title: DNA repair genes RAD52 and SRS2, a cell wall synthesis regulator gene SMI1, and the membrane sterol synthesis scaffold gene ERG28 are important in efficient Agrobacterium-mediated yeast transformation with chromosomal T-DNA.
abstract: BACKGROUND
  Plant pathogenic Agrobacterium strains can transfer T-DNA regions of their Ti plasmids to a broad range of eukaryotic hosts, including fungi, in vitro. In the recent decade, the yeast Saccharomyces cerevisiae is used as a model host to reveal important host proteins for the Agrobacterium-mediated transformation (AMT). Further investigation is required to understand the fundamental mechanism of AMT, including interaction at the cell surface, to expand the host range, and to develop new tools. In this study, we screened a yeast mutant library for low AMT mutant strains by advantage of a chromosome type T-DNA, which transfer is efficient and independent on integration into host chromosome.
  
  
  RESULTS
  By the mutant screening, we identified four mutant strains (srs2Δ, rad52Δ, smi1Δ and erg28Δ), which showed considerably low AMT efficiency. Structural analysis of T-DNA product replicons in AMT colonies of mutants lacking each of the two DNA repair genes, SRS2 and RAD52, suggested that the genes act soon after T-DNA entry for modification of the chromosomal T-DNA to stably maintain them as linear replicons and to circularize certain T-DNA simultaneously. The cell wall synthesis regulator SMI1 might have a role in the cell surface interaction between the donor and recipient cells, but the smi1Δ mutant exhibited pleiotropic effect, i.e. low effector protein transport as well as low AMT for the chromosomal T-DNA, but relatively high AMT for integrative T-DNAs. The ergosterol synthesis regulator/enzyme-scaffold gene ERG28 probably contributes by sensing a congested environment, because growth of erg28Δ strain was unaffected by the presence of donor bacterial cells, while the growth of the wild-type and other mutant yeast strains was suppressed by their presence.
  
  
  CONCLUSIONS
  RAD52 and the DNA helicase/anti-recombinase gene SRS2 are necessary to form and maintain artificial chromosomes through the AMT of chromosomal T-DNA. A sterol synthesis scaffold gene ERG28 is important in the high-efficiency AMT, possibly by avoiding congestion. The involvement of the cell wall synthesis regulator SMI1 remains to be elucidated.
authors.affiliation: Department of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Ohmine
authors.affiliation: Department of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Yukari
authors.initials: Y
authors.lastname: Satoh
authors.affiliation: Department of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Kiyokawa
authors.affiliation: Department of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Yamamoto
authors.affiliation: Department of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Moriguchi
authors.affiliation: Department of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima, Hiroshima, 739-8526, Japan. ksuzuki@hiroshima-u.ac.jp.
authors.forename: Katsunori
authors.identifier: http://orcid.org/0000-0003-1664-9835
authors.initials: K
authors.lastname: Suzuki
mesh_terms: D060054:Agrobacterium / Q000235:genetics* / Q000254:growth & development; D002473:Cell Wall / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics*; D015723:Gene Library; D008565:Membrane Proteins / Q000235:genetics; D009154:Mutation; D051721:Rad52 DNA Repair and Recombination Protein / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D004269:DNA, Bacterial; C429044:ERG28 protein, S cerevisiae; D008565:Membrane Proteins; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; C085451:SMI1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C036483:T-DNA; D014157:Transcription Factors; C061376:SRS2 protein, S cerevisiae; D004265:DNA Helicases
keywords: Agrobacterium-mediated transformation; Artificial chromosome; Cell surface interaction; DNA repair; Horizontal gene transfer; Saccharomyces cerevisiae; T-DNA; Trans-domain gene transfer; Yeast