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a Plant Biotechnology, National Centre for Natural Sciences and Technology, Institute of Tropical Biology, No 1 Mac Dinh Chi St., 1st Ho Chi Minh, Viet Nam
b Agronomy and Environment Management, Louisiana State University AgCenter, 104 Madison, Sturgis Hall, Baton Rouge, LA 70803
c Plant Breeding, Genetics and Biotechnology, International Rice Research Institute, Makati, Metro Manila, Philippines
d EMVT CIRAD, TA 30/D, Campus International de Baillar-guet, Montpellier 34398, France
* Corresponding author (nbaisakh{at}agctr.lsu.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Yellow stem borer, a lepidopterous insect that feeds inside the rice stem, causes "dead heart" in the vegetative stage, ultimately leading to "white head" in the reproductive stage. At times, severe damage can cause complete crop loss. Chemical control has proven to be ineffective because the insect larvae feed inside the stem pith and remain out of the reach of the pesticide. Moreover, the use of agrochemicals is associated with high cost and the risk of environmental and health hazards. A genotype with built-in resistance (host-plant resistance) is an environment-friendly and cost-effective component of integrated pest management (IPM). But, conventional resistance breeding, particularly for this insect pest, has been handicapped by the lack of a resistant donor in the rice gene pool, despite massive screening of 30 000 rice accessions (Teng and Revilla, 1996). Genetic engineering, on the other hand, holds great promise for transferring genes across taxa for development of transgenics resistant to this insect with the insecticidal crystal protein (ICP) genes (cry) from the soil-borne bacterium Bacillus thuringiensis (Bt). Until now, transgenics have been developed in several crops, including rice, with Bt genes. However, most earlier works document the development of transgenic rice with a single cry gene: cry1A(b) (Ghareyazie et al., 1997; Alam et al., 1998, 1999; Datta et al., 1998, 1999; Husnain et al., 2002; Wu et al., 2002), cry1A(c) (Nayak et al., 1997; Cheng et al., 1998; Khanna and Raina, 2002), cry1B (Breitler et al., 2000; Marfà et al., 2002), or cry2a (Maqbool et al., 1998). Transgenic rice with a single cry1A(b) gene was shown to confer resistance to eight lepidopterans under field conditions (Shu et al., 2000). However, some insect populations develop resistance to a single cry gene (Tabashnik et al., 2000). So, the "high-dose" and "refuge" strategies have been suggested in recent Bt rice research (Cohen et al., 2000). But, at the same time, small holder farmers in Asian countries could hardly devote their land to a refuge, and, moreover, a high dose of a foreign protein could cause a phenotypic trade-off resulting in a yield penalty (Datta et al., 2002b). Hence, efforts are being made to develop two-toxin Bt crops (otherwise known as the "pyramiding" approach), since two-toxin cultivars require smaller refuges to achieve successful resistance management and sustainable field release (Cohen et al., 2000). The use of multiple-toxin genes with different modes of action has been proposed so that cross-resistance is unlikely to occur (i.e., two cry genes for toxins with different receptors or a cry gene in combination with an altogether different unrelated toxin gene, de Maagd et al., 1996; Frutos et al., 1999).
Hybrid toxins produced through inclusion of a domain from another toxin result in increased potency of the fused protein by the shift in receptor binding (Bosch et al., 1994). Alternate receptor–ligand interaction may also be exploited to further broaden the host range of the Bt toxins (Sivasubramanian and Federici, 1994).
The translational fusion gene cry1Ab-1B encodes a single Cry1Ab-Cry1B fusion protein that provides Cry1B and Cry1Ab toxins after proteolysis in the insect midgut. To reconstitute functional Cry1B and Cry1Ab activation sites in the fusion protein, synthetic cry1B and cry1Ab genes were fused at the level of the 28th codon downstream from the Cry1B activation-site codons and the 29th codon upstream from the Cry1Ab activation-site codons (Bohorova et al., 2001). This fusion gene has been reported to confer resistance to southwest corn borer (Diatraea grandiosella Dyar), sugarcane borer [Diatraea saccharalis (Fabricius)], and fall armyworm [Spodoptera frugiperda (JE Smith)] in tropical maize (Bohorova et al., 2001).
The Bt fusion gene cry1Ab/cry1Ac consisted of 1344 bp encoding the N terminus of Cry1Ab and 486 bp encoding the C terminus of Cry1Ac. The efficacy of this fused hybrid Bt gene in transgenic indica rice has been successfully tested under both greenhouse conditions (Wu et al., 1997; Datta et al., 1998; Baisakh, 2000) that showed protection against yellow stem borer and under field conditions where a transgenic hybrid rice (Bt-Sanyou63) also showed an yield advantage of about 28% over the nontransgenic hybrid rice through protection against both yellow stem borer and leaffolder (Tu et al., 2000). Transgenic rice Bt-IR72 with this fusion gene showed consistent resistance against four lepidopteran insects, including yellow stem borer over three generations under both artificial and natural infestations (Ye et al., 2001). Transgenic rice have been produced by cotransfer of two different cry genes with resistance to two different insect pests (Maqbool et al., 2001).
In our study, we report on the resistance of transgenic indica rice cultivars to yellow stem borer conferred by a translational fusion gene, cry1Ab-1B or cry1Ab/cry1Ac, delivered through Agrobacterium- and particle gun-mediated transformations, respectively. This is the first report on the expression of the translational fusion gene cry1Ab-1B in rice.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Experimental Materials and Transformation
Altogether, four rice cultivars, Nang Huong Cho Dao (NHCD), Mot Bui (MB), and Tai Nguyen (TN) that are popular and adapted to the lowland ecosystem of Vietnam, along with aromatic cultivar Jasmine (J), were used in the present study.
Scutellum-derived embryogenic calli from mature seeds developed on MS-basal callus induction medium (Murashige and Skoog, 1962) supplemented with 2.0 mg L–1 2,4-D and embryogenic cell suspension were used as the target explants for the bombardment as well as Agrobacterium transformation. Embryogenic cell suspension was raised from the embryogenic calli on R2 medium containing 20 g L–1 maltose, with a 3- to 5-d interval subculture for 1 mo until cytoplasm-rich clusters were obtained
The preparation of explants for bombardment and Agrobacterium infection, selection of the putative transformed calli, regeneration of plantlets, rooting, and transfer of the plants to the soil in the transgenic greenhouse were essentially the same as described before (Datta et al., 1998, 2000). With hph as the selectable marker gene, hygromycin concentration in the selection medium was 50 mg L–1 and for bar as the selectable marker gene the tissues were selected on medium supplemented with 3 mg L–1 of phosphinothricin (PPT). The regenerants were transferred to rooting medium also containing 3 mg L–1 PPT. However, no hygromycin was used in the rooting medium in case of regenerants with hph as the selectable marker gene. The plantlets were transferred to the nutrient culture solution (Yoshida, 1976) for 1 wk and then planted in pots inside the containment facilities of the International Rice Research Institute (IRRI).
Polymerase Chain Reaction (PCR) and Southern Blot Analyses
A rapid microprep method (modified after Kangle et al., 1995) was followed to extract the genomic DNA from 1-mo-old seedlings for the PCR template, and PCR was performed with primers specific for hph, bar, and cry1Ab/cry1Ac. Those primers are HPH F: 5'-tacttctacacagccatc-3', HPH R: tatgtcctgcgggtaaat-3' BAR F: 5'-gtctgcaccatcgtcaacc-3', BAR R: 5'-gaagtccagctgccagaaac-3' FWW F: 5'-atcttcacctcagcgtgctt-3', FWW R: 5'-ggcacattgttgttctgtgg-3'.
The PCR conditions were the same as detailed earlier (Baisakh et al., 2000). Plant genomic DNA for Southern analysis was extracted from the freshly collected leaves or freeze-dried leaves of PCR-positive transgenics and the respective nontransformed controls following a modified miniprep method (Dellaporta et al., 1983). For Southern blot analysis, 10 µg of genomic DNA was digested overnight with appropriate restriction enzymes designated for different transgenes (see figures for details). Southern blot transfer, hybridization, washing, X-ray film exposure, and the development were done as described by Baisakh et al. (2001).
HPT, PAT, and Leaf Painting Assay, and BASTA Spray
Total soluble protein extraction from fresh leaves, protein quantification, thin layer chromatography (TLC) assays for hygromycin phosphotransferase (HPT) and phosphinothricin acetyltransferase (PAT) enzymes were performed following the procedures described by Datta et al. (1990, 1992).
A leaf assay for hygromycin resistance of the transgenic plants was done to confirm the functional expression of the hph gene. Leaves (approx. 2–3 cm long) from 1-mo-old transgenic and control plants were excised and placed in assay medium (MS + 0.5 mg L–1 BA + 50 mg L–1 hygromycin).
Similarly, the leaf assay for the bar gene was done with approximately 2 to 3 cm long leaves placed on the same medium but with PPT (3 g L–1) instead of hygromycin.
The 2- to 3-mo-old transgenics and nontransformed control plants in the greenhouse were sprayed with 0.5% (v/v) of the commercial basta [ammonium 2-amino-4-(hydroxy-methyl-phosphoryl)-butanoate] solution and observation was recorded after 1 wk.
Reverse Transcription Polymerase Chain Reaction (RT-PCR)
The total RNA was extracted from the leaves of the PCR and/or Southern positive plants carrying transgene cry1Ab-1B with the RNeasy plant minikit (Qiagen, Valencia, CA). Two micrograms of total RNA were used for the RT-PCR. The primers were designed with the forward primer (5'- gatagcaggacctatccaat-3') residing in the activation site of cry1Ab region and the reverse primer (5'- gccgaagagggaggcgtcaa-3') in the activation site of cry 1B region. The PCR profile was same as described in K Datta et al. (2002). The PCR products were resolved in a 1.5% (w/v) 1x TAE–agarose gel.
Immunoblot Analysis
For immunoblot analysis, the total protein was isolated as per Datta et al. (1998), quantified with Pierce's bicinchoninic acid (BCA) Protein Quantitation Assay kit (Pierce Products, Rockford, IL, USA) with bovine serum albumin (BSA) as standard. Fifty micrograms of total protein was separated with 12% (w/v) SDS-PAGE gel and transferred to a nitrocellulose membrane followed by blocking, hybridization, and immunodetection. The rabbit anti-Bt Cry1Ac and Cry1Ab-1B antibody was used as the primary antibody for Cry1Ab/Cry1 Ac and Cry1Ab-1B, respectively, and detection was done by an IgG anti-rabbit conjugated horseradish peroxidase following the procedures detailed earlier (Datta et al., 1998). The amount of Bt toxin was measured with an ELISA kit (Envirologix Inc., Portland, ME, USA) as per the manufacturer's instruction manual.
Quick Dip Stick Detection
Quick detection of the hybrid/fused Bt protein was done using the "cry1Ab/cry1Ac lateral flow Quickstix Strip" as per the manufacturer's instructions (Envirlogix Inc., USA). The presence of a test line (second line) on the membrane strip between the control line (common to all, including the nontransformed control) and the protective tape would indicate the expression of foreign Bt protein in the transgenics.
Insect Bioassay
The T0 and T1 transgenic plants from all the cultivars expressing the transgene at the maximum tillering stage were bioassayed for resistance to neonate larvae of yellow stem borer using the cut-stem method (Datta et al., 1998). Three to five stems (including sheath) of 8 cm in length were collected at the booting stage. These were placed on a moistened filter paper disc in a 90-mm-diameter Petri dish and infested with six neonate larvae of yellow stem borer. Petri dishes were incubated at 28°C in the dark. The percentage larval mortality was based on dead and alive larvae 4 d after their release; the missing ones were considered dead.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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PCR Analysis and HPT and PAT Assay
The PCR was performed on the DNA from the shoots when the putatively transformed plantlets were transferred to the nutrient culture solution to detect the presence of the transgene(s) and or selectable marker gene(s). The presence of the 1.1-, 0.73-, and 0.5-kb amplicons in the transformants but not in the nontransformed control indicated the transgenic status of the plants for cry1Ab/cry1Ac, hph, and bar genes, respectively (Fig. 2
a, b, and c).
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dATP phosphorylated and the C14–labeled acetylated products of hygromycin and phosphinothricin, respectively were detected due to the action of the HPT and PAT, respectively, on the substrates, which were missing in the nontransgenic controls (data not shown).
Southern Analysis
The Southern analyses of PCR-positive plants revealed the integration pattern of the transgenes in the genome. As expected from Agrobacterium-mediated transformation, the transgenics from both cultivars, NHCD and MB, showed a simple integration pattern with a single 6.0-kb fragment expected for cry1Ab-1B for most cases. However, a very faintly hybridizing band smaller in size (
2.0 kb) was observed in some of the transgenics (Fig. 3a
). The simple integration pattern of Agrobacterium-mediated gene transfer confirmed that of many earlier published results (Datta et al., 2000; Baisakh et al., 1999; Tinland, 1996; Khanna and Raina, 1999). The lower and faint fragment in Nab3, Nab4, Nab6, Mab2, and Mab4 might be a result of the transfer of a truncated T-DNA that had less homology with the intact Bt gene used as the probe in this case. The Southern blot analysis with HindIII (that cuts at one end of the coding sequence of the fusion Bt gene) and PstI (that does not cut inside the coding sequence of the T-DNA but cuts once in the transformation vector) showed only one hybridization band, meaning that the transgene had been integrated at only one site in the genome in all the transgenics produced through Agrobacterium-mediated transformation (data not shown). Further, the Southern analysis for the bar gene invariably showed only one expected size fragment (Fig. 3b). This indicates that the transgenics, where there were two copies of the Bt gene (one intact and another truncated), had an additional T-DNA insertion that contained a truncated fragment of the Bt gene without the bar gene, which is near the right border of the T-DNA. A similar type of T-DNA insertion with a truncated copy of the hph gene was observed earlier (Datta et al., 2000). Moreover, the insertion of the full-length fusion Bt gene (including the truncated one) at a preferred site in the genome supports the prediction of microhomology-mediated gene transfer into the genome (Kohli et al., 1999) as observed in transgenics derived from biolistic transformation. Nevertheless, transgene integration still remains largely a random phenomenon.
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1.5 kb) apart from the expected size 1.8 kb for the hybrid Bt gene (cry1Ab/cry1Ac), whereas others had 1.8-kb hybrid Bt gene integrated into their genome. In biolisitic transformation, integration of single and intact copy transgene(s) are reported in many species (Altpeter et al., 2005). However, the independent status of the transgenics was evident when the same Southern filters were reprobed with the hph gene as the probe. Some transgenics having an identical banding pattern for the Bt gene showed a differential pattern with an additional fragment (mostly deleted) for hph, thus indicating independent transformation events (data not shown). This could be possible during the bombardment process, in which an intact copy of both the Bt and hph gene along with a few deleted fragments of hph (possibly from mechanical shearing before integration) were targeted to the tissues, or rearrangements and deletion of the hph gene under selection pressure. Rearrangement and multiple copy number of transgene(s), especially of the selectable marker gene, have been widely shown in the biolistic transformation of rice (Christou et al., 1991; Baisakh et al., 2001).
Immunoblot Analysis
The western blot analysis showed the presence of a 66-kDa protein in the transgenics containing cry1Ab-1B, which was absent in the nontransformed control (Fig. 4a
). This confirmed the expression of the translational fusion gene (cry1Ab-1B). A 66 kDa protein expected for the cry1Ab-1B fusion gene, was also demonstrated by Bohorova et al. (2001) in transgenic maize plants. In Nab4-8-1 and Nab4-8-1-3 plants (lanes 2 and 4) there was an additional band of size 56 kDa, which might have been resulted from the proteolytic degradation of the protein during processing.
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Further, the western blot analysis showed the expression of a 60-kDa protein in the transgenics obtained from biolistic transformation expected for the hybrid Bt gene (cry1Ab/cry1Ac). Apart from the 60-kDa band, an additional band of about 42 kDa was also observed in the transgenics (Fig. 4c). This further substantiated the Bt protein expression in the transgenic plants. The 60-kDa band corresponded to the expected protein encoded by the cry1AB/cry1Ac gene and the additional lower band might be the result of either truncated mRNA transcribed by the rearranged fragments of the cry1Ab/cry1Ac gene or the posttranslational modifications of the protein by the plants' endogenous proteases. Similar cases of proteolytic degraded proteins have been reported earlier in many of our transgenic rice lines (Datta et al., 1998; Tu et al., 1998; Baisakh, 2000).
The amount of Bt protein was found to range from 0.8 to 1.3% of the total soluble protein in different transgenic lines with cry1Ab-1B, whereas this value was from 0.1 to 0.9% for transgenics with cry1Ab/cry1Ac (Chandel et al., unpublished). These variations in protein expression levels in different transgenic lines are quite frequent and common due to genotypic, developmental, and environmental control. Even such variations could be observed among the progenies of a single parental line with an identical pattern of transgene integration grown under the same environment (Aguda et al., 2001; Alinia et al., 2001; Datta et al., 1998, 2002a; Husnain et al., 2002; Marfá et al., 2002).
Insect Bioassay
The cut-stem bioassay results showed that yellow stem borer neonate larval mortality after feeding for 4 d reached 100% in more than 95% of the tested Southern- and western-positive T0 plants, whereas the mortality was 0 to 16.6% in the nontransgenic control plants (Fig. 5
). It is worthwhile to note that Bt protein even at the level of 0.1% of total soluble protein was sufficient to cause 100% mortality. The higher efficacy of the cry1Ab/cry1Ac gene was shown earlier in several different rice cultivars (Datta et al., 1998; Tu et al., 1998; Baisakh, 2000; Balachandran et al., 2002). Similarly, the fusion gene cry1Ab-1B was shown to be effective against the European shoot borer in corn (Bohorova et al., 2001). The cry1B gene itself gave a higher level of protection against the striped stem borer in transgenic Mediterranean rice (Marfá et al., 2002). The fusion of cry1B with cry1Ab seemed to increase the insecticidal property of the hybrid gene as evidenced from the complete protection of all the transgenic rice plants (Nab4 and Mab2) produced including the progenies against yellow stem borer, whereas transgenic rice with cry1Ab have been shown to confer varied protection ranging from 66.7 to 100% (Datta et al., 1998). Similarly, the transgenic plants produced with the construct cry1Ab/cry1Ac (30 plants from each NB2, NB3, NB4, NB5, MB2, MB3, MB4, TN1, and TN2) also showed protection within the range of 89.9 to 100%. Each individual progeny plant was considered a line and therefore statistical analysis was not performed over the number of progenies rather was based on the number of larvae dead or alive.
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Detached-leaf assays of these plants in PPT (3 mg L–1) showed resistance of the transgenics that remained green depending on the expression level of PAT, whereas the control plants started yellowing in 2 to 3 d after treatment and ultimately turned completely yellow within 1 wk of the experiment. These plants were also sprayed with 0.5% (v/v) solution of the PPT-based herbicide Basta. The subsequent growth and development of the resistant plants after spraying appeared normal, with green leaves and stem, whereas the nontransformed plants turned yellow within 3 d of spray and ultimately died within 1 wk of spraying (data not given).
Progeny Analysis
The T1 seed progenies from different independent transgenics with 100% protection against yellow stem borer were grown for inheritance and functionality studies of the transgene(s)–selectable marker gene(s).
At the seedling stage, the transgenics were screened by PCR followed by Southern blot analysis for the segregation pattern of the transgenes. Inheritance data (Table 2) showed that in all the transgenics from Agrobacterium transformation, Mendelian segregation (3:1) was observed, which indicates that the transgenes were inserted into one locus in the genome and this was already shown by the Southern analysis for the site of integration analysis of the primary (T0) transgenics. But in the case of transgenics derived from biolistic transformation, some transgenics (in TN and J) showed a segregation ratio other than 3:1, although in most transgenics, the transgene followed the Mendelian monogenic segregation ratio. The segregation data also correlated to the germination percentage of the seeds of transgenics in MS basal medium supplemented with PPT at 3 mg L–1 or hygromycin at 50 mg L–1. Further Southern analysis confirmed the stable integration of the Bt genes without undergoing any change in the first selfing generation. Agrobacterium-mediated transformation had the advantage of simple integration with a single or few copies of transgene at a single site (Tinland, 1996), as has been found in this study. This helped in isolating the homozygous lines from the progenies of a parental line in T2 generation (Fig. 6 ). Interestingly, in particle bombardment although multiple copies with complex rearrangements of the transgenes are not rare, several transformation events showed only the expected-size fragment without any rearrangements. We also had a few transgenics from TN and J that had rearranged copies of transgenes apart from the expected size. The nonMendelian segregation of the transgenes in these particular transgenics and the differential banding pattern among the progenies of a single parental line (data not shown) confirmed their integration in more than one locus. Such rearrangements and segregation patterns in biolistic transformation have earlier been reported from our laboratory and from many others (Bohorova et al., 2001; Datta et al., 1998; Maqbool et al., 1998), which might occur either due to mechanical shearing during bombardment process and/or during the transgene integration.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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NHH and NB contributed equally to this paper. 
Received for publication June 13, 2005.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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-endotoxins can bind to insect midgut membrane proteins on ligand blots. Appl. Environ. Microbiol. 62:2753–2757.[Abstract]
–endotoxin of Bt are resistant against yellow stem borer. Proc. Natl. Acad. Sci. USA 94:2211–2216.-endotoxin. Nat. Biotechnol. 18:1101–1104.[CrossRef][Web of Science][Medline]This article has been cited by other articles:
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  J. Bernier, A. Kumar, V. Ramaiah, D. Spaner, and G. Atlin A Large-Effect QTL for Grain Yield under Reproductive-Stage Drought Stress in Upland Rice Crop Sci., March 1, 2007; 47(2): 507 - 516. [Abstract] [Full Text] [PDF]
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