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

Abstract

Spodoptera species, representing widespread polyphagous insect pests, are resistant to Bacillus thuringiensis delta-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 flanking 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.

Figure 1

Nucleotide sequence of the 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 region coding for 630 codons starts with an ATG codon in a sequence context fitting the eukaryotic consensus (26) and terminates at a TAG stop codon. Arrowheads above the s-cryIC 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.

Figure 2

Gene synthesis by TDL–PCR. Chemically phosphorylated synthetic oligonucleotides, corresponding to adjacent segments of the s-cryIC gene, are annealed with a single-stranded template DNA, carrying partially complementary sequences of the bacterial b-cryIC gene. The oligonucleotides are ligated on the template by a thermostable Pfu ligase using repeated TDL cycles of melting, annealing, and ligation. The synthetic DNA strand is selectively amplified by short PCR end primers that represent unique terminal sequences of s-cryIC oligonucleotides located at the 5′ and 3′ termini of TDL-PCR sequence blocks (labeled by asterisks). These PCR end primers contain suitable restriction cleavage sites for cloning of the synthetic double-stranded DNA fragments.

Figure 3

Expression of cryIC genes in E. coli, Arabidopsis, alfalfa and tobacco. (A) Schematic map of plant transformation vectors. The s-cryIC gene was cloned in an optimized gene expression cassette in pNS6 between promoter (pCaMV35S) and polyadenylylation sequences (pA35S) from the 35S RNA gene of CaMV. The CaMV 35S promoter (18) contains four repeats of the upstream enhancer region (−90 to −418; marked by open boxes). The same CaMV 35S expression cassette is carried by pAEN4, a vector used for transient expression of b-cryIC and s-cryIC genes in Arabidopsis protoplasts. In addition to s-cryIC, vector pNS7 contains a phosphinothricine acetyltransferase gene (pat) under the control of a mannopine synthase (mas) 1′ promoter, and a chitinase AII (chiAII) gene driven by the mas 2′ promoter. The s-cryIC gene of pNS7 was exchanged for the bacterial b-cryIC gene in pGIF1. The structure of pGIF1 is otherwise identical with that of pNS7. ori_T and ori_V, 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; ori_pBR, replication origin of pBR322; Ap^R, bacterial ampicillin resistance gene; pg5, promoter of gene 5; pnos, nopaline synthase promoter; hpt, hygromycin phosphotransferase gene; pA4 and pA7, polyadenylylation signal sequences from the T-DNA encoded genes 4 and 7, respectively; pA_ocs, polyadenylylation signal sequence of the octopine synthase gene. Open arrowheads label plant promoters, and filled boxes mark plant polyadenylylation signal sequences. (B) Expression of b-cryIC and s-cryIC genes in E. coli and Arabidopsis. (Left) The b-cryIC and s-cryIC genes were cloned, respectively, in vectors pET-11a and pET-11d (24), and their expression in E. coli was monitored by immunoblotting with (+) or without (−) isopropyl β-thiogalactopyranoside (IPTG) induction, using a polyclonal anti-CryIC antibody. The lanes contain equal amounts of protein samples (15 μg) from E. coli extracts separated by SDS/PAGE. (Right) Arabidopsis protoplasts were transformed by polyethylene glycol-mediated DNA uptake with pAEN4 (1), and pAEN4-derived vectors carrying the b-cryIC (2) and s-cryIC (3) genes. After transient expression for 48 hr, samples containing 25 μg of soluble protein extract 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 1–756) was used as a standard (2 and 20 ng). (C) Screening for CryIC expression in alfalfa calli, carrying the transferred DNA of plant transformation vectors pNS6 and pNS7. Each lane contains 25 μg of soluble proteins from calli. For comparison, Arabidopsis protoplast extract (A.th), shown in lane 3 of B, was loaded as a standard, in addition to control protein extracts prepared from callus tissues of wild-type (wt) nontransformed alfalfa. (D) Screening for CryIC accumulation in leaf tissues of transgenic alfalfa and tobacco plants. Soluble proteins (50 μg) were prepared from NS6 (lanes 1 and 3–6) and NS7 (lane 2) alfalfa transformants, as well as from transgenic tobaccos carrying the NS7 s-cryIC gene construct (lower lanes 1–6). (E) Screening for transcripts of transgenes in leaves of soil-grown alfalfa plants carrying the transferred DNA of pGIF1, 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 μg of total RNA. The blots were hybridized, respectively, with s-cryIC, b-cryIC, and chiAII probes labeled to similar specific activity. Although several GIF1 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 of the GIF1 transformants. (The positive hybridizations 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.)

Figure 4

Screening for Spodoptera resistance of transgenic plants. (A) Insecticidal assay with neonate larvae of S. littoralis reared for 2 days on leaves from nontransformed alfalfa (M. sativa, Upper) and NS7 transgenic (Lower) plants. (B) Free choice bioassays with leaves from wild-type and transgenic alfalfa plants. In the plate to the left, 10 larvae of S. exigua (third instar) were placed on the red line located between leaves of wild-type (Left) and NS7 transgenic (Right) alfalfa plants. 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. (C and D) Leaves from tobacco (C) and alfalfa (D) plants were used for feeding of five fifth instar larvae of S. exigua for 10 hr. Petri dishes to the left in C and D contained leaves from nontransformed plants. Leaves shown in Petri dishes to the right in C and D were collected from a NS7 tobacco transgenic line producing 0.2% of soluble proteins as CryIC toxin (Fig. 3D, lane 2) and from a NS6 alfalfa transformant producing 0.1% of leaf proteins as CryIC toxin, respectively. (E) Transgenic NS7 (Left; Fig. 3D, lane 2) and nontransformed alfalfa (Right) plants were infested with 15 larvae of S. exigua (third to fourth instar stage) for 6 days.