title: In vivo formation of double-stranded T-DNA molecules by T-strand priming.
abstract: During plant genetic transformation, Agrobacterium transfers a single-stranded DNA (T-strand) into the host cell. Increasing evidence suggests that double-stranded (ds) T-DNA, converted from T-strands, are potent substrates for integration. Nevertheless, the molecular mechanism governing T-strand conversion to dsT-DNA is unknown. Integrated T-DNA molecules typically exhibit deletions at their 3' end as compared with their 5' end. We hypothesize that this may result from asymmetric polymerization of T-DNA's ends. Here we show that β-glucuronidase (GUS) expression from sense T-strands is more efficient than from antisense T-strands, supporting asymmetric conversion. Co-transfection with two partially complementary, truncated GUS-encoding T-strands results in GUS expression, which suggests functional hybridization of the T-strands via complementary annealing and supports the notion that T-strands can anneal with primers. Indeed, red fluorescent protein (RFP) expression from mutated T-strand can be restored by delivery of synthetic DNA and RNA oligonucleotides with partial wild-type RFP sequence, implying the involvement of plant DNA repair machinery.
authors: Department of Molecular, Cellular and Developmental Biology, The University of Michigan, Ann Arbor, Michigan 48109, USA.
authors: Zhuobin
authors: Z
authors: Liang
authors: Tzvi
authors: T
authors: Tzfira
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D004260:DNA Repair; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics*; D018744:DNA, Plant / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics*; D006824:Hybridization, Genetic / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D014026:Nicotiana / Q000235:genetics* / Q000382:microbiology; D014170:Transformation, Genetic / Q000235:genetics; D000097573:Red Fluorescent Protein
chemical_list: D004269:DNA, Bacterial; D018744:DNA, Plant; D005966:Glucuronidase; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C036483:T-DNA