A synthetic cryIC gene, encoding a Bacillus thuringiensis δ-endotoxin, confers Spodoptera resistance in alfalfa and tobacco

Abstract

Spodoptera species, representing widespread polyphagous insect pests, are resistant to Bacillus thuringiensis ␦-endotoxins used thus far as insecticides in transgenic plants. Here we describe the chemical synthesis of a cryIC gene by a novel template directed ligation-PCR method. This simple and economical method to construct large synthetic genes can be used when routine resynthesis of genes is required. Chemically phosphorylated adjacent oligonucleotides of the gene to be synthesized are assembled and ligated on a single-stranded, partially homologous template derived from a wild-type gene (cryIC in our case) by a thermostable Pfu DNA ligase using repeated cycles of melting, annealing, and ligation. The resulting synthetic DNA strands are selectively amplified by PCR with short specific f lanking primers that are complementary only to the new synthetic DNA. Optimized expression of the synthetic cryIC gene in alfalfa and tobacco results in the production of 0.01-0.2% of total soluble proteins as CryIC toxin and provides protection against the Egyptian cotton leafworm (Spodoptera littoralis) and the beet armyworm (Spodoptera exigua). To facilitate selection and breeding of Spodoptera-resistant plants, the cryIC gene was linked to a pat gene, conferring resistance to the herbicide BASTA.Insecticidal Cry proteins, produced as protoxins in parasporal crystals of Bacillus thuringiensis (Bt), are active as selective entomocidal agents. The crystalline Bt protoxins are solubilized and activated in the midgut of insects by 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 (1-3). Engineering of insect resistance in maize, rice, cotton, tomato, potato, and tobacco shows that a significant modification of the bacterial cry coding sequences is essential to express these Bt toxin genes in plants (4-11). Efficient transcription of recombinant cry genes in plant cell nuclei was achieved by the removal of AϩT-rich sequences that may cause mRNA instability (8-11) or aberrant splicing (12), 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 (8). Expression of bacterial cry genes in chloroplasts may overcome these technological demands; however, to date chloroplast transformation is only available in tobacco (13).The insecticidal spectrum of Bt toxins thus far expressed in transgenic plants is limited. 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 (Lepidoptera, Noctuidae) feed on over 40 different plant families world-wide, including at least 87 species of economic importance (14). Studies of Spodoptera-specific Bt isolates indicated that many of them synthesize CryIC ␦-endotoxins that are much more effective against these insects than other CryI proteins (15). Here we describe the chemical synthesi...

Protein fabrication automation

“…We found coupled PCR/ligation-based gene assembly (Strizhov et al 1996;Seyfang and Jin 2004) to be more costly, less efficient, and much less robust than typical PCR-only-based methods. Classical PCR-based assembly schemes (Dillon and Rosen 1990;Stemmer et al 1995) are often sensitive to small changes in reaction conditions and can show an unpredictable dependence on sequence (Lin et al 2002;Gao et al 2003;Young and Dong 2004).…”

“…Furthermore, the use of the SOS vectors obviates the need for PAGE-purified oligonucleotides, providing a significant savings in both cost and effort. Other gene assembly methods require PAGE-purified oligonucleotides to facilitate gene synthesis (Stemmer et al 1995;Strizhov et al 1996;Hoover and Lubkowski 2002;Gao et al 2003;Carr et al 2004;Seyfang and Jin 2004;Tian et al 2004;Xiong et al 2004;Young and Dong 2004).…”

A novel autofluorescence-based selection of calli amenable to Agrobacterium-mediated transformation in onion (Allium cepa L.)

“…This RNAi construct was cloned into the T-DNA region of the plasmid pBI121 by replacing b -glucuronidase (uidA) gene. The green fluorescent protein (GFP) expression cassette amplified from the 35Somega-sGFP (S65T) plasmid (Chiu et al 1996) and the hygromycin resistance cassette in the plant transformation vector pPCV91 (Strizhov et al 1996) were also inserted in the T-DNA region of the above-mentioned plasmid. The resulting plasmid, pLFSRNAi was introduced into Agrobacterium tumefaciens LBA4404 as described by Ditta et al (1980).…”

Two different Bacillus thuringiensis toxin genes confer resistance to beet armyworm (Spodoptera exigua Hübner) in transgenic Bt-shallots (Allium cepa L.)

“…The H04 gene is a hybrid gene which encodes domains I and II of Cry1Ab and domain III of Cry1Ca (De Maagd et al, 1996) and which was optimized for expression in plants (Carozzi et al, 2002). The H04 or cry1Ca (Strizhov et al, 1996) genes were inserted between the NcoI and BglII sites in plasmid pRBC, flanked by a chrysanthemum ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (Rubisco SSU) promoter and terminator (Outchkourov et al, 2003). The orf's with the flanking promoters and terminators were excised by HindIII digestion and inserted into the HindIII site of pCAMBIA1301 resulting in plasmids pPB34 and pPB36 which were introduced into Agrobacterium strain AGL0 by electroporation (Mattanovich et al, 1989).…”

“…Cry1 proteins are generally active against lepidopterans (larvae of moths and butterflies). For example, a synthetic cry1Ca gene, encoding a B. thuringiensis delta-endotoxin, confers resistance to S. exigua in alfalfa and tobacco (Strizhov et al, 1996) and overexpression of Bt cry2Aa2 in chloroplasts resulted in 100% killing of beet armyworm after consuming transgenic leaves (De Cosa et al, 2001). Furthermore the hybrid Bt gene H04, which encodes a fusion between the N-terminal domains I and II of Cry1Ab and the C-terminal domain III of Cry1Ca, is reported to be highly toxic to S. exigua compared to the parental Cry1Ab toxin and significantly more toxic than the Cry1Ca parental toxin (De Maagd et al, 1996.…”