wetted with distilled water and the resulting paste injected into TEFLON molds. E-64 was incorporated into the artificial seeds by dissolving it in the distilled water used for making the cowpea flour paste. E-64 was more readily integrated into the pellets by first mixing 40 weight percent of the total flour to be used for one seed with the water, and then mixing in the remaining flour. After freezing the paste on dry ice and in liquid nitrogen and lyophilization, the resulting 500 mg artificial seeds were coated with 8~ tw/v) gelatin and infested with bruchids (10 eggs/seed X 7 seeds). Artificial seeds containing N2, L1 and R1 CPI proteins were prepared in the same way as those for E-64, except the 500 mg seeds were reground and pressed into 28 mg pellets. The controls were prepared in the same manner as above, but having no cysteine proteinase inhibitor included therein. Additional controls were prepared by mixing 100 percent of the flour for a given pellet with water in one mixing step. Ten pellets from each treatment were infested with 1 viable bruchid egg/pellet. The cowpea weevil colony originated in Niger, W. Africa.
The results of this experiment are given in Tables III, IV
and V, below.

CA 02247798 l998-08-27 W097/32007 - 24 - PCT~S97103234 TABLE III
NUMBER OF FEEDING EVENTS PER MINUTE

Count Mean Std. Dev. Std. Error rN2 @ 0.1% 12 47.642 33.827 9.765 Ll @ 0.1% 12 26.042 13.829 3.992 Rl @ 0.1% 12 36.917 17.327 5.002 E-64 @ 0.1% 12 91.733 32.99 9.523 Ct (40% trtd) 12 20.3 6.673 1.926 Ct (0% trtd~ 12 24.183 10.716 3.094 TABLE IV
MEAN WITHIN SEED DEVELOPMENTAL TIME

Count Mean (days) Std. Dev. Std. Error rN2 @ 0.1% 3 37.7 8.229 4.751 rLl @ 0.1% 3 28.633 2.702 1.56 rRl @ 0.1% 3 29.933 1.069 .617 E-64 @ 0.1% 3 82.367 5.260 3.037 Ct #1 (40%) 3 28.667 2.214 1.2878 Ct #2 (00%) 3 27.5 2.946 1.701 W097/32007 - 25 - PCTrUS97103234 TABLE V
WITHIN SEED MORTALITY

Count Mean Std. Dev. Std. Error rN2 @ 0.1% 3 29.213 18.668 10.778 rLl @ 0.1% 3 21.134 15.522 8.962 rRl @ 0.1% 3 23.855 4.694 2.710 E-64 @ 0.1% 3 55.788 13.973 8.067 Ct #1 (40%) 3 3.626 ~ -Ct #2 (00%) 3 16.209 11.63 6.715 Western corn rootworm assays A dose response analysis was performed to determine the effect of inventive proteins on the growth and development of WCR. Samples were incorporated into a commercial southern corn rootworm diet, with minor modification, per manufacturer's directions. Treated diet was dispensed into bioassay trays (C-D
International, Piton, NJ) with each well serving as a treatment replicate. Each replicate (well) was infested with 3 neonate larvae, and larval weights and mortality were determined after 7 days. The experiment was replicated twice by blocking over time.
STARVIEW (Abacus Concepts, Inc.) was used for statistical analysis.
Results are shown in FIG. 3.

Colorado potato beetle assays Purified N2 protein was dissolved, to a concentration of 0.2 or 1%, in a 6% gelatin solution and painted onto potato leaves.
The leaves were then air dried. Neonate CPB larvae were placed on the leaf surface, at 10 larvae per leaf. As little as 0.2% of N2, painted onto the leaf surface, reduced CPB larval feeding, leading to reduced weight and delayed development.
Results are given in Table VI, below.

TABLEVI

DEVELOPMENTAL TIME, MORTALITY, AND LEAF DAMAGE

Treatment Larvae Avg. Larvae avg. Duration of Mortality at Leaf consumed fresh Wt. At Dry Wt. At 1 instar day 120 hr. per larvae at 120 hr. ~mg) 120 hr.(mg) 72 hr. (cm2) Leaf Only 5.95 0.943-4 0 0.27 6% gelatin 3.47 0.6225 0 0.12 1%N2 in 6% gelatin 1.37 0.17>5 50~ 0.03 0.2~N2 in 6~ gelatin 1.82 0.26 >5 0 0,03 SUBSTITUTE SHEET (RULE 26) W097/32~7 - 27 - PCTrUS97/03234 GENE SEQUENCE LISTING
General Information:
- Applicant: Suzanne Nielsen and Paul Hasegawa Title of Invention: SOYBEAN CYSTEINE PROTEINASE
INHIBITORS, NUCLEOTIDES ENCODING THE SAME, AND
METHODS
OF USE THEREOF
Number of Sequences: 6 Corresonding Address:
Addressee: Thomas Q. Henry Street: Bank One Tower, Suite 3700, 111 Monument Circle City: Indianapolis State: Indiana Country: USA
Zlp Code: 46204-5137 Computer Readable Form:
Medium Type: Diskette, 3.50 inch, 1.14 Mb storage Computer: COMPAQ
Operating System: MSDOS
Software: ASCII
Current Application Data Application Number: Not Yet Assigned Filing Date: February 28, 1997 Classification:
Priority Application Data Application Number: 60/012,423 Filed: February 28, 1996 Classification:
Attorney Information:
Name: Thomas Q. Henry Registration Number: 28,309 Reference/Docket Number: 7024-135/PUR-046-PCT
Telecommunication Information:
Telephone: 317-634-3456 Telefax: 317-634-7561 Information for SEQ ID NO:l Sequence Characteristics Length:
Type:
Strandedness:
Topology:
Molecule Type:

CA 02247798 l998-08-27 W097/32007 - 28 - PCT~US97103234 Sequence Description: SEQ ID NO:l Gly Asn Arg Asp Val Thr Gly Ser Gln Asn Ser Val Glu Ile Asp Ala Leu Ala Arg Phe Ala Val Glu Glu His Asn Lys Lys Gln Asn Ala Leu Leu Glu Phe Glu Lys Val Val Thr Ala Lys Gln Gln Val Val Ser Gly Thr Leu Tyr Thr Ile Thr Leu Glu Ala Lys Asp Gly Gly Gln Lys Lys Val Tyr Glu Ala Lys Val Trp Glu Lys Ser Trp Leu Asn Phe Lys Glu Val Gln Glu Phe Lys Leu Val Gly Asp Ala Pro Ala Information for SEQ ID NO:2 Sequence Characteristics Length:
Type:
Strandedness:
Topology:
Molecule Type:
Sequence Description: SEQ ID NO:2 Leu Gly Gly Phe Thr Asp Ile Thr Gly Ala Gln Asn Ser Ile Asp Ile Glu Asn Leu Ala Arg Phe Ala Val Asp Glu His Asn Lys Lys Glu Asn Ala Val Leu Glu Phe Val Arg Val Ile Ser Ala Lys Lys Gln Val Val Ser Gly Thr Leu Tyr Tyr Ile Thr Leu Glu Ala Asn Asp Gly Val Thr Lys Lys Val Tyr Glu Thr Lys Val Leu Glu Lys Pro Trp Leu Asn Ile Lys Glu Val Gln Glu Phe Lys Pro Ile Thr Val Ala Val Asn Pro Leu Ser Val Thr Val W097/32007 - 29 - PCT~US97/03234 Information for SEQ ID NO:3 Sequence Characteristics Length:
Type:
Strandedness:
Topology:
Molecule Type:
Sequence Description: SEQ ID NO:3 Ala Ala Leu Glu Lys Val Gln Glu Leu Gly Gly Ile Thr Asp Val His Gly Ala Ala Asn Ser Val Glu Ile Asn Asn Leu Ala Arg Phe Ala Val Glu Glu Gln Asn Lys Arg Glu Asn Ser Val Leu Glu Phe Val Arg Val Ile Ser Ala Lys Gln Gln Val Val Ala Gly Val Asn Tyr Tyr Ile Thr Leu Glu Ala Lys Asp Gly Leu Ile Lys Asn Glu Tyr Glu Ala Lys Val T~ Val Arg Glu Trp Leu Asn Ser Lys Glu Leu Leu Glu Phe Lys Pro Val Asn Val Ser Ser Thr Gln W097/32007 _ 30 _ PCT~S97/03234 Information for SEQ ID NO:4 Sequence Characteristics Length:
Type:
S Strandedness:
Topology:
Molecule Type:

Sequence Description: SEQ ID NO:4 15 CTAGCTCGCT TTGCTGTTGA AGAACACAAC AAAAAACAGA ATGCCCTTTT lO0 GGAGTTTGAA AAGGTGGTAA CTGCGAAACA GCAAGTGGTT TCTGGTACCT l50 GATATATCAA AGGATCCTAT ATGTATAAAA TAAATGTTGC llllllTTCG 400 25 TATGTGTTTC TTTCAAAAAA A~A~AAAAAA AAAAAAAAAA AAAAA 495 Information for SEQ ID NO:5 Sequence Characteristics Length:
Type:
Strandedness:
Topology:
Molecule Type:
Sequence Description: SEQ ID NO:5 GAAAATCTCG CTCGCTTTGC TGTTGATGAA CACAACAAAA AAGAGAATGC lO0 Information for SEQ ID NO:6 Sequence Characteristics Length:
Type:
Strandedness:

W097/32007 - 31 - PCT~S97/03234 Topology:
Molecule Type:

Sequence Description: SEQ ID NO:6

Claims (35)

What is claimed is:

1. A protein comprising an amino acid sequence having substantial identity to the amino acid sequence of SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO .

2. The protein according to claim 1, said protein having a C80 of at most about 10-4 with respect to one or more cysteine proteinases.

3. The protein according to claim 1, wherein the protein has a C80 of at most about 10-4 with respect to digestive enzymes of Western corn rootworm.

9. An isolated DNA fragment comprising a nucleotide sequence having substantial identity to the nucleotide sequence of SEQ ID
NO 4, SEQ ID NO 5 or SEQ ID NO 6.

5. A vector useful for transforming a cell, said vector comprising a nucleotide sequence having substantial identity to the nucleotide sequence of SEQ ID NO 4, SEQ ID NO 5 or SEQ ID NO
6, and regulatory elements flanking the nucleotide sequence, the regulatory elements being effective to control expression of the sequence in a plant.

6. A plant transformed with the vector of claim 5, or progeny thereof, the plant being capable of expressing the nucleotide sequence.

7. The plant of claim 6, the plant being selected from the group consisting of corn, potato and cowpea.

8. The plant according to claim 7, the plant being a corn plant.

9. A microorganim transformed with the vector of claim 5, the microorganism being capable of expressing the nucleotide sequence.

10. The microorganism of claim 9 wherein the microorganism is Escherichia coli.

11. A method for improving a plant's ability to defend against herbivorous pests comprising:
providing a vector comprising a nucleotide sequence encoding a cysteine proteinase inhibitor, and regulatory elements flanking the nucleotide sequence, the regulatory elements being effective to control expression of the nucleotide sequence in a target plant; and transforming the target plant with the vector to provide a transformed plant, wherein the transformed plant is capable of expressing the nucleotide sequence.

12. The method according to claim 11, wherein the target plant is a corn plant.

13. The method of claim 11 wherein the nucleotide sequence has substantial identity to the nucleotide sequence of SEQ ID NO 4, SEQ ID NO 5 or SEQ ID NO 6.

19. The method of claim 11, wherein the nucleotide sequence encodes a protein having an amino acid sequence having a C80 of at most about 10-4 with respect to one or more cysteine proteinase enzymes.

15. The method of claim 11, wherein said transforming comprises transforming by a direct transformation methodology selected from the group consisting of viral transformation, electroporation, microinjection, and microprojectile bombardment.

16. The method of claim 11, wherein the regulatory elements include a plant promoter.

17. The method of claim 11, wherein the target plant is selected from the group consisting of corn, potato and cowpea.

18. A transgenic plant prepared according to the method of claim 11 or progeny thereof.

19. A method for producing a cysteine proteinase inhibitor protein comprising:
providing a DNA sequence vector comprising a nucleotide sequence encoding a cysteine proteinase inhibitor, and regulatory elements flanking the nucleotide sequence, the regulatory elements being effective to allow expression of the nucleotide sequence in a target cell; and transforming the target cell with the vector to provide a transformed cell, wherein the transformed cell is capable of expressing the nucleotide sequence.

20. The method of claim 19 wherein the nucleotide sequence has substantial identity to the nucleotide sequence of SEQ ID NO 4, SEQ ID NO 5 or SEQ ID NO 6.

21. The method of claim 19, wherein the nucleotide sequence encodes a protein having a C80 of at most about 10 -4 with respect to one or more cysteine proteinase enzymes.

22. The method of claim 19, wherein s~ transforming comprises transforming by a direct transformation methodology selected from the group consisting of viral transformation, electroporation, microinjection, and microprojectile bombardment.

23. The method of claim 19, wherein the regulatory elements include a promoter.

24. The method of claim 19, wherein the target cell is an E.
coli cell.

25. A transgenic cell prepared according to the method of claim 19.

26. A method for controlling a herbivorous pest comprising causing the pest to ingest an insecticidal amount of a protein having an amino acid sequence substantially similar to the amino acid sequence of SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3.

27. The method of claim 26 wherein the pest is an insect or a larva thereof.

28. The method of claim 26 which includes causing the pest to ingest an insecticidal amount of a transgenic plant which expresses the protein.

29. The method of claim 26 wherein the pest is selected from a cowpea weevil, a Western corn rootworm larva, and a Colorado potato beetle.

30. The method of claim 29 wherein the protein exhibits a C80 of at most 10-4 with respect to a digestive cysteine proteinase of the pest.

31. A corn plant which expresses recombinant DNA encoding a cysteine proteinase inhibitor.

32. The plant of claim 31 wherein the cysteine proteinase inhibitor is from soybean.

33. The plant of claim 31 wherein the cystein proteinase inhibitor has an amino acid sequence which is substantially identical to that of SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3.

34. A method of producing a transformed plant, comprising incorporating into the nuclear genome of the plant an isolated nucleotide sequence which encodes a cysteine proteinase inhibitor to provide a transformed plant capable of expressing the cysteine proteinase inhibitor in an insecticidally-effective amount.

35. The method according to claim 28, wherein the cysteine proteinase inhibitor has an amino acid sequence having substantial identity to the amino acid sequence of SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3.