AU775719B2 - Synthetic bacillus thuringiensis gene encoding cryica (cryic) toxin - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): RAMOT UNIVERSITY FOR APPLIED RESEARCH AND INDUSTRIAL DEVELOPMENT LTD and MAX-PLANCK GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN E.V.

Invention Title: SYNTHETIC BACILLUS THURINGIENSIS GENE ENCODING CRYCA (CRYIC) TOXIN The following statement is a full description of this invention, including the best method of performing it known to me/us: i SYNTHETIC BACILLUS THURINGIENSIS GENE ENCODING CRYLCA (CRYLC) TOXIN The entire disclosure in the complete specification of our Australian Patent Application No. 46354/97 is by this cross-reference incorporated into the present specification.

FIELD OF THE INVENTION The present invention relates to a synthetic version of a gene isolated from Bacillus thuringiensis 0 (hereinafter "Bt" or thuringiensis" encoding an insecticidal crystal protein designated CryIC, plants Stransformed with the gene, and the insecticidal crystal' protein toxin expressed by the gene, all of which are used to control insects of the Spodoptera species as well as those of the Mamestra species.

BACKGROUND OF THE INVENTION Insect.infestation is responsible for millions of dollars of losses to commercially valuable agricultural crops each year. More than three billion dollars is 20 spent worldwide annually to control insect pests.

Traditionally, crops have been controlled from insect pests primarily through the use of toxic sprays.

Unfortunately, residues of the sprays remaining on the fruits and vegetables have accumulated in human tissues, often with adverse effects, while at the same time many insects have become immune or resistant to the toxins.

Additionally, the sprays often kill useful organisms, and precipitation runoff washes the toxins into streams and other bodies of water often killing fish.

Because of these and other disadvantages of using toxic sprays, alternative means of crop protection have been developed. One approach is the use of biological pesticides. One such agent is the bacteria B.

thuringiensis which has been very effective against a variety of caterpillars and worms. This bacteria has been traditionally sold in the form of a dust containing millions of spores. When the spores are sprayed on plants, they are harmless to humans and animals other than the target insect. During its sporulation cycle, Bt produces proteins toxic to certain pests in crystal form known as crystal delta-endotoxins. When the insect ingests any plant tissue with Bt spores on it, the bacteria quickly becomes active and multiplies within the insect's digestive tract, soon paralyzing the gut. The insect stops feeding within two or three hours.

The delta-endotoxin are encoded by crystal protein genes. Thus far, over 100 Cry proteins were identified and classified according to their sequence 15 homology and insect specificity (reviewed in H6fte and Whiteley, 1989; Aronson 1993, Schnepf 1995). The cry genes have been divided into six classes and several subclasses based on structural similarities and insecticidal specificity. The major classes are as 20 follows: Class Insect SDecificity cryl Lepidoptera (butterflies, moths) cryll Lepidoptera and Diptera (flies, mosquitos) cryIII Coleoptera (beetles, weevils) 25 cryIV Diptera (flies, mosquitos) cryV Coleoptera and Lepidoptera cryVI Nematode (roundworms) With particular regard to the lepidoptera specific crystal proteins to which the present invention is directed, six subclasses having different gene types have been identified. Subclasses of the cryl genes include the following: cryIA(a), crylA(b), cryIA(c), cryIB, cryIC, and cryID. CryIC endotoxin is the most active B.

thuringiensis crystal protein against the Spodoptera species which includes the following pests: S.

-2littoralis, S. exempta, S. exigua, S. frugiperda, S.

litura and others.

Unfortunately, production of the bacterial spores for commercial use is limited and the protective effect is short-lived. Accordingly, plant molecular biologists have developed transgenic plants that express the Bt toxin within their cells and tissues which have been effective against pests which feed on the leaves of the plant. For example, U.S. Patent No. 5,187,091 to Donovan et al. describes incorporating into a plant a cryIIIC gene thereby rendering the plant more resistant to insect attack. Additionally, tobacco and tomato plants expressing the Bt toxin gene reportedly have killed larvae of tobacco hornworms. However, the wild-type.

crystal gene is poorly expressed in transgenic plants.

Hence, protection is not attained against less sensitive, "but agronomically important, insect pests like the cotton bullworm. (Watson et al. Recombinant DNA, 2d ed. 1992).

The expression of the full-length lepidopteran specific Bt gene (cryl in particular has been reported to be unsuccessful in expressing insecticide in some plants.

(Vaeck et al., 1987) To increase expression in plants, truncated and synthetic genes containing codons preferred in plants 25 have been successfully employed.

U.S. Patent No. 5,380,831 to Adang et al. discloses a synthetic B. thuringiensis gene designed to be expressed in plants at a level higher than naturally occurring BC genes. The gene utilizes codons preferred in plants. The modifications described include the elimination of CUUCGG hairpins and plant polyadenylation signal ,-an m oifring the A+T rontent tto that found in plants.

U.S. Patent No. 5,500,365 to Fischoff et al.

discloses synthetic plant genes which encode insecticidal proteins of Bt for plant transformation wherein the genes express their protein product at higher levels than the -3wild-type genes. In particular, they removed regions with many consecutive A+T bases or G+C bases as well as ATTA sequences and putative polyadenylation signals, and the condon usage was changed according to plant preferences.

The insecticidal spectrum of Bt thus far expressed in transgenic plants is limited. Genes encoding the processed forms of CryIA(a), and have been expressed in plant-associated bacteria and transgenic plants to control major insect pests of maize, rice, cotton, tomato, potato and tobacco. Nonetheless, insects of the Spodoptera species, which cause severe agricultural damage, have thus far escaped efficient control because of problems preventing a high level 15 expression of CryIC toxins in transgenic plants.

Therefore, the engineering of Bt toxins with novel specificity is essential for the biological control of recalcitrant plague insects, such as Spodoptera. Members of the Spodoptera genus feed on over 40 different plant 20 families world-wide, including at least 87 species of economic importance. Armyworms, most of which fall within the genus Spodoptera, march in swarms from field to field devastatingly defoliating entire crops. In the United States corn, sorghum and peanut are crops upon which fall armyworm (Spodoptera frugiperda), infestations often reach devastating levels. In one year, for example, losses in the state of Georgia alone were estimated at over 20 million dollars. Corn yield losses attributed to the fall armyworm for the United States have been estimated at 2% annually. In the southeastern United States, S. frugiperda, is a major pest of corn, sorghum and peanut, causing more than $60 million in damages per year. From the various insecticidal crystal proteins of B. thuringiensis expressed in transgenic plants that have been disclosed in prior art none showed activity required for plant protection against Spodoptera species. Moreover, no significant differences in leaf area consumed, mortality or pupal weights of S. exigua larvae were detected between transgenic B. thuringiensis Monsanto cotton line and non-transformed plants (Burris et al., 1994).

The Spodoptera species are polyphagous cutworms and armyworms, that may amplify to enormous numbers and devastate huge agricultural areas. The wide-spread beet armyworm S. exigua attacks rice, sugarbeet, alfalfa, cotton, corn, tobacco, tomato, potato, onions, peas, citrus, sunflower, and many grasses. The Egyptian cotton leafworm S. littoralis, a major pest in African and Mediterranean countries, favors fodder crops, such as alfalfa and clover, but also feeds on may vegetables, industrial crops, medical plants, ornamentals, and trees Young Spodoptera larvae may be controlled by pyrethroids, DDT, chlorinated hydrocarbons and organophosphorus insecticides. However, because the eggs are laid on grassland, the efficiency of chemical insecticides, including the most efficient compounds methomyl and 20 Pirate (AC303630), is rather limited. During the last decades a considerable effort was therefore invested into the development of safe insecticides to control armyworms in an environmentally friendly fashion.

Despite the significant damage caused by Spodoptera insects, safe and efficient pest control through the genetic engineering of plants is lacking because of the :difficulty of achieving a high level of expression of CryIC toxin in transgenic plants. It would therefore be most desirable to have a gene encoding CryIC toxin that can be expressed in transgenic plants thereby safeguarding them against Spodoptera pests in an effective yet environmentally friendly manner.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

SUMMARY OF THE INVENTION The present invention relates to a synthetic Bt gene that expresses CryIC delta-endotoxins against Spodoptera insects when expressed in plants transfected by the gene.

*o* 5a H:Juania\Keppaternt10087-22doc 11/03/04 More specifically, it comprises a chemically synthesized gene coding for a truncated CryIC protoxin of 630 amino acids which has been expressed in alfalfa, tobacco, and potato plants and has proven to provide resistance to S.

littoralis and S. exigua.

To improve the engineering of CryIC toxins, the gene according to the present invention establishes a consensus CryIC sequence within the boundaries of the entomocidal fragment of CryIC toxin that confers resistance to midgut proteases and larvae of Spodoptera littoralis. Insecticidal Cry proteins, produced as protoxins (65-140 kDa) in parasporal crystals of Bacillus thuringiensis are active as selective entomocidal agents. The crystalline Bt protoxins are solubilized and 15 activated in the midgut of insects of proteolysis. The activated toxins (60-70 kDa) bind to the membrane of midgut columnar cells and form ion-channels, inducing osmotic lysis of the epithelium. Engineering of insects resistance in maize, rice, cotton, tomato, potato, and 20 tobacco shows that a significant modification of the bacterial cry coding sequences is essential to express these Bt toxin genes in plants.

Various features of the natural Bt genes differ from those of plants and heterologous genes expressed in plants. Bt genes are rich in adenine and thymine T (more than 62%) while plant exons have about 45% A+T content. Fortuitous plant processing signals present in Bt genes drastically diminish the level of their expression in plant cells. Efficient transcription of the synthetic crylC gene according to the present invention in plant cell nuclei was achieved by the removal of AT rich sequences that may cause mRNA instability or aberrant splicing, and the translation of cry mRNAs is enhanced by modification of their codon usage to make it more similar to that of the host plant.

In addition, the sequence context around the translation start was modified to conform to the eukaryotic consensus.

-6- Synthesis of the synthetic gene herein was accomplished using a unique method "TDL-PCR" described herein and in more detail in our co-pending U.S.

application which is incorporated herein by reference.

The synthetic gene according to the present invention may be employed to transform most plants thereby protecting them against pests which are members of the Spodoptera genus (Lepidoptera, Noctuidae) which feed on over 40 different plant families world-wide, including at least 87 species of economic importance (Hill, 1983). For example, the widespread beet armyworm S. exigua attacks rice, sugarbeet, alfalfa, cotton, corn, tobacco, tomato, potato, onions, peas, citrus, sunflower, and many grasses. The Egyptian cotton leafworm S..

littoralis, a major pest in African and Mediterranean countries, favors fodder crops, such as alfalfa and clover, but also feeds on may vegetables, industrial crops, medical plants, ornamentals and trees.

Additionally, the CryIC toxin that is expressed by transgenic plants according to the present invention can be collected and used, for example, as an insecticidal spray due to the fact that the protein is water soluble.

CryIC toxins produced by bacteria, in contrast, are water insoluble rendering them undesirable for a variety of industrial and agricultural applications.

With the foregoing and other objects, advantages and features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed 30 description of the invention, the figures, and the appended claims.

-7- In the claims which follow and in the description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic plan for the gene synthesis method according to the present invention.

Fig. 2 shows the nucleotide sequence of the synthetic cryIC gene. Where different, the native *o oo* o* *o ooo *o o* 7a H:Uuania\Keep\pateI087-2.d 110304 (bacterial) sequence is shown above. The amino acid sequence is shown below.

Fig. 3a is a schematic map of plant transformation vectors.

Fig. 3b is a photograph of Western blots showing expression of b-cryIC and s-cryIC genes in E. coli and Arabidopsis.

Fig. 3c is a photograph of Western blots showing screening for cryIC expression in alfalfa.

Fig. 3d is a photograph of Western blots showing screening for cryIC accumulation in leaf tissue of transgenic alfalfa and tobacco plants.

Fig. 3e is a photograph of a Northern blot showing screening for transcripts of transgenes in leaves of 15 soil-grown alfalfa plants.

Fig. 3f is a Western blot analysis of leaf protein extracts from transgenic potato plants expressing the cryIC gene. 10 ng of truncated CryIC produced in E. coli (positive control), total proteins (50 jg) from plant 20 var. Desiree (negative control) and transformed plant number 1,3,5 and 6 containing 2,2,1 and 1 copies of the cryIC gene respectively, were electrophoresed on 12% polyacrylamide gels, then transferred to PVDF membrane.

The blot was incubated with rabbit-anti CryIC, and then with horseradish peroxidase conjugated to anti-rabbit immunoglobulin. Enzymatic visualization of immunoreactive CryIC was then carried out.

Fig. 3g is a Northern blot analysis of cryIC gene transcripts in transgenic potato plants. Total RNA Ag) extracted from untransformed Desiree plant and transgenic plants ARI (1,2,5 and 6) resulted from transformation with the binary vector pAR1, were electrophoresed with glyoxal in 1 agarose TBE gels', then transferred onto nylon membrane and probed with "P labeled yic gene. The number of introduced cry copies per plant is indicated below.

-8- Fig. 4 is a photograph showing screening for Spodoptera resistance in transgenic plants.

neonate larvae of S. littoralis reared on transgenic (bottom) and nontransformed (top) alfalfa (M.

sativa) plants.

"free choice" bioassays with leaves from transgenic (right) and nontransgenic (left) alfalfa plants and larvae of S. exigua (3rd instar).

leaf (C-from tobacco; D-from alfalfa) bioassays with 5th instar larvae of S. exigua reared on leaves taken from transgenic (right) and nontransgenic (left) plants.

bioassays with alfalfa soil grown transgenic (left) and non-transgenic (right) plants and larvae of S.

15 exigua.

Fig. 5 is a Bioassay of transformed potato plants (Desiree). Spodoptera littoralis larvae of the 2nd to 4th instars were fed on leaves of Desiree (control) and primary transformants. 1-plant No. AR1(2), 2-plant No.

20 AR1(1), 3-plant No. AR1(3), 4-plant No. ARI(5), No. AR1(6). Leaves were photographed 48h after being exposed to the larvae.

Fig. 6 is a Southern blot analysis which confirms the integration of the cryIC gene into plant genome.

Plant DNA (20,Ag) was digested with the restriction enzyme EcoRI in panel A and XbaI in panel B and electrophoresed on 0.8 agarose TAE gels. DNA was then transferred onto nylon membranes and probed with 32

P

labeled hph cryIC(b) and with Amp resistance gene Lane I control Desiree plant, lanes 2-8 transformed potato plants No. 1,2,3,5,6,7,8 carrying 2, 8, 2,1,1,3 and 1 copies of the cryIC gene respectively.

Partial digestion with XbaI (which is sensitive to methylation) of plant DNA extracted from potato plant carrying at least 8 copies of the synthetic cryIC gene (panel B, lane suggests possible involvement of plant hyper-methylation.

Fig. 7 is a Western blot analysis of total proteins extracted from the transgenic plant AR1(1) and incubated in vitro with gut proteases from 4th instar larvae.

Similar AR1(1) protein samples were subjected to sequential denaturation and renaturation which completely abolished their resistance to proteolysis (last mixed with long E. coli produced CryIC (630aa) were incubated with gut proteases. No resistance of bacterial CryIC to proteases was observed. Following incubation the protein samples were separated on 12% SDS-acrylamide gel, blotted onto PVDF membrane and probed with ani-CryIC polyclonal antibodies.

is 0 From left to right: Lane 1 AR1(1) proteins, no gut proteases.

Lane 2-4 AR1(1) proteins incubated with gut proteases (o.2/g proteins, depicted as enzIV) for 2, and 20 min. respectively.

Lane 5 Proteins of non-transformed Desiree plant.

Lane 6 Proteins of non-transformed Desiree plant mixed with long E. coli produced CryIC (630aa).

Lane 7-9 as in 6 but incubated with gut proteases for 1, 10 and 20 min. respectively.

Lane 10 AR1(1) proteins after denaturation and renaturation as detailed below.

Lane 11-13 AR1(1) proteins subjected to 6M Guanidinium hydrochloride (denaturation), dialyzed against Tris-HCI, pH8 over-night (renaturation) and then incubated with gut proteases for 2, and 20 min. respectively.

DETAILED DESCRIPTION OF THE INVENTION The process for gene synthesis is described in detail in Example I and is described in even greater detail in our co-pending U.S. patent serial number 6,043,415 titled "Gene Synthesis Method" filed on December 23, 1996, which is incorporated herein by reference. In short, chemically synthesized and phosphorylated oligonucleotides of the gene to be created are assembled on a single-stranded partially homologous template DNA derived from the natural or wild-type gene.

After annealing, the nicks between adjacent oligonucleotides are closed by a thermostable DNA ligase followed by repeated cycles of melting, annealing, and ligation. This template directed ligation ("TDL") results in a new single-stranded synthetic DNA product which is subsequently amplified and isolated from the wild type-template strand by the polymerase chain reaction (PCR) with short flanking primers that are complementary only to the new synthetic strand. These PCR end-primers contain suitable restriction cleavage sites for cloning of the synthetic double-stranded DNA fragments. This process is illustrated schematically in Fig. 1.

Although the gene according to the present invention was used to successfully transform alfalfa, Arabidopsis, tobacco and potato plants, it can be used to transform any other dicot in a similar manner so as to render the plant resistant to insect attack. Genetic engineering of plants with the cryIC gene (Fig.2) may be accomplished by 25 introducing a vector containing the gene into the plant cells using one of a variety of vectors known to those in the plant genetic engineering art. The synthetic cryIC :gene according to the present invention may be delivered into the plant cells by Agrobacterium mediated 30 transformation, by microinjection, by bombardment with DNA-coated microparticles, by PEG medicated transformation, by electroporation and by other techniques known by those skilled in plant genetic engineering.

The CryIC 6-endotoxin, and transgenic plants which express this insecticide, may be employed to safeguard against all members of the Spodoptera species. Important -11- Spodoptera pests include S. exigua (Beet armyworm),

S.

litura (Rice cutworm, Common cutworm), S.maurita (Paddy armyworm), S. eridania (Southern armyworm), S. praefica (Western yellow-striped armyworm), S. ornithogalli (Cotton cutworm), and others. The toxin according to the present invention is also effective against species of Mamestra genus, including M.brassica (Cabbage mo_, M.configurata (Bertha armyworm), M.illoba (Mulberry Caterpiller), M. persicariae (Beet Caterpiller) and others. The effective CryIC LC, 5 doses for M.brassica were reported at levels even 5 fold lower than those required for S.littoralis (H6fte, Whitely, 1989).

M.brassica is a serious pest on many crops, mainly Brassica crops, totally polyphagous, abundant and.

widespread. M. configurata is an important economic pest on oil seed crops such as canola, B.napus and B.rapae in Canada and the United States.

The present inventors have confirmed the insecticidal activity of CryICaS against M. Brassica and 20 M. Configurata. The crystal protein was used in feeding assays on potato tuber slices and provided efficient control.

In other experiments, the crystal protein showed activity against Phthorimaea operculella. Furthermore, 25 P. operculella was controlled also by feedings with tuber slices from transgenic plants which expressed the truncated synthetic CryIC gene of the invention.

Although CryIC 6-endotoxin is the most active Bt toxin against Spodoptera and Mamestra species, it has insecticidal activity towards other important pests of Lepidoptera order, such as Trichoplasia ni (Cabbage semilooper), Plutella xylostella (Diamondback moth) Pieris brassica (Large white butterfly), Pieris rapae (Small white butterfly) with the LC50 doses, that are comparable or even lower of those required for protection against Spodoptera insects. Therefore, synthetic cryC gene of present invention can be used not only as a -12monotransgene, but it can also be included in various strategies with multiple Be genes in order to fight or to avoid an appearance of BC resistant insect pests.

In addition to the activity towards Lepideptera, the CryIC 6-endotoxin is toxic to the larvae of several dipteran insects, such as Aedes aegypti, Anopheles gambia, Culex quinquefasciatus (Smith et al., 1996).

This fact opens a possibility to use the synthetic cryIC gene for creation of transgenic mammals in order to protect cattle and other suffering animals from dipteran vectors of various diseases as well as to protect livestock from irritating attacks of swarm of midges to increase, for example, milk or meat production.

Bt 6-endotoxins are accumulated in bacteria as insoluble inclusions, which upon ingestion by insect larvae must be activated by midgut proteases. Truncated Bt 6-protoxins are produced in E. coli as insoluble inclusion bodies, consisting of misfolded proteins, that in turn greatly reduces toxicity. However, the truncated 20 CryIC produced in transgenic plants expressing the synthetic cryIC gene according to the present invention is highly soluble which renders it useful in a variety of industrial and agricultural applications. In transgenic Arabidopsis, containing the synthetic cryIC gene of the 25 present invention, crylC protein was accumulated up to 1% 'of total soluble protein, 25 ng per microliter in contrast to the solubility of the truncated CryIC produced in E. coli (0.8 ng per microliter). Whole amount of CryIC protein is deposited in a soluble fraction of the plant cell. The fact that the plant produced CryIC 6-endotoxin is soluble permits its use as a new product exploiting its solubility properties a water based spray). The soluble CryIC protein produced by transgenic plants may be employed in insecticidal formulations either in an isolated form or with an agriculturally acceptable carrier that are well known to those skilled in insecticide formulation.

-13- One disadvantage of microbial Bt formulations is a high price of the production requiring the marginally economic use of fermenters and media for bacterial growth. However, transgenic plants with synthetic cryIC according to the present invention, for examples alfalfa, are free from these limitations. Insect self-protected plant material can be collected during several years by cutting plants in fields. Due to its water solubility, the plant produced CryIC insecticide can be easily extracted from the collected plant material.

EXAMPLES:

The following Examples are provided to illustrate the tractice of the invention and are not intended to limit the scope thereof.

EXAMPLE I--Gene Synthesis Gene Construction Figure 2 shows the nucleotide sequence of the 20 synthetic cryIC gene (s-cryIC). Nucleotides of the bacterial cryIC sequence (b-cryIC) exchanged in the synthetic gene are shown in the upper lanes. The nucleotide sequence of the s-cryIC coding region for 630 codons starts with an ATG codon in a sequence context fitting the eukaryotic consensus and terminates at a TAG stop codon. Arrowheads above the s-crylC sequence indicate the boundaries of adjacent synthetic oligonucleotides used for TDL-PCR gene synthesis. HincII and BglII cleavage sites used for the assembly of three TDL-PCR blocks are indicated by boxes above the sequences. The amino acid sequence of the truncated CryIC 5-endotoxin is displayed in single letter code below the s-cryIC sequence.

The designed DNA sequence of the s-crylC gene (Fig.

2) was divided into three blocks separated by HincII and BglII cleavage sites. The BamHI-HincII block-I was constructed from eight, the HincII BglII block-II from five, and the BglII-BamHI block-III from seven Oligonucleotides. The oligonucleotides were assembled on a single-stranded DNA template of phagemid pR1, carrying the 630 N-terminal codons of the wild-type B.

thuringiensis cryIC gene (Fig. 1 and Terminal oligonucleotides in each TDL-PCR block carried unique sequences on their 5' and 3' ends, which were not complementary with the template, but were matched to short PCR primers for selective amplification of the synthetic DNA strand. These PCR primers contained unique restriction enzyme cleavage sites used for cloning of the amplified double-stranded DNA fragments into pBluescript.

The TDL-PCR block-I was PCR amplified by a 5'-primer AAGAGGATCCACCATGGAGGAGAAC-3'), carrying a BamHI site and.

a 3'-primer (5'-ATGATCTAGATGCAGTAGCG-3'). The 3'-primer was complementary to an oligonucleotide

GTCAACTAACAAGGGAAGTTTATACGGACCCACGCT

ACTGCATCTAGATCAT-3') at the 3'-end of block-I, that carried cryIC sequences with the HinclI site, and 20 unrelated overhang sequences with an XbaI site. The oligonucleotide at the 5'-end of block-II

GATAACTCGAGCGAGCCTAAACTATGACAATAGG

•AGATATCCAATTCAGCCAGTTG-3') added uniaue DNA sequences with an XhoI site to the cryIC sequences upstream of the HincII site and matched a PCR primer GATAACTCGAGCGAGCCTA-3'). The 3' -terminal oligonucleotide in block-II carried cryIC sequences extending to the BglII site and downstream overhang sequences with an Xbal site that were complementary to a PCR primer CCTGACTCTAGAAGATC-3'). In the oligonucleotide located at the 5'-end of block-III an EcoRI site was added upstream o the 1-%Mr7TT c i f r-"rTT rc fi--inrr t a PCR primpr (5'-CTGTCTGAATICAAAGATC-3'). The oligonucleotide at the 3'-end of block-III carried a BamHI site, following the position of TAG stop codon in the pR1 phagemid, as well as adjacent unique sequences with a NotI site that were complementary to a PCR primer TDL Technique Template directed ligation (TDL) reactions were carried out at a template to oligonucleotide ratio 1:200 (a total of 0.05 pM of template versus 13 pM of each oligonucleotide) in a final volume of 504p using a reaction buffer (20 mM Tris.HCl (pH 20 mM KC1, mM McCI 2 0.1% NP-40, 0.5 mM rATP, 1 mM DTT)-and 4 U Pfu ic DNA ligase (Stratagene), or any other similar thermostable DNA ligase.

Thirty cycles of TDL reactions were used to obtain a desirable amour. of a TDL product. The temperature range during melting step is between 90 t) 98 0 C with 'a 5 preferable temperature cf 920, with 1 minute of requiredstep time. Annealing and ligation were performed at a temperature range of 45 to 60 0 C with a preferable temperature of 52°C during required step time from 3 to 10 minutes. Melting step was followed by annealing and 2C ligation step to obtain a TDL cycle which was repeated at least 30 times. To increase the number of TDL cycles for every additional 30 cycles a new portion cf rATP (0.5 mM and 4 U of Pfu ligase was added. Temperature cycling during TDL step was done on a Perkin-Elmer thermal cycler 25 (Norwalk, CT) PCR Selective Amplification of Synthetic TDL-PCR Blocks pl from the TDL reaction mix served as template for PCR amplification with 100 pM of primers, 250 pM dNTP and 2.5 U Ampli-Taq or any other similar thermostable DNA polyer.rase such as UlTma (Perkin-Elmer) polymerase in 100 p1 buffer (10 mM Tris.HCl (pH 50 mM KC1 and 0.1 Triton- X100), using 30 cycles at 92 0 C for I min, for min, and at 72 0 C for 1.5 minutes, with final extension for 10 minutes, at 72° C. PCR amplifications were performed on a Perkin-Elmer (Norwalk, CT) thermo 16 cycler. The amplified DNA fragments were gel purified, digested with BamHI-XbaI (block-I), XhoI-XbaI (block-II), and EcoRI-NotI (block-III), then cloned in pBluescript SK to verify their DNA sequences.

Desian of Synthetic cryIC Gene Aimed to be Transferred to Plants a) Identification of the minimal entomocidal fragment of Spodoptera specific BC toxin CryIC. From prior art it is known that expression of the truncated Bt genes in transgenic plants was superior compared to the full-length genes of protoxins. Moreover, identification of the minimal toxic fragment of a protoxin would significantly reduce efforts and costs involved in construction of a synthetic gene. In CrylA proteins the 15 tryptic fragment, which is still responsible for toxicity, composes a half of the protoxin. To improve the production of recombinant CryIC toxins, we mapped the minimal toxic fragment of the protoxin by testing of various truncated proteins for the stability and protease 20 sensitivity to trypsin and Spodoptera midgut protease as well as by the measurement of the insecticidal activity to S. littoralis larvae. In contrast to previous data, the boundaries of trypsin-resistant CryIC core toxin were Mapped to amino acid residues 128 and R627. Proteolysis 25 of the truncated CryIC proteins showed that Spodoptera midgut proteases may further shorten the C-terminus of CryIC toxin to residue A615. However, C-terminal truncation of CryIC to residue L614, and a mutation causing amino acid replacement I610T abolished the insecticidal activity of CryIC toxin to S. littoralis larvae, as well as its resistance to trypsin and Spodoptera midgut proteases. Because no CryIC toxin carrying a proteolytically processed N-terminus could be stably expressed in bacteria, our data indicate that in contrast to other CryIC proteins an entomocidal fragment located between amino acid positions 1 and 627 is -17required for stable production of recombinant CryIC toxins.

b) Establishment of the CryICa5 protein sequence.

Design of the synthetic cryIC gene was based on the sequence of the corresponding wild-type gene EMBL X96682), which we established after sequencing of three independent crylC genes isolated from three different B. thuringiensis strains K26-21, MR1-37 (new isolates, collected from the soil samples in Kenya and Israel, respectively) and subsp. aizawai 7.29, all selected from high insecticidal activity against larvae of the S. littoralis. We have found that all the three strains contain the identical cryIC gene sequence, which has certain discrepancies with all the cryIC sequences 15 known in prior art (cryICal Honee et al.; crylCa2 Sanchis et al.; crylCa3 U.S. Patent 5,246,852, 1993; crylCa4 0400246, 1995). Moreover, in fact, the sequence represents, a consensus of all 12 known cryIC genes.

20 Corresponding sequence of CryICa5 protein differs by amino acid replacement A124E, A294R, and H453D from the CryICal, by a T450Q exchanges from the CryICa3, and by the A124E from the CryICa4. Similarly, multiple sequence shifts resulting in N366I, V386G and 376WPAPPEN382 to 376CQRHHFN382 amino acid replacements were found in the .previously published CryIC sequence from Subsp. aizawai 7.29 (Sahchis et al, 1989). The occurrence of glutamate in position 124 and glutamine in position 405 were clearly due to previous errors, since A124 and T405 were found to be conserved in all CryIC proteins. Similarly, sequence variations detected between Positions 366 and 386 of the Cry C sequence from S-bp. aizawai 7.29 could safely be excluded because they would wither create a new tryptic cleavage site, such as the R378 residue, or affect the activity and insect specificity of the toxin, such as the W376C replacement and the 374QPWP377 motive that are located in a surface exposed loop of the -18variable toxin domain II. Therefore, we believe that the CryIC sequence, known in prior art, contain critical errors with negative consequences either for function or stability of CryIC protein, had the protein a corresponding synthetic gene designed on the basis of the wild-type DNA sequences known in prior art.

Modifications of the synthetic CryIC gene (s-CryIC) sequence of the present invention did not alter the amino acid sequence of the minimal toxic fragment of the CryICa5 protoxin, containing N-terminal fragment with the length of 630 amino acid residues.

The synthetic cryIC gene coding for an N-terminal protoxin fragment of 630 amino acids was designed (Fig.

2) by exchanging 286 bp of the bacterial cryIC sequence 15 (EMBL X96682; 1890 bp) such that 249 out of 630 codois were modified according to preferential codon usage in dicotyledonous plants. These exchanges removed 21 potential plant polyadenylation signals, 12 ATTTA motifs, 68 sequence blocks with 6 or more consecutive A/T's, and all motifs containing 5 or more G+C or A+T nucleotides.

Sequences around the translation initiation site were changed to conform to the eukaryotic consensus sequence, and a TAG stop codon was introduced downstream of amino acid codon 630. The G+C content of the cryIC gene was 25 thus increased from 36.6% to 44.8%. The s-cryIC gene was synthesized from oligonucleotides of 70-130 bases that were chemically phosphorylated at their 5'-ends. Since chemical phosphorylation is performed as the last step of automated DNA synthesis, only full-length oligonucleotides contain the 5'-phosphate group.

Bacterial cryIC sequences coding for the 630 N-terminal codons were cloned in a pBluescript vector to generate a single-stranded DNA template for ordered annealing of 5-8 synthetic oligonucleotides by partial basepairing. The adjacent oligonucleotides were assembled and ligated on this single- stranded template by a thermostable Pfu-ligase using 30-60 cycles of repeated -19melting, annealing and ligation. In combination with chemical phosphorylation this template directed ligation (TDL, Fig. 1) method provided a sequence specific selection for phosphorylated full-length oligonucleotides from a complex mixture of nonphosphorylated failure synthesis products, and yielded a linear amplification of single-stranded synthetic crylC DNA segments generated by ligation. Therefore, except for desalting, no additional purifications of a crude oligonucleotide mixture after chemical DNA synthesis were necessary. The TDL ligation at high temperatures also circumvented potential problems of erroneous annealing. The synthetic cryIC sequences were converted to double-stranded DNA fragments and specifically amplified by PCR using short end-primers that did not anneal to the bacterial cryIC template carried by the pBluescript vector. The s-crylC gene was thus synthesized from three sequence blocks that were combined by ligation of HinclI and BglII digested DNA fragments, and cloned in pBluescript.

20 With further reference to the Figures relating to this Example, Fig. 2 shows the nucleotide sequence of the synthetic cryIC gene (s-cryC) Nucleotides of the bacterial cryIC sequence (b-crylC) exchanged in the synthetic gene are shown in the upper lanes. The 25 nucleotide sequence of the s-cryIC region coding for 630 codons starts with an ATG codon in a sequence context fitting the eukaryotic consensus and terminates at a TAG stop codon. Vertical black arrows above the s-crylC sequence indicate the boundaries of adjacent synthetic oligonucleotides used for TDL-PCR gene synthesis. HincII and BglII cleavage sites used for the assembly of three TDL.-PCr blocks are framed.

It will be understood by an artisan of average skill in the art that variations of the gene or of the toxin domain protein sequence can be created by well known molecular biology techniques. Biological assays in line with assays described herein allow for testing of the variants to determine bio-suppression of insects. It is therefore to be understood that functional variants of the synthetic gene or protein are within the scope of the invention.

For example a variant gene can be produced by sitedirected mutagenesis, whereby individual nucleotides are replaced, or short stretches of nucleotides are added or deleted in a manner that would not change the coding frame of the remaining sequence. Expression of the variant gene would produce a variant protein sequence.

changes in the amino acid sequence would be particularly tolerated if these changes comprise conservative substitutions of amino acids. For example, leucine and isoleucine can be substituted for each other and would be considered a conservative amino acid substitution which would not be expected to substantially affect the folding, toxicity, or insect range of the toxin.

conservative amino acid substitutions are well known in the art.

u -21- TABLE 1 Summary of Changes Introduced in the Truncated Synthetic crvlC Gene (s-crvlC Compared to the Natural Counterpart (n-crvlC) s-crvIC n-crvIC G+C content 44.8% (exon like) 36.6% (intron like) Bases different from wild type 285 of 1890 (15.1%) Codons different from wild type 249 of 630 (39.4%) Potential plant polyadenylation -21 sequences (Dean et al., 1986) ATTTA sequences 12 10 A+T rich regions 68 6 Consecutive A and/or T) All codons rarely used in plants and present in the wild type crYlC were substituted by the most preferred codons in alfalfa and dicots plants.

G+C runs of 5 or more and A+T runs of 5 or more were avoided in the synthetic crylC.

The sequences upstream of the translation initiation site was changed according to the eukaryotic consensus sequences.

o EXAMPLE II Plant aene exoression constructs and transformation of alfalfa and tobacco.

20 The plant expression vector pPCV91 was constructed S by modification of pPCV720. A NotI site in the RK2domain was eliminated by filling in with DNA polymerase Klenow fragment, and a CaMV35S promoter with four repeats of the enhancer domain (-90 to -418), was introduced into the HindIII site of pPCV720. Upstream of a BamHI cloning site this cassette contained 20 bp from the 3'-end of the traslated 0 leader sence o f tobacco mosicr virus (TMV) RNA, whereas downstream of the BamHI site it carried a polyadenylation signal sequence derived from the CaMV 35S RNA gene. A BamHI site present in the mannopine synthase dual promoter (pmas) of pPCV720 was -22replaced by a NotI site using a Sau3A-NotI adaptor GATCTGCGGCCGCA-3'). The resulting vector pPCV91 carried three plant gene expression cassettes with unique BamHI, NotI and SalI cloning sites. To construct pNS6, the synthetic crylC gene was cloned as a BamHI fragment downstream of the CaMV35S promoter. In pNS7, a synthetic pat gene, encoding phosphinothricine acetyltransferase and a chiAII gene from Serratia marcescens were inserted into the SalI and NotI sites located respectively downstream of the mas 1' and 2' promoters. In pAR1, the bacterial signal peptide of chiAII was substituted by the plant leader peptide derived from potato proteinase inhibitor. A bacterial cryIC gene from B.thuringinesis sub so.. aizawai 7.29 (EMBL X96682), carrying the 756 N- 1i terminal codons of cryIC, was cloned in pGIF1 in which it replaced the synthetic crylC gene of pNS7. Vectors pNS6, pNS7, pGIF1 and pAR! were conjugated to Agrobacterium tumefacines GV3101(pMP90RK), and used for transformation of alfalfa (Medicago sativa L. var. Regen S clone RA3) 20 and tobacco (Nicotaina tabacum SRI) and potato (Solanum tuberosum var. Desiree) as described (D'Halluin et al., 1990; Koncz et al., 1994). To select for transformed explant, alfalfa, tobacco, and potato tissue culture media contained 40 pg /ml for alfalfa and 15 pg/ml of hygromycin for the last two were used.

Seeds of tobacco transformed according to the procedure set forth herein were deposited as patent ~deposits on December 23, 1996, under the Budapest Treaty on the International Recognition of the Deposit of 30 Microorganisms for Patent Purposes at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland under Accession Number ATCC 97837.

EXAMPLE III Monitoring the expression of CrvIC in transcenic plants Bacterial and synthetic cryIC genes, coding for the 630 N-terminal amino acids of the CryIC toxin (Fig. 2), were cloned into the BamHI site of a pAEN4 vector carrying the CaMV35S gene expression cassette of pPCV91.

Arabidopsis thaliana protoplast were isolated from root cultures and transformed by PEG-mediated DNA uptake, using 1.5 x 106 protoplast and 35 gg plasmid DNA in each experiment. The protoplast were harvested 48 hours after DNA uptake and lysed in SDS--sample buffer to separate proteins on 10% SDS-PAGE before immunoblotting. A.

olyclonal antibody used for immunoblotting was raised 15 against a truncated CryIC 6-endotoxin carrying 756 Nterminal amino acids. Expression of CryIC in E.coli strains, carrying bacterial or synthetic crylC genes respectively in pET-lla or lid, was monitored-by a second alkaline phosphatase conjugated goat anti-rabbit antibody. Immunoblott analysis of proteins synthesized in plant cells was performed using an ECL kit (Amersham).

RNA (20 gg) samples isolated from leaves and petioles of alfalfa plants were separated on agaroseformaldehyde gels. BamHI fragments (1.9 kb), carrying '25 either with synthetic or bacterial CryIC sequences (Fig.2), and a NotI fragment with the chiAII gene (1.8 kb) were labeled by random-priming and used as hybridization probes. Similarly, RNA (20 pg) extracted from transgenic potato plants transformed with pAR1 was also subjected to Northern analysis, using separately scryIC as well as rDNA sequence as a specific and general probe respectively.

-24- EXAMPLE IV Insect bioassay Leaf bioassay were performed with the Egyptian cotton leafworm (Spodoptera littoralis) and the beet armyworm (Spodoptera exigua) using neonate, 2-3rd, 3-4th, and 4-5-6th instar larvae. Ten larvae of a selected developmental stage were placed on a moistened filter disc in Petri dishes with detached leaves from greenhouse grown plants. The assays were repeated 2-3 times for each plant. The mortality of neonate larvae was scored after 3 days, whereas the mortality of larvae from 2-4th and from 4-6th instar stages were evaluated respectively after 5 and 7 days. For the insect assays with whole p. olants, transgenic greenhouse grown alfalfa iines 15 producing 0.02 to 0.1% of total soluble protein as Cr-yIC and S. exigua larvae of the 3-4th instar stage were used.

Three NS7 and three NS6 transgenic, as well as wild-type plants were infested with 15-20 larvae each. In "freechoice" experiments, 25 larvae were placed in a Petri 20 dish located between transgenic NS6 or NS7 and nontransgenic alfalfa plants in the greenhouse. Leaf damage was evaluated after 6 days.

Potato leaves expressing about 0.02-0.05% of their total proteins as CryIC were assayed for their toxicity 25 to S.littoralis. Only a single primary transformant out of 10 was less resistant to Instar III and IV larvae.

This plant was later shown to contain at least 8 copies of s-crylC that probably co-suppress each other.

EXAMPLE V Expression of crvlC genes in E.coli Arabidoesis, alfalfa, tobacco, and potato Fig. 3(A) comprises a schematic map of plant transformation vectors. The synthetic s-cryIC gene is cloned in an optimized gene expression cassette in pNS6 between promotor (pCaMV35S) and polyadenylation sequences from the 35S RNA gene in Cauliflower Mosaic Virus. The CaMV35S promotor contains 4 repeats of the upstream enhancer region (-90 to -418) marked by open boxes. The same CaMV35S expression cassette is carried by a pAEN4, a vector used for transient expression of bacterial b-cryIC and synthetic s-cryIC genes in Arabidopsis protoplasts. In addition to crylC, vector pNS7 contains a phosphinothricine acetyltransferase gene (pat) under the control of mannopine synthase (mas) 1'promoter, and chitinase AII (chiAII) gene driven by the mas 2' promoter. The chiAII was a plant leader peptide instead of the native bacterial substitutes the original .chiAII in pARI, the rest of the plasmid sequence is similar to pNS7, pAR1, the rest of the plasmid sequence.

15 is similar to pNS7. pAR1 was introduced into potato and tobacco using Agrobacterium tumefaciens GV3101, The s-cryIC gene of pNS7 was exchanged for the bacterial b-cryIC gene in pGIFl. The structure of pGIF1 is otherwise identical with that of pNS7. Abbreviations: 20 ori T and oriv, conjugational transfer and vegetative replication origins of plasmid RK2; LB and RB, the left and right 25 bp border repeats of the T-DNA, respectively; oripBR, replication origin of pBR322; Ap

R

bacterial ampicillin resistance gene; pg5, promoter of 25 gene 5; pnos, nopaline synthase promoter; hpt, hygromycin phosphotransferase gene; pA4 and pA7, polyadenylation signal sequences of the T-DNA encoded genes 4 and 7, respectively; pAg polyadenylation signal sequence of the octopine synthase gene. Open arrows label plant promoters, black boxes mark plant polyadenylation signal sequences.

Fig. 3 relates to expression of b-cryzC and scryIC genes in E. coli and Arabidopsis. Left. The bacterial b-cryIC and synthetic s-cryIC genes were cloned respectively in vectors pET-lla and lid, and their expression in E. coli was monitored with or without IPTG (isopropyl-f-thiogalctopyranoside) induction by -26immunoblotting, using a polyclonal anti-CryIC antibody.

The lanes contain equal amounts of protein samples gg) from E. coli extracts separated by SDS-PAGE. Right.

Arabidopsis protoplasts were transformed by PEG-mediated DNA uptake with pAEN4 and pAEN4- derived vectors carrying the b-cryIC and s-cryIC genes.

Following transient expression for 48 hrs. 25 Ag of soluble protein extracts prepared from protoplasts were separated by SDS-PAGE and subjected to immunoblotting.

To estimate the amount of CryIC toxin in plant samples, purified CryIC protein of 86 kDa (carrying amino acid residues 1 to 756) was used as standard (2 and 20 ng.) Fig. 3(C) relates to screening for CryIC expression in alfalfa calli, carrying the T-DNA of plant transformation vectors pNS6 and pNS7. Each lane contains 25 Ag of soluble proteins from calli. For comparison, Arabidopsis protoplast extract th), shown in lane 3 of was loaded as standard, in addition to control protein extracts prepared from callus tissues of wild 20 type (wt) nontransformed alfalfa.

Fig. 3(D) Screening for CryIC accumulation in leaf tissues of transgenic alfalfa and tobacco plants.

Soluble proteins (50 Ag) were prepared from NS6 (lanes 1 and 3-6) and NS7 (lane 2) alfalfa transformants, as well 25 as from transgenic tobaccos carrying the NS7 s-cryIC gene Sconstruct (bottom lanes 1-6) Fig. 3(E) Screening for transcripts of transgenes in leaves of soil-grown alfalfa plants carrying the T-DNA of pGIFI, pNS6 and pNS7 vectors (three lanes each for NS6 and NS7 reflect three independent transgenic plants).

Each lane in the three identical. blots contains 20 Ag total RNA. The blots were hybridized respectively with s-cryIC, b-cryIC and chiAII probes labeled to similar specific activity. Although several GIFI transgenic plants expressing the chiAII gene were found during this screening (data not shown), no expression of the b-cryIC gene was detected in any GIFI transformant. (The positive -27hybridizations with the b-cryIC probe are due to the partial homology between the synthetic and natural cryIC genes and the difference in the intensity of hybridizations with the s-cryIC and b-cryIC probes reflects differences between these cryIC sequences.) Fig. 3F is a Western blot of total proteins Ag/lane) extracted from leaves of transgenic potato plants transformed with pAR1 and probed with anit-CryIC polyclonal antibodies. The left lane contains 10ng E.

coli produced CryIC (first 630 amino acids). CryIC constituted for about 0.02 to 0.05% of the total leaf proteins.

Fig. 3G is a Northern blot analysis of leaf RNA (16gg) extracted from transgenic potato plants and probed.

15 with the coding region of s-cryIC (shown in Fig. 3G) and then with rDNA probe to evaluate the total RNA amount loaded on each lane. Transgenic plants AR1-6, AR1-7, and AR1-8 revealed equal levels of s-cryIC transcripts while 2 fold level was found in AR1-1 and 20 1/1- level in AR1-2 that also revealed less production of CryIC and low resistance to the larvae.

Bacterial and synthetic crylC genes were cloned respectively in vectors pET-lla and lld, and expressed in E.coli. The synthesis of CryIC protein was monitored by 25 immunoblotting. In comparison to cells harboring the bacterial crylC gene, the expression of the synthetic gene in E. coli yielded a significantly lower toxin level (Fig. 3B).

The native and synthetic cryIC genes were inserted between promoter and polyadenylation signal sequences of the Cauliflower Mosaic Virus (CaMV) 35S RNA gene in the plant gene vector pAEN4. In pAEN4 the 5'-ends of cryIC genes were fused to untranslated 2 leader sequences of the Tobacco Mosaic Virus (TMV) to enhance the translation of mRNAs, whereas the upstream CaMV 35S promoter was supplemented with 4 repeats of the enhancer domain to -418), to stimulate the transcription of chimeric -28genes in plants The cryIC genes were introduced by PEG-meditated transformation into Arabidopsis protoplasts, and the accumulation of CryIC toxin was monitored by immunoblotting following transient gene expression (Fig. 3B). In protoplasts carrying the bacterial gene no toxin was detectable, whereas cells transformed with the synthetic gene accumulated significant amount of CryIC protein (Fig. 3B).

The cryIC genes were transferred into pPCV91, a T- DNA-based transformation vector carrying a selectable hygromycin resistance (hpt) gene. In the dual gene expression cassette of pPCV91 (Fig. 3A), a synthetic phosphinothricine acetyltransferase (pat) gene was cloned downstream of the mannopine synthase (mas) 1' promoter, 15 to link the cryIC genes to a genetic marker allowing field-selection of transgenic plants by the herbicide BASTA. A chitinase AII (chiAII) gene from Serratia marcescens was inserted downstream of the mas 2' promoter, because our previous studies indicated that 20 chitinases may enhance the insecticidal activity of Bt toxins by destroying the chitinous peritrophic membrane of insect midgut. The pPCV91 constructs, carrying the native, or synthetic, crylC genes either alone, or in combination with a pat and chiAII genes, were introduced 25 by Agrobacterium-mediated transformation into alfalfa, tobacco, and potato. From tobacco and potato calli and somatic embryos of alfalfa selected on hygromycin, transformed shoots were regenerated. Transgenic plants derived from each transformation were assayed for the synthesis of CryIC toxin in leaves by immunoblotting, and for cryIC gene expression using RNA hybridization. In calli or in plants carrying the bacterial cryIC gene (confirmed by DNA hybridization, data not shown), neither stable steady-state crylC mRNA (Fig. 3E) nor toxin could be detected (data not shown). In contrast, transformed calli (Fig. 3C) as well as shoots carrying the synthetic gene (Fig. 3D), synthesized the CryIC toxin and -29accumulated significant amounts of steady-state crylC mRNA (Fig. 3E). Shoots producing detectable amounts of CryIC toxin (0.01-0.2% of soluble leaf proteins) were vegetatively propagated and, if they carried the pat and chiAII genes, were further exposed to BASTA selection in the greenhouse and tested by RNA hybridization (Fig. 3E) using the corresponding genes as probes.

EXAMPLE VI Assaying Resistance of cryIC Transgenic Plants to the Eqvotian Cotton Leafworm and Beet Armvworm Transgenic alfalfa plants obtained by transformation with the pNS6 and pNS7 constructs (Fig. 3A) were tested for insect tolerance by feeding leaves to neonate larvae of the Egyptian cotton leaf worm littoralis) 15 out 15 of 27 NS6 transformants, and 14 out of 32 NS7 transformants produced 100% mortality of larvae (Fig. 4A, Table Immunoblotting of leaf protein extracts showed that these plants produced 0.01-0.1% of total soluble protein as CryIC toxin in leaves (Fig. 3D). Leaves from these plants used in the diet of beet armyworm (S.

exigua) also caused 100% mortality of larvae throughout their development (Fig. 4C-D), Table Screening of the NS7 transgenic alfalfa demonstrated that 15 out of 32 tested plants exhibited the high level CryIC 25 production (0.02-0.1% of -total soluble protein), 2 plants had low toxin levels (less than 0.02%) and in plants CryIC levels were below the detection limit of immunoblotting with 50 mg of soluble protein.

NS6 transgenics consisted of 5/15 of high level 7/15 low level and 3/15 undetectable CryIC expressors.

About 80 hygromycin resistant potato plants (cultivars Desiree) were regenerated and 9 of them were subjected to molecular analysis and bioassays. Two out of 9 plants did not express CryIC and were sensitive to S.littoratis larvae. The rest of the 7 plants displayed resistance to all instar larvae. While the plant AR1-2 was less resistant, the other 6 plants were totally resistant to all instar larvae (Fig. 5) and contain 1-3 inserted copies of s-cryIC. At least eight copies of scryIC were detected in the plant AR1-2. The potatoproduced CryIC was less susceptible to proteolysis by the S. littoralis gut proteases (Fig. but denaturation and renaturation in vitro render it susceptible to proteolysis.

From 63 NS7 tobacco transformants 42 lines (66.6%) were resistant to 1.0% BASTA, Proper Mendelian segregation of a BASTA and hygromycin resistance markers was confirmed after selfing 11 transgenic tobacco lines.

From these BASTA resistant plants 10 stocks were assayed by immunoblotting and found to produce 0.1-0.2% of leaf soluble proteins as CryIC toxin (Fig. 3D) and resulted in 100% mortality of S. exigua larvae. 3 from these lines were used in bioassays with S. exigua and found to cause 100 mortality of larvae from different developmental stage.

The insecticidal assays showed no difference between plants carrying the cryIC gene alone or in combination with the chiAII gene. A synergistic effect between chitinase AII and CryIC toxin could have escaped our detection because toxin levels as low as 0.01% of total plants were sufficient to kill all larvae. To imitate field conditions, CryIC expressing plants were infested by 15-20 larvae of 3rd-4th instar stage in the greenhouse. After 6 days, no viable insect escapes were detected and the transgenic plants suffered barely detectable leaf damage; on average less than 1% of the leaf area was affected. Infestation of a mixed population of wild-type and transgenic plants carrying the synthetic cryIC gene resulted in devastation of wildtype, but yielded no apparent colonization of worms on the CryIC toxin expressing plants in the population.

-31- Similar results were obtained by infesting detached leaves from these plants with larvae of S. exigua.

EXAMPLE VII Screening for Spodoptera Resistance of Transqenic Plants Fig. 4(A) shows an insecticidal assay with neonate larvae of S. littoralis reared for 2 days on leaves from non-transformed alfalfa saliva, top) and NS7 transgenic (bottom) plants.

Fig. 4(B) shows "free choice" bioassays with leaves from wild-type and transgenic alfalfa plants. In the plate to the left 10 larvae of S. exigua (3rd instar) *were placed on the red line located between leaves of wild-type (left) and NS7 transgenic (right) alfalfaplants. In the plate to the right, the larvae were placed between leaves from wild-type (left) and NS6 (right, Fig. 3D, lane 6) transgenic alfalfas. For 5 days the larvae failed to colonize leaves from the transgenic plants in both assays.

20 Fig. 4(C-D) shows leaves from tobacco and alfalfa plants were used for feeding of five fifth oo instar larvae of S. exigua for 10 hrs. Petri dishes to the left in contained leaves from nontransformed plants. Leaves shown in Petri dishes to the right in (C- 25 D) were collected from a NS7 tobacco transgenic line ~producing 0.2% of soluble proteins as CryIC toxin (Fig.

3D, lane and from a NS6 alfalfa transformant producing 0. 1 of leaf proteins as CryIC toxin, respectively.

Fig. 4(E) shows transgenic NS7 (left, Fig. 3D, lane 2) and nontransformed alfalfa. (right) plants were infested with 15 larvae of S. exigua (3-4th instar stage) for 6 days.

Fig. 5 demonstrates the resistance of transgenic potato leaves resulted from pAR1 introuction to 2nd, 3rd and 4th instar larvae of S.littoralis. Leaves of plant -32- AR1-2 less resistant while the control leaves were partially or totally consumed by the young and older larvae respectively. The leaves were photographed after 24h of exposure to the larvae.

Fig. 6 is a Southern analysis of transgenic potato plants showing the integration of 1 to at least 8 copies of CryIC. The plant number is indicated above the lanes, probes-below the panels.

Fig. 7 is a Western blot performed with total proteins extracted from transgenic potato leaves or from E.coli expressing the 630aa CryIC, and incubated for 2, 20 min. with gut juice of the 4th instar larvae of S.liCoralis. The E.coli produced CryIC is more susceptible to proteolysis. Denaturation of the plant CryIC analysis its resistance to proteolysis.

The control of Spodoptera (armyworms) by transgenic alfalfa plants shown in Table 2.

TABLE 2 Mortality of S. littoralis neonate larvae 95-100% 30-90% NS6 15/27 5/27(18.5%) 7/27 (33.3%) NS7 14/32 5/32 13/32 (40.6%) Mortality of S. Exigua larvae fed on leaves of plants with 0.02-0.1% CrvIC toxin level Transgenics NS6 NS7 Time of scoring (number of plants tested) (days) Instar 1 100% 100%(3) 3 2-3 100%(5) 100%(7) 3-4 100%(2) 100%(3) 4-5-6 100%(3) 100%(2) 7 Out of 60 NS7 transgenic alfalfa plants 14 lines were found to be resistant to 0.1-0.2% BASTA. From these plants 9 lines displayed high levels of CryIC -33toxin production in leaves and caused 100% mortality of both S. littoralis and S. exigua larvae.

*The figures show the ratio between the numbers exhibiting the corresponding mortality rate to the total number of transgenic plants tested; in parenthesis this fraction in Although only preferred embodiments are specifically described herein, it will be appreciated that many modifications and variations of the present invention are possible in light of the above teachings and within the purview of the appended claims without departing from the spirit and intended scope of the invention.

*a a.

-34-

Claims (8)

1. An isolated DNA sequence comprising the nucleotide sequence shown in Figure 2 (SEQ ID NO.1), which codes for a Bacillus thuringiensis CryIC toxin having between 615 and 630 amino acids.

2. The isolated DNA sequence of claim 1, wherein said nucleotide sequence codes for a Bacillus thuringiensis CryIC toxin having 630 amino acids.

3. A vector comprising the isolated DNA sequence of claim 1 or claim 2.

4. A plant cell comprising the isolated DNA sequence of claim 1 or claim 2. C. A transgenic plant comprising the isolated DNA 15 sequence of claim 1 or claim 2.

6. A transgenic plant according to claim 5, wherein said plant is selected from the group consisting of potato, tomato, cotton, sunflower, corn, alfalfa, rice and rapeseed. 20 7. A method of increasing plant resistance to an insect pest, comprising the steps of: transforming a plant cell with an isolated DNA sequence consisting of the nucleotide sequence shown in Figure 2 (SEQ ID NO:1), 25 which codes for a Bacillus thuringiensis CryIC toxin having between 615 and 630 amino acids; and regenerating a fertile transgenic plant from said transformed plant cell, which expresses said CryIC toxin.

8. A method according to claim 7, wherein said nirl1 Pnt- ide seence codes f-r RBacil 7 7 ius th- r'in-'i ns CryIC toxin having 630 amino acids.

9. A method according to claim 7, wherein said transgenic plant is selected from the group consisting of potato, tomato, tobacco, cotton, sunflower, alfalfa, rice, rapeseed and corn. H:\Emma\Keep\Specis\46354.97 .doc 6/04/00 36 A method according to claim 7, wherein said insect pest is in a genus selected from the group consisting of Spodoptera, Mamestra, Phtorimea, Trichoplusia, Plutella, Pieris, Chilo and Sciropophage.

11. An isolated DNA sequence according to claim 1, substantially as hereinbefore described with reference to the examples and drawings. Dated this 2 3 rd of June 2004. RAMOT UNIVERSITY FOR APPLIED RESEARCH AND INDUSTRIAL DEVELOPMENT LTD and MAX-PLANCK GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN E.V. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H:Unita\Keppwm\10087.02 caims dox 23/06/04