Michael J. Adang

The reconstruction and expression of a Bacillus thuringiensis cryIIIA gene in protoplasts and potato plants

A Bacillus thuringiensis (B.t.) cryIIIA δ-endotoxin gene was designed for optimal expression in plants. The modified cry gene has the codon usage pattern of an average dicot gene and does not contain AT-rich nucleotide sequences typical of native B.t. cry genes. We assembled the 1.8 kb cryIIIA gene in nine blocks of three oligonucleotide pairs. For two DNA blocks, the polymerase chain reaction was used to enrich for correctly ligated pairs. We compared modified cryIIIA gene with native gene expression by electroporation of dicct (carrot) and monocot (corn) protoplasts. CryIIIA-specific RNA and protein was detected in carrot and corn protoplasts only after electroporation with the rebuilt gene. Transgenic potato lines were generated containing the redesigned cryIIIA gene under the transcriptional control of a chimeric CaMV 35S/mannopine synthetase (Mac) promoter. Out of 63 transgenic potato lines, 58 controlled first-instar Colorado potato beetle (CPB) larvae in bioassays. Egg masses which produced ca. 250 000 CPB larvae were placed on replicate clones of 56 transgenic potatoes. No CPB larvae developed past the second instar on any of these plants. Plants expressing high levels of δ-endotoxin were identified by their toxicity to more resistant third-instar larvae. We show there was good correlation between insect control and the levels of δ-endotoxin RNA and protein.

The Heliothis virescens 170 kDa aminopeptidase functions as “Receptor A” by mediating specific Bacillus thuringiensis Cry1A δ-endotoxin binding and pore formation

The relationship between Bacillus thuringiensis Cry1Aa, Cry1Ab and Cry1Ac delta-endotoxin binding and pore formation was investigated using a purified 170 kDa aminopeptidase N (APN) from Heliothis virescens brush border membranes. Aminopeptidases with molecular sizes of 110, 140 and 170 kDa were eluted from a Cry1Ac toxin affinity column using N-acetylgalactosamine. The 140 kDa aminopeptidase has a cross-reacting determinant typical of a cleaved glycosyl-phosphatidylinositol anchor. After mild base treatment to de-acylate the glycosyl-phosphatidylinositol linkage and incubation in phosphatidyl inositol phospholipase C, anti-cross-reacting determinant antibody recognized the 170 kDa protein. Kinetic binding characteristics of Cry1A toxins to purified 170 kDa APN were determined using surface plasmon resonance. Cry1Aa, Cry1Ab and Cry1Ac, but not Cry1C and Cry1E toxins recognized 170 kDa APN. Each Cry1A toxin recognized two binding sites: a high affinity site with KD ranging from 41 to 95 nM and a lower affinity site with KD in the 325 to 623 nM range. N-acetylgalactosamine inhibited Cry1Ac but not Cry1Aa and Cry1Ab binding to 170 kDa APN. When reconstituted into phospholipid vesicles, the 170 kDa APN promoted toxin-induced 86Rb+ release for Cry1A toxins, but not Cry1C toxin. Furthermore Cry1Ac, the Cry protein most toxic to H. virescens larvae, caused 86Rb+ release at lower concentrations, and to a greater extent than Cry1Aa and Cry1Ab toxins. The correlation between toxin-binding specificity and 86Rb+ release strongly suggests that the purified 170 kDa APN is the functional receptor A in the H. virescens midgut epithelial cell brush border membranes.

Identification of novel Bacillus thuringiensis Cry1Ac binding proteins in Manduca sexta midgut through proteomic analysis

The crystal proteins of Bacillus thuringiensis are widely used in transgenic crops and commercially available insecticides. Manduca sexta, the tobacco hornworm, is the model insect for B. thuringiensis studies. Although brush border vesicles prepared from larval M. sexta midgut have been used in numerous mode-of-action studies of B. thuringiensis toxins, their protein components are mostly unknown. Vesicles prepared from the brush border of M. sexta midgut were analyzed using one- and two-dimensional gel electrophoresis to establish a midgut brush border proteome. Sub-proteomes were also established for B. thuringiensis Cry1Ac binding proteins and glycosylphosphatidyl inositol (GPI) anchored proteins. Peptide mass fingerprints were generated for several spots identified as Cry1Ac binding proteins and GPI-anchored proteins and these fingerprints were used for database searches. Results generally did not produce matches to M. sexta proteins, but did match proteins of other Lepidoptera. Actin and alkaline phosphatase were identified as novel proteins that bind Cry1Ac in addition to the previously reported aminopeptidase N. Aminopeptidase N was the only GPI-anchored protein identified. Actin, aminopeptidase N, and membrane alkaline phosphatase were confirmed as accurate protein identifications through western blots.

Analysis of unstable RNA transcripts of insecticidal crystal protein genes of Bacillus thuringiensis in transgenic plants and electroporated protoplasts.

We have examined expression of several insecticidal crystal protein (ICP) genes of Bacillus thuringiensis in transgenic tobacco plants and electroporated carrot protoplasts. We determined that low levels of lepidopteran toxin cryIA(b) ICP gene expression in plants and electroporated carrot cells is due to RNA instability. We used a series of 3′ deleted cryIA(b) constructs directed by the cauliflower mosaic virus 35S promoter to demonstrate that this instability is minimally contained in the first 579 bases of the gene in both systems. This instability may result from 5′ → 3′ as well as 3′ → 5′ RNA metabolism. The coleopteran toxic cryIIIA gene was also examined in electroporated carrot cells, and found to be poorly expressed. A model for improvement of ICP RNA stability in plants is presented.

Ion channels formed in planar lipid bilayers by Bacillus thuringiensis toxins in the presence of Manduca sexta midgut receptors

A purified, GPI‐linked receptor complex isolated from Manduca sexta midgut epithelial cells was reconstituted in planar lipid bilayers. CryIAa, CryIAc and CryIC, three Bacillus thuringiensis insecticidal proteins, formed channels at much lower doses (0.33–1.7 nM) than in receptor‐free membranes. The non‐toxic protein CryIB also formed channels, but at doses exceeding 80 nM. The channels of CryIAc, the most potent toxin against M. sexta, rectified the passage of cations. All other toxin channels displayed linear current–voltage relationships. Therefore, reconstituted Cry receptors catalyzed channel formation in phospholipid membranes and, in two cases, were involved in altering their biophysical properties.

Expression of a Bacillus thuringiensis cryIA(c) gene in transgenic peanut plants and its efficacy against lesser cornstalk borer

The invasion of peanut (Arachis hypogaea L.) pods and seeds by aflatoxin-forming species of Aspergillus is linked to injury by the lesser cornstalk borer and frequently causes a severe reduction in crop quality. The lesser cornstalk borer is susceptible to the lepidopteran-active Bacillus thuringiensis insecticidal crystal protein. We have introduced a codon-modified Bacillus thuringiensis cryIA(c) gene into peanut using microprojectile bombardment. The toxin-coding region of a Bt cryIA(c) gene was reconstructed for expression in plants and the resulting 3.4 kb gene cassette (promoter: 1.8 kb coding: 3′) was directly cloned into the BglII site of plant transformation vectors. The vectors contained the hph gene, conferring resistance to the antibiotic hygromycin. Somatic embryos initiated from immature peanut cotyledons of two cultivars were used as the target for bombardment. DNA from hygromycin-resistant embryogenic cell lines, regenerated plants, and a progeny plant showed the presence and integration of hph and Bt genes by PCR and/or Southern blot analyses. ELISA immunoassay of the CryIA(c) protein from the hygromycin-selected plants showed the expression of CryIA(c) protein up to 0.18% of total soluble protein. Insect feeding bioassay of transformed plants indicated various levels of resistance to the lesser cornstalk borer, from complete larval mortality to a 66% reduction in larval weight. A negative correlation between percent survival or larval weight and the amount of Bt CryIA(c) protein was recorded indicating in general that the higher the protein level the lower the survival or larval weight of the insect. Based on leaf bioassay, transformation of peanut with vectors containing the Bt cryIA(c) gene may be effective in protecting the peanut plants from damage by lepidopteran insect larvae of lesser cornstalk borer

Importance of Cry1 δ-Endotoxin Domain II Loops for Binding Specificity in Heliothis virescens (L.)

ABSTRACT We constructed a model for Bacillus thuringiensis Cry1 toxin binding to midgut membrane vesicles from Heliothis virescens. Brush border membrane vesicle binding assays were performed with five Cry1 toxins that share homologies in domain II loops. Cry1Ab, Cry1Ac, Cry1Ja, and Cry1Fa competed with 125I-Cry1Aa, evidence that each toxin binds to the Cry1Aa binding site in H. virescens. Cry1Ac competed with high affinity (competition constant [Kcom] = 1.1 nM) for 125I-Cry1Ab binding sites. Cry1Aa, Cry1Fa, and Cry1Ja also competed for125I-Cry1Ab binding sites, though theKcom values ranged from 179 to 304 nM. Cry1Ab competed for 125I-Cry1Ac binding sites (Kcom = 73.6 nM) with higher affinity than Cry1Aa, Cry1Fa, or Cry1Ja. Neither Cry1Ea nor Cry2Aa competed with any of the 125I-Cry1A toxins. Ligand blots prepared from membrane vesicles were probed with Cry1 toxins to expand the model of Cry1 receptors in H. virescens. Three Cry1A toxins, Cry1Fa, and Cry1Ja recognized 170- and 110-kDa proteins that are probably aminopeptidases. Cry1Ab and Cry1Ac, and to some extent Cry1Fa, also recognized a 130-kDa molecule. Our vesicle binding and ligand blotting results support a determinant role for domain II loops in Cry toxin specificity for H. virescens. The shared binding properties for these Cry1 toxins correlate with observed cross-resistance in H. virescens.

Reduced Levels of Membrane-Bound Alkaline Phosphatase Are Common to Lepidopteran Strains Resistant to Cry Toxins from Bacillus thuringiensis

Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Identification of biomarkers would assist in the development of sensitive DNA-based methods to monitor evolution of resistance to Bt toxins in natural populations. We report on the proteomic and genomic detection of reduced levels of midgut membrane-bound alkaline phosphatase (mALP) as a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to susceptible larvae. Reduced levels of H. virescens mALP protein (HvmALP) were detected by two dimensional differential in-gel electrophoresis (2D-DIGE) analysis in Cry-resistant compared to susceptible larvae, further supported by alkaline phosphatase activity assays and Western blotting. Through quantitative real-time polymerase chain reaction (qRT-PCR) we demonstrate that the reduction in HvmALP protein levels in resistant larvae are the result of reduced transcript amounts. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived strains of S. frugiperda resistant to Cry1Fa. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest that reduced mALP expression should be targeted for development of effective biomarkers for resistance to Cry toxins in lepidopteran pests.

Regeneration of transgenic peanut plants from stably transformed embryogenic callus

Abstract Embryogenic tissue cultures of Arachis hypogaea L. (peanut or groundnut), have been transformed via microprojectile bombardment. We introduced a gene ( hph ) conferring resistance to the antibiotic hygromycin under the control of the CaMV 35S promoter. Selection for resistant callus was initiated 4–5 weeks post-bombardment on medium containing 10–20 mg/l hygromycin. Twelve percent of the bombardments resulted in recovery of a transgenic cell line. An average of two transgenic embryogenic cell lines was isolated per bombardment experiment over 4–6 months of continuous selection. Each bombardment experiment consisted of 11–19 plates, and each plate contained approximately fourteen 25 mm 2 embryogenic callus pieces. Thus, nearly 1% of the bombarded callus pieces produced a stably transformed cell line. Over 100 plants have been regenerated collectively from all of the transformed cell lines. The presence and integration of foreign DNA in hygromycin-resistant callus lines and regenerated plants has been confirmed by polymerase chain reaction amplification of a defined portion of the chimeric gene and by Southern hybridization analysis. Hygromycin resistance was expressed in leaflets from transformed plants which remained green when cultured on basal medium containing hygromycin. Leaflets from control, non-transformed plants turned brown within 3 weeks on the hygromycin-containing medium.

Bt-R1a Extracellular Cadherin Repeat 12 Mediates Bacillus thuringiensis Cry1Ab Binding and Cytotoxicity

The cadherin protein Bt-R1a is a receptor for Bacillus thuringiensis Cry1A toxins in Manduca sexta. Cry1Ab toxin is reported to bind specific epitopes located in extracellular cadherin repeat (CR) 7 and CR11 on Bt-R1 (Gomez, B., Miranda-Rios, J., Riudino-Pinera, E., Oltean, D. I., Gill, S. S., Bravo, A., and Soberon, M. (2002) J. Biol. Chem. 277, 30137–30143; Dorsch, J. A., Candas, M., Griko, N., Maaty, W., Midboe, E., Vadlamudi, R., and Bulla, L. (2002) Insect Biochem. Mol. Biol. 32, 1025–1036). We transiently expressed CR domains of Bt-R1a in Drosophila melanogaster Schneider 2 (S2) cells as fusion peptides between a signal peptide and a terminal region that included membrane-proximal, membrane-spanning, and cytoplasmic domains. A domain consisting of CR11 and 12 was the minimal 125I-Cry1Ab binding region detected under denaturing conditions. Only CR12 was essential for Cry1Ab binding and cytotoxicity to S2 cells when tested under native conditions. Under these conditions expressed CR12 bound 125I-Cry1Ab with high affinity (Kcom = 2.9 nm). Flow cytometry assays showed that expression of CR12 conferred susceptibility to Cry1Ab in S2 cells. Derivatives of Bt-R1a with separate deletions of CR7, 11, and 12 were expressed in S2 cells. Only deletion of CR12 caused loss of Cry1Ab binding and cytotoxicity. These results demonstrate that CR12 is the essential Cry1Ab binding component on Bt-R1 that mediates Cry1Ab-induced cytotoxicity.