title: Blocking single-stranded transferred DNA conversion to double-stranded intermediates by overexpression of yeast DNA REPLICATION FACTOR A.
abstract: Agrobacterium tumefaciens delivers its single-stranded transferred DNA (T-strand) into the host cell nucleus, where it can be converted into double-stranded molecules. Various studies have revealed that double-stranded transfer DNA (T-DNA) intermediates can serve as substrates by as yet uncharacterized integration machinery. Nevertheless, the possibility that T-strands are themselves substrates for integration cannot be ruled out. We attempted to block the conversion of T-strands into double-stranded intermediates prior to integration in order to further investigate the route taken by T-DNA molecules on their way to integration. Transgenic tobacco (Nicotiana benthamiana) plants that overexpress three yeast (Saccharomyces cerevisiae) protein subunits of DNA REPLICATION FACTOR A (RFA) were produced. In yeast, these subunits (RFA1-RFA3) function as a complex that can bind single-stranded DNA molecules, promoting the repair of genomic double strand breaks. Overexpression of the RFA complex in tobacco resulted in decreased T-DNA expression, as determined by infection with A. tumefaciens cells carrying the β-glucuronidase intron reporter gene. Gene expression was not blocked when the reporter gene was delivered by microbombardment. Enhanced green fluorescent protein-assisted localization studies indicated that the three-protein complex was predominantly nuclear, thus indicating its function within the plant cell nucleus, possibly by binding naked T-strands and blocking their conversion into double-stranded intermediates. This notion was further supported by the inhibitory effect of RFA expression on the cell-to-cell movement of Bean dwarf mosaic virus, a single-stranded DNA virus. The observation that RFA complex plants dramatically inhibited the transient expression level of T-DNA and only reduced T-DNA integration by 50% suggests that double-stranded T-DNA intermediates, as well as single-stranded T-DNA, play significant roles in the integration process.
authors.affiliation: Golan Research Institute, University of Haifa, Qatzrin 12900, Israel (M.D.-Y., R.D.);Noga AgroTech Desert Agriculture, Kmehin 85511, Israel (A.L.);Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109 (R.D., T.T.); andDepartment of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel (T.T.) merydy@gmail.com.
authors.forename: Mery
authors.identifier: http://orcid.org/0000-0001-9652-0964
authors.initials: M
authors.lastname: Dafny-Yelin
authors.affiliation: Golan Research Institute, University of Haifa, Qatzrin 12900, Israel (M.D.-Y., R.D.);Noga AgroTech Desert Agriculture, Kmehin 85511, Israel (A.L.);Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109 (R.D., T.T.); andDepartment of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel (T.T.).
authors.forename: Avner
authors.initials: A
authors.lastname: Levy
authors.affiliation: Golan Research Institute, University of Haifa, Qatzrin 12900, Israel (M.D.-Y., R.D.);Noga AgroTech Desert Agriculture, Kmehin 85511, Israel (A.L.);Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109 (R.D., T.T.); andDepartment of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel (T.T.).
authors.forename: Raz
authors.initials: R
authors.lastname: Dafny
authors.affiliation: Golan Research Institute, University of Haifa, Qatzrin 12900, Israel (M.D.-Y., R.D.);Noga AgroTech Desert Agriculture, Kmehin 85511, Israel (A.L.);Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109 (R.D., T.T.); andDepartment of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel (T.T.).
authors.forename: Tzvi
authors.initials: T
authors.lastname: Tzfira
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000502:physiology*; D004269:DNA, Bacterial / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D015870:Gene Expression; D030821:Plants, Genetically Modified / Q000378:metabolism; D012318:RNA Polymerase I / Q000378:metabolism / Q000502:physiology; D011995:Recombination, Genetic; D051716:Replication Protein A / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism / Q000382:microbiology*; D020798:Two-Hybrid System Techniques
chemical_list: D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; C082728:RFA1 protein, S cerevisiae; C082727:RFA2 protein, S cerevisiae; C093847:RPA14 protein, S cerevisiae; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; C036483:T-DNA; D012318:RNA Polymerase I