title: Inducible Expression of Agrobacterium Virulence Gene VirE2 for Stringent Regulation of T-DNA Transfer in Plant Transient Expression Systems.
abstract: Agrotransfection with viral vectors is an effective solution for the transient production of valuable proteins in plants grown in contained facilities. Transfection methods suitable for field applications are desirable for the production of high-volume products and for the transient molecular reprogramming of plants. The use of genetically modified (GM) Agrobacterium strains for plant transfections faces substantial biosafety issues. The environmental biosafety of GM Agrobacterium strains could be improved by regulating their T-DNA transfer via chemically inducible expression of virE2, one of the essential Agrobacterium virulence genes. In order to identify strong and stringently regulated promoters in Agrobacterium strains, we evaluated isopropyl-β-d-thiogalactoside-inducible promoters Plac, Ptac, PT7/lacO, and PT5/lacOlacO and cumic acid-inducible promoters PlacUV5/CuO, Ptac/CuO, PT5/CuO, and PvirE/CuO. Nicotiana benthamiana plants were transfected with a virE2-deficient A. tumefaciens strain containing transient expression vectors harboring inducible virE2 expression cassettes and containing a marker green fluorescent protein (GFP) gene in their T-DNA region. Evaluation of T-DNA transfer was achieved by counting GFP expression foci on plant leaves. The virE2 expression from cumic acid-induced promoters resulted in 47 to 72% of wild-type T-DNA transfer. Here, we present efficient and tightly regulated promoters for gene expression in A. tumefaciens and a novel approach to address environmental biosafety concerns in agrobiotechnology.
authors: 1 Nomads UAB, Graičiūno 8, LT-02241, Vilnius, Lithuania;|3 Institute of Biotechnology of Vilnius University, Graičiūno 8, LT-02241, Vilnius, Lithuania.
authors: Erna
authors: E
authors: Denkovskienė
authors: 1 Nomads UAB, Graičiūno 8, LT-02241, Vilnius, Lithuania;
authors: Šarūnas
authors: Š
authors: Paškevičius
authors: 2 Nomad Bioscience GmbH, Biozentrum Halle, Weinbergweg 22, D-06120 Halle (Saale), Germany;
authors: Stefan
authors: S
authors: Werner
authors: 2 Nomad Bioscience GmbH, Biozentrum Halle, Weinbergweg 22, D-06120 Halle (Saale), Germany;
authors: Yuri
authors: Y
authors: Gleba
authors: 1 Nomads UAB, Graičiūno 8, LT-02241, Vilnius, Lithuania;
authors: Aušra
authors: A
authors: Ražanskienė
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000472:pathogenicity; D001426:Bacterial Proteins / Q000235:genetics*; D001565:Benzoates / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D005453:Fluorescence; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007473:Ion Channels / Q000235:genetics*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008163:Luminescent Measurements / Q000379:methods; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D014162:Transfection / Q000379:methods; D014774:Virulence / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D001565:Benzoates; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D007473:Ion Channels; C036483:T-DNA; C051583:virE2 protein, Agrobacterium; D049452:Green Fluorescent Proteins; D007544:Isopropyl Thiogalactoside; C055607:cumic acid