Expression of a synthetic cry1EC gene for resistance against Spodoptera litura in transgenic peanut (Arachis hypogaea L.)

The tobacco cutworm (Spodoptera litura) is a polyphagous foliage insect and a major pest on peanut (Arachis hypogaea L.). S. litura is susceptible to the chimeric delta-endotoxin Cry1EC reported earlier. De-embryonated cotyledon explants of peanut were transformed using Agrobacterium tumefaciens strain EHA101 harboring a synthetic cry1EC gene driven by the CaMV 35S promoter. Transgenic plants of peanut with a single copy insertion of cry1EC were selected in the T(0) generation by Southern blot hybridization. Real-time PCR, Western blot and ELISA analysis indicated that expression of the cry1EC gene was higher in single copy T(1) plants. Immunoassay showed expression of Cry1EC up to 0.13% of total soluble protein in T(1) plants. Leaf feeding bioassay on highly expressing transgenic lines showed 100% killing of larvae at the 2(nd) instar stage of S. litura. This is the first report of transgenic peanut plants with resistance to S. litura.     

Transgenic crop plants expressing synthetic cry9Aa gene are protected against insect damage

A synthetic gene sequence of cry9Aa was made to achieve high expression levels in a plant cell. Tobacco, potato, cauliflower and turnip rape plants were transformed with this synthetic gene driven by the double 35S promoter using Agrobacterium tumefaciens LBA4404. The presence and expression of the synthetic cry9Aa gene was evaluated in Southern, Northern and Western analysis and with insect bioassays. The expression of the gene in tobacco plants reached a level of 5 pg of mRNA per 1 μg of total RNA and 0.3% of soluble protein or 1.4 μg of Cry9Aa protein per 1 g of leaf material. The expression level in the other species was three to ten times lower. Tobacco plants were also transformed with a truncated native cry9Aa gene construct and with a translational fusion construct of the truncated native cry9Aa and the uidA (GUS) gene sequence. The constructs were transformed in tobacco plants under the control of the same promoter as the synthetic cry9Aa. The expression level of the native cry9Aa gene constructs ranged from 0.03 to 1 pg of cry9Aa mRNA per 1 μg of total RNA. The protein was undetectable in Western analysis. In comparison to the native constructs the expression level of the synthetic cry9Aa gene was five to ten times higher at the mRNA level and at least 50 times higher at the translational level. Bioassays against Plutella xylostella performed with transgenic cauliflower showed high insecticidal activity of the plants expressing the synthetic cry9Aa gene.     

Transgenic broccoli with high levels of Bacillus thuringiensis Cry1C protein control diamondback moth larvae resistant to Cry1A or Cry1C

A synthetic Bacillus thuringiensis (Bt) cry1C gene was introduced into broccoli (Brassica oleracea ssp. italica) by Agrobacterium-mediated transformation. Twenty-one Cry1C transgenic plants were regenerated from 400 hypocotyl and petiole explants. Variable amounts of stable steady- state cry1C mRNA accumulated in different transgenic plants. Cry1C protein (up to 0.4% of total soluble protein) was produced in correlation with the cry1C mRNA levels. Leaf section and whole-plant bioassays were done using diamondback moth (DBM) larvae from lines susceptible to Bt or resistant to Cry1A or Cry1C proteins (Cry1AR or Cry1CR, respectively). Plants with high levels of Cry1C protein caused rapid and complete mortality of all three types of DBM larvae with no defoliation. Plants with lower levels of Cry1C protein showed an increasing differential between control of susceptible of Cry1AR DBM. This study demonstrated that high production of Cry1C protein can protect transgenic broccoli not only from susceptible or Cry1AR DBM larvae but also from DBM selected for moderate levels of resistance of Cry1C. The Cry1C- transgenic broccoli were also resistant to two other lepidopteran pests of crucifers (cabbage looper and imported cabbage worm). These plants will be useful in studies of resistance management strategies involving multiple transgenes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Signal Peptide of Potato PinII Enhances the Expression of Cry1Ac in Transgenic Tobacco

Insect-resistant plants have been developed throughexpression of insecticidal proteins from Bacillusthuringiensis (Bt) in the early 1980s [1,2]. However, forcontrol of insect pests, it is necessary to increase theexpression of Bt protein overall or in specific plant tissues.To increase the expression level, synthetic Bt genes havebeen developed and used to produce transgenic plants[2–5]. A number of approaches have been taken to increasethe expression level of foreign proteins in transgeni…     

Transgenic Rice Plants Expressing a Modified <Emphasis Type="Italic">cry1Ca1</Emphasis> Gene are Resistant to <Emphasis Type="Italic">Spodoptera litura</Emphasis> and <Emphasis Type="Italic">Chilo</Emphasis><Emphasis Type="Italic">suppressalis</Emphasis>

Nucleotide sequence encoding the truncated insecticidal Cry1Ca1 protein from Bacillus thuringiensis was extensively modified based on the codon usage of rice genes. The overall G + C contents of the synthetic cry1Ca1 coding sequence were raised to 65% with an additional bias of enriching for G and C ending codons as preferred by monocots. The synthetic gene was introduced into the Chinese japonica variety, Xiushui 11, by Agrobacterium-mediated transformation. Transgenic rice plants harboring this gene were highly resistant to Chilo suppressalis and Spodoptera litura larvae as revealed by insect bioassays. High levels of Cry1Ca1 protein were obtained in the leaves of transgenic rice, which were effective in achieving 100% mortality of S. litura and C. suppressalis larvae. The levels of Cry1Ca1 expression in the leaves of these transgenic plants were up to 0.34% of the total soluble proteins. The larvae of C. suppressalis and S. litura could consume a maximum of 1.89  and 4.89 mm² of transgenic leaf area whereas the consumption of non-transgenic leaves by these larvae was significantly higher; 58.33 and 61.22 mm², respectively. Analysis of R₁ transgenic plants indicated that the cry1Ca1 was inherited by the progeny plants and provided complete protection against C. suppressalis and S. litura larvae.     

Signal peptide of potato PinII enhances the expression of Cry1Ac in transgenic tobacco

The modified Cry l Ac was expressed in transgenic tobacco plants. To allow secretion of the CrylAc protein into the intercellular space, the signal peptide sequence of potato proteinase inhibitor II (pinII) was N-terminally fused to the CrylAc encoding region. Expression of Cry 1 Ac in transgenic tobacco plants was assayed with ELISA. The results showed that pinII signal peptide sequence enhanced the expression of Cry lAc protein and led to the secretion of the Cry 1 Ac protein in transgenic tobacco plants. GFP gene was also fused to the signal peptide sequence and transformed to tobacco. The results of fluorescent detection showed that GFP had localized in the apoplast of transgenic plants.     

The Bt gene <Emphasis Type="Italic">cry2Aa2</Emphasis> driven by a tissue specific ST-LS1 promoter from potato effectively controls <Emphasis Type="Italic">Heliothis virescens</Emphasis>

Expression of the Cry2Aa2 protein was targeted specifically to the green tissues of transgenic tobacco Nicotiana tabacum cv. Xanthi plants. This deployment was achieved by using the promoter region of the gene encoding the Solanum tuberosum leaf and stem specific (ST-LS1) protein. The accumulated levels of toxin in the leaves were found to be effective in achieving 100 mortality of Heliothis virescens larvae. The levels of Cry2Aa2 expression in the leaves of these transgenic plants were up to 0.21 of the total soluble proteins. Bioassays with R₁ transgenic plants indicated the inheritance of cry2Aa2 in the progeny plants. Tissue-specific expression of the Bt toxin in transgenic plants may help in controlling the potential occurrence of insect resistance by limiting the amount of toxin to only predated tissues. The results reported here validate the use of the ST-LS1 gene promoter for a targeted expression of Bt toxins in green tissues of plants.     

Expression of Cry1Ac in Transgenic Tobacco Plants Under the Control of a Wound-Inducible Promoter (AoPR1) Isolated from Asparagus officinalis to Control Heliothis virescens and Manduca sexta

Expression of cry1Ac gene from Bacillus thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1 promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of Cry1Ac protein increased during 6–72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay.     

Cloning and characterization of a novel gene cry9Ec1 encoding lepidopteran-specific parasporal inclusion protein from a Bacillus thuringiensis serovar galleriae strain

A novel delta-endotoxin gene from a lepidopteran-specific Bacillus thuringiensis serovar galleriae strain was cloned, and the full sequence of the cry gene was determined. The cloned 6.5-kb DNA fragment included the full sequence of the cry gene and three open reading frames located upstream of the cry gene. The gene, designated cry9Ec1, encodes a polypeptide of 1154 amino acid residues with a predicted molecular weight of 130 237. The deduced amino acid sequence of the Cry9Ec1 protein had the highest homology (77.7%) with the Cry9Ea1 protein when compared with existing Cry proteins. The expression, in an acrystalliferous B. thuringiensis strain, of the cry9Ec1 gene was high when controlled by the cyt1A2 promoter, leading to the formation of large spherical inclusions. The purified crystals from the recombinant strain were toxic when tested against two lepidopteran species, Bombyx mori and Plutella xylostella. However, the Cry9Ec1 protein gave no toxicity against Spodoptera litura, Spodoptera exigua, Plodia interpunctella, Helicoverpa zea, and Culex pipiens molestus.     

Cry1F protein not detected in soil after three years of transgenic Bt corn (1507 corn) use

To evaluate the potential of Bacillus thuringiensis (Bt) Cry1F protein accumulation in soil, transgenic corn containing event DAS-01507-1 encoding the cry1F gene was grown in three field sites for 3 consecutive yr, and the corn plants were incorporated into the soil through postseason tillage or no tillage each year. Soil samples were collected from these fields, and the level of Cry1F protein in these samples was determined using an enzyme-linked immunosorbent assay (ELISA) with a synthetic invertebrate gut fluid as an extraction buffer. The ELISA was validated in soil matrices over the concentration range of 18-180 ng/g dry weight, with a limit of detection of 4.5 ng/g dry weight. The assay was shown to have good accuracy and precision. No detectable Cry1F protein was found in any of the soil samples collected from the Cry1F corn fields. Soil also was bioassayed, and no biological activity was observed against Heliothis virescens neonates. These results indicate that the level of Cry1F protein accumulated in soil after 3-yr continuous planting of transgenic Cry1F corn is negligible.     

Some peculiarities of steroid metabolism in transgenic <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> plants bearing the <Emphasis Type="Italic">CYP11A1</Emphasis> cDNA of cytochrome P450<Subscript>SCC</Subscript> from the bovine adrenal cortex

In the mitochondria of animal steroidogenic tissues, cytochrome P450_SCC encoded by the CYP11A1 gene catalyzes the conversion of cholesterol into pregnenolone—the general precursor of all steroid hormones. In this work we study the steroid metabolism in transgenic tobacco plants carrying the CYP11A1 cDNA encoding cytochrome P450_SCC from the bovine adrenal cortex. The transgenic plants under investigation markedly surpass the control wild-type plants by size and are characterized by a shortened period of vegetative growth (by rapid flowering); their leaves contain pregnenolone—the product of a reaction catalyzed by cytochrome P450_SCC. The level of progesterone in transgenic tobacco leaves is higher than in the control plants of the wild type. The seeds of the transgenic plants contain less (24R)-brassinosteroids than the wild-type tobacco plants. The results obtained indicate that the synthesis of an active P450_SCC cytochrome in transgenic Nicotiana tabacum plants has a profound effect on steroid metabolism and is responsible for the specific phenotypic features of transgenic plants bearing CYP11A1 cDNA.     

Insect Control and Dosage Effects in Transgenic Canola Containing a Synthetic Bacillus thuringiensis cryIAc Gene

Zygotic hypocotyls of canola (Brassica napus L.) cv Oscar, cv Westar, and the breeding line UGA188-20B were transformed with a truncated synthetic Bacillus thuringiensis insecticidal crystal protein gene (Bt cryIAc) under the control of the cauliflower mosaic virus 35S promoter using Agrobacterium tumefaciens-mediated transformation. Fifty-seven independently transformed lines were produced, containing 1 to 12 copies of the transgenes. A range of cry expressors was produced from 0 to 0.4% Cry as a percentage of total extractable protein. The Brassica specialists, the diamondback month (Plutella xylostella L.) and the cabbage looper (Trichoplusia ni Hubner), were completely controlled by low-, medium-, and high-expressing lines. Whereas control of the generalist lepidopteran, the corn earworm (Helicoverpa zea Boddie), was nearly complete, the other generalist caterpillar tested, the beet armyworm (Spodoptera exigua Hubner), showed a dose response that had a negative association between defoliation and cry expression. These plants were produced as models for an ecological research assessment of the risk involved in the field release of naturalized transgenic plants harboring a gene (Bt) that confers higher relative fitness under herbivore-feeding pressure.     

Seasonal expression of Bt proteins in transgenic rice lines and the resistance against Asiatic rice borer Chilo suppressalis (Walker)

Laboratory bioassays and field surveys were carried out to compare the resistance of three transgenic rice (Oryza sativa L.) lines including Bt-DL expressing a single gene cry1Ab, Bt-KF6 expressing stacked genes cry1Ac and CpTI genes and Bt-SY63 expressing a fusion gene cry1Ab/cry1Ac, respectively, to an important rice pest Chilo suppressalis (Walker). In addition, enzyme-linked immunosorbent assays (ELISA) were conducted to monitor the Bt protein expressions in rice leaves and stems at different rice growth stages. Results showed that all the transgenic rice lines exhibited significantly high resistance to the pest compared with their corresponding nontransformed isolines. Among the transgenic rice lines, Bt-SY63 and Bt-KF6 had higher resistance to C. suppressalis at early growth stage, but lower resistance at late stages, while the pest resistance of Bt-DL was relatively stable throughout the growing season. The results were consistent with ELISA results showing that Bt protein levels in Bt-SY63 or Bt-KF6 leaves decreased in late growth stages, but were relatively stable in Bt-DL at all growth stages. This demonstrates that the resistance to a pest by Bt plants is positively correlated with Cry protein expression levels in plant tissues. Compared with Bt-SY63 and Bt-KF6, the Bt protein expression levels were significantly lower in Bt-DL, while its resistance to C. suppressalis was the highest. This may suggest that C. suppressalis is more susceptible to Cry1Ab than to Cry1Ac. The data from the current study are valuable for decision-making for commercial use of Bt rice lines and development of appropriate pest control and resistance management strategies for the transgenic rice lines.     

The −689/+197 region of the maize protease inhibitor gene directs high level,wound-inducible expression of the cry1B gene which protects transgenic rice plants from stemborer attack

To investigate the activity of the regulatory region of the maize (Zea mays L.) proteinase inhibitor (mpi) gene, we transferred into rice (Oryza sativa L.) plants the –689/+197 (C1) fragment of the mpi genomic clone fused to either theuidA gene or a synthetic Bacillus thuringiensis cry1B gene. Although uidA and cry1B encode very different proteins consistent results were obtained from their respective histochemical and fluorometric and immunoblot detections in T₃ transgenic rice lines. In response to mechanical wounding, a 4–5 fold increase in GUS activity and a Cry1B accumulation reaching 0.1–0.2% of total soluble proteins were observed from basal and undetectable levels respectively in leaf tissue. The establishment of the time-course of wound response in both systems revealed a maximum induction level 12–16 h after treatment. From both systems we also deduced that the C1 region is not active in pollen and seed endosperm. Three independent transformation events expressing cry1B under the control of the C1 region exhibited protection against striped stem borer damage and showed 100% mortality of second instar larvae 8 days after release. These results illustrate the first evidence that wound-inducible expression of a Bacillus thuringiensis endotoxin gene affords full protection to transgenic rice plants.     

Broccoli plants with pyramided cry1Ac and cry1C Bt genes control diamondback moths resistant to Cry1A and Cry1C proteins

This study was undertaken to determine the effects of pyramiding two Bacillus thuringiensis (Bt) genes in the same plant on the production of Bt proteins and the control of diamondback moths (DBM, Plutella xylostella) resistant to one or the other protein. Broccoli lines carrying both cry1Ac and cry1C Bt genes were produced by sexual crosses of cry1Ac- and cry1C-transgenic plants. Plants containing both genes were selected by tests for resistance to kanamycin and hygromycin, and confirmed by PCR analysis for the Bt genes. Both cry1Ac and cry1C mRNAs were detected in the hybrid lines, and Cry1Ac and Cry1C proteins were stably produced at levels comparable to the parental plants. Plants producing both Cry1Ac and Cry1C proteins caused rapid and complete mortality of DBM larvae resistant to Cry1A or Cry1C, and suffered little or no leaf damage. These plants, in combination with the resistant DBM populations available, will allow greenhouse or field studies of resistance management strategies involving gene pyramiding.     

Bt rice harbouring cry genes controlled by a constitutive or wound-inducible promoter: protection and transgene expression under Mediterranean field conditions

Seven homozygous transgenic lines of two European commercial cultivars of rice (Ariete (A) and Senia (S)), harbouring the cry1B or cry1Aa Bacillus thuringiensis (Bt) delta-endotoxin genes, were field evaluated for protection from striped stem borer (SSB) (Chilo suppressalis) damage during the 2001 and 2002 summer crop seasons in the Delta de l'Ebre region, Spain. The plant codon-optimized toxin gene was placed under the control of the promoter of either the constitutive ubi1 gene or the wound-inducible mpi gene from maize. Stable, high-level, insecticidal protein accumulation was observed throughout root, leaf and seed tissues of field-grown plants harbouring the cry1B (lines A64.1, A33.1, A3.4 and S98.9) or cry1Aa (lines S05.1 and A19.14) genes under the control of the ubi1 promoter. Conversely, no toxin was detected in unwounded vegetative tissues of the A9.1 line harbouring the cry1B gene controlled by the mpi promoter, indicating that natural environmental stresses did not trigger the activity of the wound-inducible promoter. However, the toxin accumulated at 0.2% total soluble proteins in A9.1 sheath tissue exhibiting brown lesions resulting from SSB damage. The agronomical traits and performance of the transgenic lines were generally comparable with parental controls, except in the two lines accumulating Cry1Aa, which exhibited a high frequency of plants non-true to type. Natural infestation was assisted with manual infestations of L2/L3 SSB larvae in border control plants surrounding the experimental plots, which served as a reservoir for the second-cycle SSB population. The observation of damage (brown lesions and dead hearts) during the crop season and dissection of plants at harvest stage revealed a range of protection amongst the transgenic lines, which was highly consistent with the level of toxin accumulation and with previous experience in greenhouse assays. Lines A3.4 and S05.1 were found to exhibit stable and full protection against SSB attacks, mediated by the accumulation of Cry1B and Cry1Aa toxin, respectively, which was comparable with that afforded by the spraying of chemical insecticides on control plants. The wound-induced A9.1 line exhibited a satisfactory level of protection, with a notably low level of penetration of SSB larvae in the stems, but higher external symptoms than constitutive lines, probably due to the time lag to benefit from the protective effect of Cry1B.