J. Biosci., Vol. 21, Number 5, September 1996, pp 673685. Printed in India.

Cloning of a cryIIIA endotoxin gene of Bacillus thuringiensis var. tenebrionis and its transient expression in indica rice
TONY M JOHNSON*, A S RISHI**, PRITILATA NAYAK and SOUMITRA K SEN
Plant Molecular and Cellular Genetics and Centre for Plant Molecular Biology, Bose Institute, Centenary Building, P1/12, CIT Scheme VII-M, Calcutta 700 054, India Present address: *Department of Biochemistry, College of Biological Sciences, 140 Gortner Laboratory, 1479 Gortner Avenue, St. Paul, MN 55108-1022, USA **Biotechnology Division, Rallis India Limited, Rallis Agrochemical Research Station, Bangalore 560 058, India MS received 1 September 1995; revised 13 June 1996 Abstract. Damage caused to rice production by coleopteran insects like rice weevil (Sitophilus oryzae), a stored grain insect pest and rice hispa (Dicladispa armigera), a pest of the growing plant is quite high. In order to combat the damage, generation of insect resistant transgenic rice plant was considered desirable. CryIIIA endotoxin of Bacillus thuringiensis var. tenebrionis, a 65 kDa protein toxic to coleopteran insects, figured as the candidate gene product. Thus, the cryIIIA gene was isolated from a local isolate of Bacillus thuringiensis var. tenebrionis. The gene was tailored at the N-terminal end to its minimal size by using a synthetic ATG codon which replaced the first codon next to ATG of threonine to proline. This modification did not affect the functional property of the gene product. A chimeric construct of the modified cryIIIA gene was developed containing CaMV35S promoter and nos terminator for plant expression. The expressibility of the cryIIIA gene in indica rice was judged through test for transient expression in indica rice protoplasts. Keywords. Bt cryIIIA gene; coleopteran rice pest; N-terminal truncation; sandwich ELISA; transient gene expression.

1. Introduction Postharvest loss due to storage grain pests in cereals and seed legumes is enormously high in developing countries due to inadequate protection measures practised for economic reasons against these pests. Researchers have focused on the possible ways to endow plants with improved survival in field by generating transgenics with insecticidal crystal protein (ICP) genes of Bacillus thuringiensis (Bt) (Delannay et al 1989; Gasser and Fraley 1992; Wilson et al 1992), proteinase inhibitor genes (Hilder et al 1987; Johnson et al 1989), genes for lectins (Chrispeels and Raikhel 1991) and -amylase inhibitors (Huesing et al 1991; Shade et al 1994). It is known that rice weevil (Sitophilus oryzeae L.) is the most destructive insect pest of grains in the world. Additionally, rice hispa (Dicladispa armigera), another coleopteran insect pest of growing rice plants causes considerable damage (Herdt 1991). The strategy to develop insect resistant crop plants by making Bt gene express in plants has already yielded useful results in certain crop plants including cotton, rape and corn. The ICP produced by B. thuringiensis var. tenebrionis (Btt) is a 65 kDa -endotoxin of CryIIIA

Corresponding author (Fax, 091-33-343886; Email, soumitra@boseinst.ernet.in).

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type (Hofte and Whitely 1989), toxic to coleopteran insects. A strain of Btt producing crystal protein of CryIIIA type was isolated locally. On feeding tests, the grubs of rice weevil were found to be sensitive to the crystal protein produced by this isolate. In pursuance of our interest in the ultimate development of Bt transgenic crop plants, we report in this communication our success in cloning a cryIIIA gene from the bacterial strain that we isolated. The peptide expressed by the Bt gene in Escherichia coli was found to be toxic to the larvae of rice weevil. Furthermore, it was observed that the gene could transiently express in rice protoplasts. 2. Materials and methods 21 Isolation and characterization of the local isolate of Btt

A strain of Btt was isolated from rice grain dust. The identification and characterization of the strain was carried out on the basis of tests carried out at Kyoshu University, Japan; and on the basis of crystal protein character, entomocidal property and immunological analysis as revealed through ELISA using a reference protein of CryIIIA (Krieg et al 1983), as standard. 2.2 Purification of ICP from Btt Crystal proteins were purified from the sporulated culture of the isolated strain of Btt, following the procedure of McPherson et al (1988). SDS-PAGE analysis revealed that the purified CryIIIA protein contained a single polypeptide of 65 kDa (figure 1). Polyclonal antibodies were raised in rabbit against the 65 kDa protein and the specificity of the polyclonal antibody was confirmed by immunoblot analysis. 23 Production of monoclonal antibodies Five monoclonal antibodies (Mab) against the 65 kDa CryIIIA protein were produced in the laboratory following essentially the method of Kohler and Milstein (1975). Spleen cells from immunized mice and the non-immunoglobulin producing mouse myeloma cells (P3 63.Ag8.653) were fused at the ratio of 3:1 using polyethylene glycol (PEG, MW 1500, Boehringer Mannheim). Hybrids were selected in Dulbecco modified eagles medium (Nissui Pharmaceutical Co. Ltd) containing hypoxanthine-aminopterine-thymidine (GIBCO-BRL). Antibody secreting hybrid cells were selected by indirect enzyme-linked immunosorbent assay (ELISA). Cell lines producing antibodies were cloned by limiting dilution (Harlow and Lane 1988), using peritoneal macrophage from unimmunized Balb/c as feeder layer. Expanded cells were injected intraperitoneally with pristane (2,6,10,14-tetramethyl pentadecane) in Balb/c mice for ascitis fluid. Monoclonal IgG was purified by ammonium sulphate (45%) precipitation followed by DEAE-cellulose column chromatography (Harlow and Lane I988). 2.4 Indirect ELISA Indirect ELISA was performed according to Harlow and Lane (1988). Culture supernatants were used as primary antibody. Goat ant-mouse IgG + IgM + IgA alkaline

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phosphate conjugate and p-nitrophenyl phosphate (p-NPP) were used as secondary antibody and substrate, respectively. Optical density was measured at 450 nm in an ELISA reader (Anthos). 2.5 Isolation of the cryIIIA gene Based on the earlier report (Sekar et al 1987), we took a chance by assuming that the cryIIIA gene in the local isolate is also contained in a 59 kb BamHI fragment of the plasmid DNA. Thus, the total plasmid DNA of the Btt strain was digested with BamHI endonuclease and the ~ 59 kb fragments were shotgun cloned into pUC18 and transformed into E. coli strain DH5. Bacterial colonies containing recombinant plasmids were screened through antigen-antibody assay system against the toxin for the presence of the cryIIIA gene through colony hybridization using rabbit polyclonal antibodies as probe. Fortunately, one positive clone (pBTT1) could be isolated (figure 2). The cloned BamHI fragment revealed that the restriction cleavage pattern (figure 3) of the BamHI fragment and its partial nucleotide sequences were similar to the earlier known cryIIIA gene (Sekar et al 1987; McPherson et al 1988). The 59 kb BamHI cleaved fragment containing the cryIIIA gene cloned in pUC18 (pBTT1) produced two bands of 72 kDa and 65 kDa (figure 4) in E. coli. The two proteins were generated quite likely through the help of the Bt promoter already contained in the cloned fragment and the two ATG codons present in the same open reading frame of the cryIIIA gene (McPherson et al 1988). It has already been reported

Figures 13. (1) SDS-PAGE analysis of the crystal protein purified from the local isolate of Bacillus thuringiensis var. tenebrionis. Lane 1, molecular weight markers in kDa and lane 2, the Cry protein (4 g) purified from the local isolate. (2) Colony hybridization using rabbit polyclonal antibody as probe for the identification of the clone (arrow) containing the ICP gene. (3) Agarose gel electrophorogram of pBTT1 DNA cleaved with restriction endonucleases: lane 1, BamHI; lane 2, HindIII; lane 3, PstI, lane 4, EcoRI cleaved pBTT2; lane 5, HindIII cleaved -DNA.

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Figure 4. Immunoblot analysis: lane 1, the purified CryIIIA protein from B. thuringiensis var tenebrionis; lane 2, purified protein from E. coli cells containing the cloned cryIIIA gene.

Figure 5. (A) Strategy adopted for truncation of the N-terminal end of the cryIIIA gene. (B, BamHI, Bg, BglII, E, EcoRI, H, HindIII, P, PstI, Sp, SphI). (B) Details of N-terminal modification with the sequences involved.

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that the Bt promoters can express in E. coli albeit poorly (Adang et al 1985), which however, can be immunologically detected. Both the polypeptides are known to be equally toxic to insects (McPherson et al 1988). In order to facilitate plant expression of the cryIIIA gene, the following experimental approach was followed (figure 5A) to truncate the N-terminal end of the cryIIIA gene near the second ATG codon. A 27 kb Pstl fragment of the ICP gene contained in pBTT1 was isolated and cloned into pUC18 at the PstI site to generate pBTT2. The PstI site is situated 6 nucleotides downstream to the second ATG codon of the gene (figure 5A, B). The cloning of the cryIIIA gene took advantage of the ATG sequences of the SphI site in the multiple cloning sites of pUC18 as shown in figure 5B. This in effect caused replacement of one codon for threonine to proline, next to ATG. Proper orientation of the ligation product was ascertained through restriction with HindIII. 2.6 Expression of the cryIIIA gene in E. coli Expression of the cloned cryIIIA gene was tested in E. coli by utilising pET-3, a bacterial expression vector (Studier et al 1990). The pET-3 is a transcriptional expression vector containing a T7 promoter situated between 23 to + 26 followed by the unique BamHI cloning site with no potential translational initiation site. It also has a transcriptional terminator T downstream to BamHI site. A HindIII cleaved ~ 23 kb fragment of the gene derived from pBTT2 was cloned into BamHI site of pET-3. The cloning was carried out after all the cut ends of the DNA were endfilled with Klenow, followed by blunt end ligation. The recombinant DNA was transformed into E. coli strain BL-21 (DE3). Induction of the transformed colonies with 01 mM IPTG resulted in the synthesis of T7 RNA polymerase which eventually transcribed the modified cryIIIA gene cloned under the control of T7 promoter. Screening of the transformants was carried out through colony hybridization using rabbit polyclonal antibodies. The transformants further showed that the gene product contained normal immunological properties, as revealed when challenged individually with five different Mabs (figure 6).

Figure 6. Immunoblot analysis of the protein expressed in E. coli by the truncated cryIIIA gene using 5 different Mabs raised against the 65 kDa CryIIIA protein (lane 1, Mab/Btt1; lane 2, Mab/Btt2; lane 3, Mab/Btt3; lane 4, Mab/Btt4; lane 5, Mab/Btt5).

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E. coli cells of strain BL-21 (DE3) were grown overnight in L-broth, pelleted and concentrated by suspending in 10 mM NaCl, 10 mM Tris HCl (pH 80) and 1mM EDTA containing PMSF at 200 ng/ml. The suspension was sonicated on ice with three 30 s bursts at maximum power (Labsonic 2000, B. Braun) and the extracts were stored at 20C. Protein concentration was determined following Bradford (1976) method. Crystals and E. coli extracts were then solubilized in cracking buffer (1% SDS, 2% 2-mercaptoethanol, 6 M urea, 001 M sodium phosphate, pH 72) by heating at 95C for 5 min. SDS-PAGE was carried out following the method of Laemmli (1970) and immunoblot analysis was done following the method of Towbin et al (1979) where goat-antirabbit-HRP (GIBCO-BRL) was used as the secondary antibody. 2.8 Transient expression of the ICP gene in the rice protoplasts 28a PEG uptake of DNA into rice protoplasts: Embryogenic cell suspension cultures of Oryza sativa sub sp. indica ev. Heera were used as the experimental material. Three days after subculture, 1 g of the suspension cells were mixed with 10 ml of enzyme mixture containing 2% cellulase Onozuka R10, 01% pectolyase Y23, CPW salts (Fearson et al 1973), 04 M mannitol (pH 60) and incubated for 34 h in dark at 28C. The isolated protoplasts were sieved through a 30 m nylon mesh, centrifuged at 100 g for 5 min and the pellet was resuspended in 1 ml of CPW medium (Fearson et al 1973), floated on top of 5 ml of 20% sucrose and centrifuged for 10 min at 100 g. Protoplasts forming a ring on top of the sucrose layer were collected and washed once with CPW medium. The viability of the isolated protoplasts were tested with FDA staining. The density of the viable protoplasts was adjusted to 1 107 protoplasts/ml. PEG (40%, MW 8000) was prepared by dissolving in Krens F medium (Krens et al 1982) without CaCl2.2H2 O. 2M CaCl22H2 O was prepared separately, autoclaved and then added to the PEG solution to bring CaCl2 to a final concentration of 125 mM. One ml of protoplast suspension was taken in a 50 ml conical bottom tube, 50 l of pCNMBTT DNA (1 g/l) was added to the protoplasts. One ml of the PEG solution was added dropwise to the protoplasts and mixed slowly, incubated at 45C for 5min followed by 20 s in ice and 25 min in 30C. Krens F medium was added dropwise initially to the protoplast-PEG mixture and then 500 l to 1 ml fractions over a period of 30 min to bring the total volume to 30 ml. The protoplasts were then centrifuged at 100 g for 15 min. The resultant pellet was washed with Kpr medium (modified Kao medium, Lee et al 1989) once and resuspended in 1 ml of the same medium. The viability of the protoplasts was tested and the final density of the viable protoplasts was adjusted to 12 106/ml prior to plating. 2XKpr medium was mixed with an equal volume of autoclaved 16% SeePlaque agarose in water in 45C. Six ml compact volume of suspension cells were suspended in the above medium and poured into 70 mm petri-plates and allowed to solidify. Membrane filters of 08 m pore size were laid on top of the agarose feeder layer. Protoplast suspension (200 l) was spread on top of the membrane filter, the petriplates were sealed with parafilm and incubated in dark at 24C.

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2.8b Protein isolation: After 48 h of incubation, the protoplasts were salvaged from the membranes and suspended in extraction buffer (1:1w/v) containing 50 mM Tris (pH 95),100 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 M leupeptin. The samples were sonicated briefly and homogenized for 30 s. The materials were then incubated for 1 h at 4C and the pH was equilibrated to 85 with 01 N HCl followed by centrifugation (16,000 g) at 4C for 30 min. The supernatant was aspirated out and subjected to a 20% (w/v) (NH4)2 SO4 precipitation followed by centrifugation (37,000 g) at 4C for 30 min. The supernatant was collected and dialysed against 50 mM Tris buffer (pH 85) containing 100 mM NaCl. The dialysed samples were affinity purified by mixing with rabbit polyclonal antibody (anti-CryIIIA)-Sepharose (50 l/m1) and incubated overnight at 4C. The material was then centrifuged (1,000g) and the agarose pellet was treated with minimum volume of 200 mM Gly (pH 25), for 25 min followed by centrifugation (1,000g) for 1 min and the suprnatant was immediately treated with 1/10 th volume of 1 M Tris buffer (pH 85). The samples were then advanced for ELISA. The treatment samples were utilized for tests after determining the soluble protein concentration in each sample by the method of Bradford (1976) using BSA as the protein standard. 2.8c Sandwich ELISA: Polystyrene microtitre plates were coated with rabbit polyclonal antibodies (1:300 dilution), resuspended in 10 mM PBS and kept overnight at 4C. The wells were blocked with 150l 1% BSA, incubated for 1 h at 37C and washed with 10 mM PBS. Proteins extracted from the cultured rice protoplasts were added to each well and incubated for 2 h at 37C. After washing away the unbound proteins, single Mab/Btt2 (Johnson 1993) was added to each well and incubated for 1 h at 37C. Goat anti-mouse (IgG + IgM + IgA) alkaline phosphate conjugate was added and the presence of antigen was determined by using p-nitrophenyl phosphate as substrate dissolved in diethanolamine buffer (pH 95). Detection limit of ELISA was determined by making a standard curve using the CryIIIA protein. The concentration of the antigen present from treatment samples, judged in 5 replications in each case of three separate experimental attempts, was determined from the standard curve drawn from the known antigen as control. 2.9 Insect bioassay The rice weevils were reared in the laboratory for 34 generations on rice flour. Laboratory grown first instar larvae were collected immediately after their eclosion. They were then cultured in small tubes with provison for aeration in incubator at 28C and 75% relative humidity. Each tube contained 10 larvae and 10 g of rice flour as food. Each treatment lines for bioassay was represented by 3 replicates. The toxin expressed in Bt and E. coli were isolated separately as described previously except that PMSF was excluded from the sonication buffer. Dehusked rice grains were powdered to homogeneity to which the toxin was added. The mixing was carried out in a manner so that the food offered to the larvae contained 0 and 40 ng of the toxin peptide per mg of the total soluble rice flour protein. The LC50 of ICP of Btt of a standard strain (Kreig et al 1983) was estimated to be ~ 48 ng/mg of the rice flour protein. The duration of each bioassay was fixed for 1 month. Effects of the toxin on the survival and developmental cycle of the feeding insect grubs were recorded (table 1).

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Table 1. Effect on insect survival and metamorphosis when the insects were fed with 40 ng ICP/mg RFP*.

*RFP, Rice flour protein

3. Results 3.1 Cloning of the cryIIIA gene into plant expression vector The isolated cryIIIA gene was cloned into a plant expression vector, pCN18 (figure 7A). The pCN18 had been developed by transferring the EcoRI-HindIII fragment of the plant vector pBI121 (Jefferson et al 1987) into pUC18 to generate pCGN18 and the uidA gene contained in this transferred EcoRI-HindIII fragment was eliminated through restriction at the SmaI and SstI sites followed by blunt end ligation after end-filling with Klenow (figure 7A). The cryIIIA gene contained in pBTT2 was then restricted with HindIII and the ~ 23 kb fragment was blunt end ligated into the BamHI site of pCN18 after end-filling with Klenow (figure 7B). We selected a clone (pCNBTT) where the cryIIIA gene insert could be recovered by digestion with BamHI and BglII (figure 8A). That this 22 kb BamHI-BglII fragment contained the cryIIIA gene was ascertained by cloning it into the bacterial expression vector pET-3 (pETB). Immuno blot analysis (figure 9) confirmed that the 22 kb BamHI-BglII fragment contained the functional cryIIIA gene which produced a 65 kDa protein. This 22 kb fragment was then cloned into the BamHI site of pCNM18 (figure 7B), a modified form of pCN18. The modification was carried out in the manner, as shown in figure 7A. A 10 kb PstI-EcoRI fragment containing the CaMV35S promoter and the nos terminator from pCN18 was eluted and endfilled with Klenow. Simultaneously, pUC18 was digested with PvuII, the 24 kb fragment was eluted from the gel and ligated with the endfilled 10 kb PstI-EcoRI fragment from pCN18 to create pCNM18. This vector contained BamHI and XbaI sites downstream to CaMV35S promoter and an EcoRI site downstream to the nos terminator. The EcoRI site could be recovered by endfilling with Klenow followed by ligation at PvuII site. Subsequently, the 22 kb fragment of DNA generated from pCNBTT after digestion with BglII + BamHI, was cloned into pCNM18 to generate pCNMBTT (figure 7B). Restriction analysis of the cloned modified cryIIIA gene in pCNMBTT confirmed the presence of the characteristic 07 kb EcoRI fragment of the cryIIIA gene. Orientation of the modified cryIIIA gene with respect to CaMV35S promoter was checked by digestion with BamHI-EcoRI (figure 8B). The right orientation of the cryIIIA gene to the promoter was confirmed through the evidence for generation of 07, 085, 085 and 32 kb fragments, as in case of opposite orientation, production of 02, 07, 085 and 385 kb DNA fragments would have resulted.

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Figure 7. Cloning scheme for generation of (A) pCNM18 and (B) pCNMBTT (B, BamHI, Bg, BglII, E, EcoRI, H, HindIII P, PstI, Pv, PvuII, S, SphI, Sm, SmaI, Ss, SstI, X, XbaI, Mcs, multiple cloning site).

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Figures 8 and 9. (8) Agarose gel electrophoresis of pCNMBTTDNA cleaved with restriction endonucleases. (A) Lane 1 and lane 3, EcoRI, lane 2, BamHI-BglII and lane 4, PstI. (B) Lane 1, uncut DNA; lane 2, EcoRI cleaved DNA; lane 3, cleaved with BamHI-EcoRI showing 32, 085, 085 and 07 kb fragments; lane 4, HindIII cleaved -DNA. (9) Immunoblot analysis of the protein purified from the cryIIIA gene contained in 22 kb BamHI-BglII fragment in pET-3 (lane 1, 65 kDa; lane 2, negative control).

3.2 Transient expression of the cryIIIA gene in rice protoplasts Transient expression of the cryIIIA gene contained in pCNMBTT in rice was studied by introducing the gene into viable rice protoplasts through PEG uptake. Polyclonalmonoclonal Sandwich ELISA was used for monitoring the CryIIIA protein in five replicate experiments. It could be evidenced that the chimeric construct containing the cryIIIA gene was capable of expressing in rice protoplasts. The levels of transient expression after 48 h of incubtion of protoplasts ranged between 00008 to 0001% of its total soluble protein.

4. Discussion Truncation of the N-terminal end of the cryIIIA gene was carried out in the present study to clone a shorter form of the gene to facilitate its expression in plant (Rhim et al 1990). The cryIIIA gene, when expressed in E. coli directs the synthesis of a 72 kDa protein. However, in nature the 72 kDa protein is further processed to a 65 kDa protein by spore-associated bacterial proteases which remove the first 57 N-terminal amino acids (McPherson et al 1988). On the contrary, it is known that the 3' end of the gene can not be truncated, as it causes loss of the toxic activity (Hofte and Whiteley 1989). In the present study, modification of the gene by deletion at the N-terminal end, addition of a synthetic ATG codon and replacement of the codon, threonine to proline, has been

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seen not to alter the immunological as well as entomocidal property of the expressed protein (table 1). A truncated form of the cryIIIA gene with a deletion at the 5' end has shown not to affect protein function (Rhim et al 1990). Polyclonal-monoclonal antibody sandwich ELISA was adopted in the present study for detecting the presence of the CryIIIA protein. The Mab/Btt2 was used for the purpose its efficiency was similar to pooled Mabs, for detecting the CryIIIA protein in a mixture of antigens (Johnson 1993). We could detect as low as 2 ng of the CryIIIA protein in a given sample of protein. The two antibody sandwich ELISA is known to be fairly accurate and sensitive (Harlow and Lane 1988). That use of monoclone based technique can be used for detection and quantification of Cry protein has been demonstrated earlier (Vaeck et al 1987). The past workers could detect as low as 100 pg to 1 ng of the protein by the use of a specific Mab. The transient expression by the rice protoplasts indicated that the chimeric construct of the cryIIIA gene is expressive in rice. The levels of transient expression of the native gene in the present case could be immunologically detected. Bacterial cry genes are known to be inefficient for expression in plant. The slow rate and/or low level of nuclear production of full-length mRNA of Bt gene is an important cause (Murray et al 1991) for the relatively low expression levels of Cry proteins in plant cells. The Bt genes are excessively AT-rich as compared to normal plant genes. The bias in nucleotide composition of the DNA could have a number of deleterious conesquences on gene expression, since AT-rich regions are often found in introns or have a regulatory role in determining polyadenylation. Plants use G and C in the third position of degenerate codons, A or T being rare. Bt genes have the opposite codon usage. Codon usage is reported to be related to the abundance of the corresponding tRNAs (Bennetzen and Hall 1982). Frequent use of rare codons would decrease the rate of synthesis of a Cry protein in plant cells. Keeping this in view, reconstruction of cryIIIA gene has been achieved and has shown to significantly improve the expression (Sutton et al 1992; Adang et al 1993; Perlak et al 1993). Additional evidences also exist in favour of expression of other reconstructed cry genes in plants (Perlak et al 1991; Fujimoto et al 1993; Koziel et al 1993; Nayak et al 1995; Wunn et al 1996). Bacterial cry gene has been reported to show very high expression when made to express in chloroplasts (McBride et al 1995). Considering the above, an argument thus can come up on the merit of our attempts to transfer the native form of the cry gene to plants. Native cryIA gene, when transferred to tomato was reported to confer tolerance against highly sensitive lepidopteran insect larvae (Fischhoff et al 1987; Delannay et al 1989). Additionally, a truncated form of the native cryIIIA gene with a deletion at the 5' end when driven by CaMV35S promoter could effect high expression in tomato plants (Rhim et al 1995) and confer resistance against Colorado potato beetle. Bacterial cry gene when used with 5' UTR regulatory elements was shown to effect high expression in plant (Adang et al 1988; Carrozi et al 1992; Kar et al 1996). Thus, what would be the required level of expression of the native cryIIIA gene in rice to offer the desired protection against the target coleopteran insect pests remains to be ascertained in further through development of transgenics.

Acknowledgements The authors thank Drs Debabrata Basu and Sampa Das for their suggestive interactions. Grants from the Rockefeller Foundation, New York (RF 89026#39) and the

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Department of Biotechnology, New Delhi to SKS; the University Grants Commission, New Delhi to PN in the form of an Associateship and the Department of Atomic Energy, New Delhi to ASR in the form of a Fellowship are acknowledged gratefully.

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