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EMBO Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007
Sciences III, Quai Ernest-Ansermet 30, CH-1211 Genève, Switzerland We describe an
experimental system which is currently under development in our laboratory to address
to which extent epigenetic modifications affect gene targeting rates in Arabidopsis
thaliana. For this we use PPOX gene encoding protoporphyrinogen oxidase, an enzyme
involved in chlorophyll biosynthesis. Inhibition of PPOX by the herbicide Butafenacil
results in rapid plant death due to oxidative stress. However, the combination of two
mutations in PPOX gene confers resistance towards Butafenacil. It was shown that the
introduction of these mutations to the chromosomal copy of PPOX gene by gene targeting
is possible and can be used to assess gene targeting frequencies. We first examined
epigenetic marks at the PPOX locus. DNA methylation analyses indicate that in the wild
type plants PPOX locus is associated with substantial levels of DNA methylation,
therefore, using mutants impaired in maintenance of DNA methylation may alter the
epigenetic status of this locus. We developed an EpiRILs population comprising F8 lines
from a cross between Col WT and met1-3, a mutant affected in a methyltransferase
responsible for CpG methylation maintenance. These EpiRILs have the same genetic Col
background, but differ in their cytosine methylation profiles. We will describe the interest
of using this epigenetic material to assess the role of chromatin structure in homologous
recombination rates. This research is supported by the European Community in the frame
of the TAGIP project (No. 018785).
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P - 22. Genetic modification of rice plants by using Zinc Finger technology.
Keishi Osakabe, Kiyomi Abe, Masaki Endo, Hiroaki Saika, Seiichi Toki. Division of Plant
Sciences, National Institute of Agrobiological Sciences, 305-8602 Tsukuba, Ibaraki, Japan
Zinc finger nucleases (ZFNs) are synthetic nucleases consisting of a zinc finger DNAbinding
domain fused to the cleavage domain of the restriction endonuclease, Fok I. ZFNs
can be used to introduce double-strand DNA breaks (DSBs) in specific DNA sequences,
and promote site-specific homologous recombination (gene targeting) and targeted
genome modification at the desired genomic loci including deletion- and insertion-type
mutations. To apply this technology to the genetic modification of rice, we made a
system to identify potential zinc-finger target sites in a gene of interest with the FASTA
program based on the finding by Barbas's group (1). We already established the efficient
transformation system of rice (2) and the gene targeting system with the rice acetolactate
synthase (OsALS) gene locus (3). Thus, we chose the OsALS gene for the initial gene
targeting experiment with ZFNs. We identified a ZFN recognition sequence at the Cterminal
region of OsALS using our searching system, and designed ZFNs (ZFNosals_L
and ZFNosals_R). We also confirmed that the combination of ZFNosals_L and ZFNosals_R
could digest its recognition site on the OsALS gene in in vitro. We are further confirming
digestion in in vivo by the ZFNs, and testing whether the introduction of the ZFNs can
increase the efficiency of gene targeting at the OsALS gene locus. References 1. Mandell,
J.G. and Barbas III, C.F. (2006) Nucleic Acids Res., 34, W516-W523. 2. Toki, S., et al. (2006)
Plant J., 46, 969-976. 3. Endo, M., et al. (2006) 8th International Congress of Plant
Molecular Biology, Book of Abstracts, p153.
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P - 23. Two unlinked double-strand breaks can induce reciprocal exchanges in
plant genomes via homologous recombination and non-homologous endjoining
Michael Pacher, Waltraud Schmidt-Puchta, Holger Puchta. Molecular Biology &
Biochmeistry of plants / Prof. Puchta, University of Karlsruhe / Institute of Botany II, 76128
Karlsruhe, Germany
Using the rare-cutting endonuclease I-SceI we were able to demonstrate before that the
repair of a single DSB in a plant genome can be mutagenic due to insertions and
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