Modification of Vip3Ab1 C-Terminus Confers Broadened Plant Protection from Lepidopteran Pests.

Vegetative insecticidal proteins (Vips) from Bacillus thuringiensis (Bt) are unique from crystal (Cry) proteins found in Bt parasporal inclusions as they are secreted during the bacterial vegetative growth phase and bind unique receptors to exert their insecticidal effects. We previously demonstrated that large modifications of the Vip3 C-terminus could redirect insecticidal spectrum but results in an unstable protein with no lethal activity. In the present work, we have generated a new Vip3 protein, Vip3Ab1-740, via modest modification of the Vip3Ab1 C-terminus. Vip3Ab1-740 is readily processed by midgut fluid enzymes and has lethal activity towards Spodoptera eridania, which is not observed with the Vip3Ab1 parent protein. Importantly, Vip3Ab1-740 does retain the lethal activity of Vip3Ab1 against other important lepidopteran pests. Furthermore, transgenic plants expressing Vip3Ab1-740 are protected against S. eridania, Spodoptera frugiperda, Helicoverpa zea, and Pseudoplusia includens. Thus, these studies demonstrate successful engineering of Vip3 proteins at the C-terminus to broaden insecticidal spectrum, which can be employed for functional expression in planta.

Insecticidal Activity of a Vip3Ab1 Chimera Is Conferred by Improved Protein Stability in the Midgut of Spodoptera eridania.

Vip3A proteins are important for the control of spodopteran pests in crops, including Spodoptera frugiperda (fall armyworm). Native Vip3Ab1 controls S. frugiperda, but it is ineffective against S. eridania (southern armyworm), a major pest of soybean in South America. Recently, a Vip3Ab1 chimera with a modified C-terminus was described, Vip3Ab1-740, which has increased potency against S. eridania while maintaining activity against S. frugiperda. As S. frugiperda and S. eridania are differentially susceptible to Vip3Ab1, experiments were conducted to identify and understand the mechanism by which this expanded potency is conferred. The role of protein stability, processing, and in vivo effects of Vip3Ab1 and Vip3Ab1-740 in both of these species was investigated. Biochemical characterization of the midgut fluids of these two species indicated no obvious differences in the composition and activity of digestive enzymes, which protease inhibitor studies indicated were likely serine proteases. Histological examination demonstrated that both proteins cause midgut disruption in S. frugiperda, while only Vip3Ab1-740 affects S. eridania. Immunolocalization indicated that both proteins were present in the midgut of S. frugiperda, but only Vip3Ab1-740 was detected in the midgut of S. eridania. We conclude that the gain of toxicity of Vip3Ab1-740 to S. eridania is due to an increase in protein stability in the midgut, which was conferred by C-terminal modification.

Expression characterization of the herbicide tolerance gene Aryloxyalkanoate Dioxygenase (aad-1) controlled by seven combinations of regulatory elements.

BACKGROUND: Availability of well characterized maize regulatory elements for gene expression in a variety of tissues and developmental stages provides effective alternatives for single and multigene transgenic concepts. We studied the expression of the herbicide tolerance gene aryloxyalkanoate dioxygenase (aad-1) driven by seven different regulatory element construct designs including the ubiquitin promoters of maize and rice, the actin promoters of melon and rice, three different versions of the Sugarcane Bacilliform Badnavirus promoters in association with other regulatory elements of gene expression. RESULTS: Gene expression of aad-1 was characterized at the transcript and protein levels in a collection of maize tissues and developmental stages. Protein activity against its target herbicide was characterized by herbicide dosage response. Although differences in transcript and protein accumulation were observed among the different constructs tested, all events were tolerant to commercially relevant rates of quizalafop-P-ethyl compared to non-traited maize under greenhouse conditions. DISCUSSION: The data reported demonstrate how different regulatory elements affect transcript and protein accumulation and how these molecular characteristics translate into the level of herbicide tolerance. The level of transcript detected did not reflect the amount of protein quantified in a particular tissue since protein accumulation may be influenced not only by levels of transcript produced but also by translation rate, post-translational regulation mechanisms and protein stability. The amount of AAD-1 enzyme produced with all constructs tested showed sufficient enzymatic activity to detoxify the herbicide and prevent most herbicidal damage at field-relevant levels without having a negative effect on plant health. CONCLUSIONS: Distinctive profiles of aad-1 transcript and protein accumulation were observed when different regulatory elements were utilized in the constructs under study. The ZmUbi and the SCBV constructs showed the most consistent robust tolerance, while the melon actin construct provided the lowest level of tolerance compared to the other regulatory elements used in this study. These data provide insights into the effects of differing levels of gene expression and how these molecular characteristics translate into the level of herbicide tolerance. Furthermore, these data provide valuable information to optimize future designs of single and multiple gene constructs for maize research and crop improvement.

Canola engineered with a microalgal polyketide synthase-like system produces oil enriched in docosahexaenoic acid.

Dietary omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5) are usually derived from marine fish. Although production of both EPA and DHA has been engineered into land plants, including Arabidopsis, Camelina sativa and Brassica juncea, neither has been produced in commercially relevant amounts in a widely grown crop. We report expression of a microalgal polyketide synthase-like PUFA synthase system, comprising three multidomain polypeptides and an accessory enzyme, in canola (Brassica napus) seeds. This transgenic enzyme system is expressed in the cytoplasm, and synthesizes DHA and EPA de novo from malonyl-CoA without substantially altering plastidial fatty acid production. Furthermore, there is no significant impact of DHA and EPA production on seed yield in either the greenhouse or the field. Canola oil processed from field-grown grain contains 3.7% DHA and 0.7% EPA, and can provide more than 600 mg of omega-3 LC-PUFAs in a 14 g serving.