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Engineered glyphosate resistance is the most widely adopted genetically modified trait in agriculture, gaining widespread acceptance by providing a simple robust weed control system. However, extensive and sustained use of glyphosate as a sole weed control mechanism has led to field selection for glyphosate-resistant weeds and has induced significant population shifts to weeds with inherent tolerance to glyphosate. Additional weed control mechanisms that can complement glyphosate-resistant crops are, therefore, urgently needed. 2,4-dichlorophenoxyacetic acid (2,4-D) is an effective lowcost, broad-spectrum herbicide that controls many of the weeds developing resistance to glyphosate. We investigated the substrate preferences of bacterial aryloxyalkanoate dioxygenase enzymes (AADs) that can effectively degrade 2,4-D and have found that some members of this class can act on other widely used herbicides in addition to their activity on 2,4-D. AAD-1 cleaves the aryloxyphenoxypropionate family of grass-active herbicides, and AAD-12 acts on pyridyloxyacetate auxin herbicides such as triclopyr and fluroxypyr. Maize plants transformed with an AAD-1 gene showed robust crop resistance to aryloxyphenoxypropionate herbicides over four generations and were also not injured by 2,4-D applications at any growth stage. Arabidopsis plants expressing AAD-12 were resistant to 2,4-D as well as triclopyr and fluroxypyr, and transgenic soybean plants expressing AAD-12 maintained field resistance to 2,4-D over five generations. These results show that single AAD transgenes can provide simultaneous resistance to a broad repertoire of agronomically important classes of herbicides, including 2,4-D, with utility in both monocot and dicot crops. These transgenes can help preserve the productivity and environmental benefits of herbicide-resistant crops.

Demand for the commercial use of genetically modified (GM) crops has been increasing in light of the projected growth of world population to nine billion by 2050. A prerequisite of paramount importance for regulatory submissions is the rigorous safety assessment of GM crops. One of the components of safety assessment is molecular characterization at DNA level which helps to determine the copy number, integrity and stability of a transgene; characterize the integration site within a host genome; and confirm the absence of vector DNA. Historically, molecular characterization has been carried out using Southern blot analysis coupled with Sanger sequencing. While this is a robust approach to characterize the transgenic crops, it is both time- and resource-consuming. The emergence of next-generation sequencing (NGS) technologies has provided highly sensitive and cost- and labor-effective alternative for molecular characterization compared to traditional Southern blot analysis. Herein, we have demonstrated the successful application of both whole genome sequencing and target capture sequencing approaches for the characterization of single and stacked transgenic events and compared the results and inferences with traditional method with respect to key criteria required for regulatory submissions.

In higher plants, three subfamilies of sucrose nonfermenting-1 (Snf1)-related protein kinases have evolved. While the Snf1-related protein kinase 1 (SnRK1) subfamily has been shown to share pivotal roles with the orthologous yeast Snf1 and mammalian AMP-activated protein kinase in modulating energy and metabolic homeostasis, the functional significance of the two plant-specific subfamilies SnRK2 and SnRK3 in these critical processes is poorly understood. We show here that SnRK2.6, previously identified as crucial in the control of stomatal aperture by abscisic acid (ABA), has a broad expression pattern and participates in the regulation of plant primary metabolism. Inactivation of this gene reduced oil synthesis in Arabidopsis (Arabidopsis thaliana) seeds, whereas its overexpression increased Suc synthesis and fatty acid desaturation in the leaves. Notably, the metabolic alterations in the SnRK2.6 overexpressors were accompanied by amelioration of those physiological processes that require high levels of carbon and energy input, such as plant growth and seed production. However, the mechanisms underlying these functionalities could not be solely attributed to the role of SnRK2.6 as a positive regulator of ABA signaling, although we demonstrate that this kinase confers ABA hypersensitivity during seedling growth. Collectively, our results suggest that SnRK2.6 mediates hormonal and metabolic regulation of plant growth and development and that, besides the SnRK1 kinases, SnRK2.6 is also implicated in the regulation of metabolic homeostasis in plants.

Demand for the commercial use of genetically modified (GM) crops has been increasing in light of the projected growth of world population to nine billion by 2050. A prerequisite of paramount importance for regulatory submissions is the rigorous safety assessment of GM crops. One of the components of safety assessment is molecular characterization at DNA level which helps to determine the copy number, integrity and stability of a transgene; characterize the integration site within a host genome; and confirm the absence of vector DNA. Historically, molecular characterization has been carried out using Southern blot analysis coupled with Sanger sequencing. While this is a robust approach to characterize the transgenic crops, it is both time- and resource-consuming. The emergence of next-generation sequencing (NGS) technologies has provided highly sensitive and cost- and labor-effective alternative for molecular characterization compared to traditional Southern blot analysis. Herein, we have demonstrated the successful application of both whole genome sequencing and target capture sequencing approaches for the characterization of single and stacked transgenic events and compared the results and inferences with traditional method with respect to key criteria required for regulatory submissions.

Aminopeptidases and cadherin-like proteins have been identified as Bacillus thuringiensis Cry toxin-binding proteins from several insect species. In the Cry1A-susceptible lepidopteran insects, Heliothis virescens and Manduca sexta, aminopeptidases are preferentially associated with Triton X-100 insoluble lipid rafts membrane microdomains. Unlike aminopeptidases, cadherin-like proteins are not raft-associated. However, binding of Cry1A toxins to cadherin-like proteins caused relocation of cadherin-like protein to the lipid rafts microdomain and further activation of Cry1A toxins by removal of toxin domain I α-helix 1. Association of these toxin-binding proteins as well as Cry1A toxins with lipid rafts indicates an important role of lipid rafts in Cry1A toxicity. Moreover, it is showed that lipid rafts integrity is essential for Cry1A toxin pore formation, possibly by providing a platform for toxin aggregation and pore formation. The importance of cadherin-like protein in Cry toxicity has been implied by its correlation with a Cry1A resistant H. virescens strain. H. virescens and M. sexta cadherin-like proteins are present in insect midgut columnar cell apical membranes, which are the Cry toxin target sites in the susceptible insects. Cry1A treatment of the H. virescens cadherin-like protein expressing stable mammalian cells HEK293 affects cell-cell contact and cell membrane integrity, indicating direct involvement of cadherin-like protein in Cry1A toxin action. Moreover, the Cry1A-binding region in H. virescens cadherin-like protein is mapped to amino acids 1422–1461, and this region is not only involved in toxin binding, but is also important in toxicity. Another intracellular protein, Hvp85, also binds Cry1Ac specifically. Binding of Hvp85 to Dor suggests that Hvp85 may be involved in lysosomal delivery and its potential involvement in Cry1A action. In addition to the known receptors aminopeptidases and cadherin-like proteins, several smaller proteins with sizes of 45–90 kDa bound Cry1Ac. Our study suggests that Cry protein action is complex and the role of p85 in Cry1Ac toxicity remains unclear but warrants further investigation.