15th International Conference on Arabidopsis Research - TAIR

T09-001
Brca2 is essential to meiosis in Arabidopsis
Dray E(1), Siaud N(2), Richaud A(3), Doutriaux MP(4)
1-PhD student
2-PhD student
3-Research assistant
4-CNRS
Mutations in theBRCA2 gene are major cause of breast cancer susceptibility
in human. As observed for RAD51, disruption of the BRCA2 gene causes an
accumulation of double-strand breaks and is embryo-lethal in mice. Since
the Brca2 and Rad 51 proteins were shown to interact in vivo, they are considered
to be implicated in the same pathway of DNA repair in mammals.
Two BRCA2-like sequences are present in the Arabidopsis genome. Both
genes are expressed in flower buds and encode nearly identical proteins
which contain four BRC motifs. In a yeast two hybrid assay, the Arabidopsis
Brca2 proteins interact with Rad51 and Dmc1. These results were confirmed
in vitro by co-IP. RNAi constructs aimed at silencing the BRCA2 genes at
meiosis triggered a reproducible sterility phenotype which was associated
with dramatic meiosis alterations (1). We obtained the same phenotype
upon introduction of RNAi constructs aimed at silencing the RAD51 gene at
meiosis in dmc1 mutant plants (2). The meiotic figures we observed strongly
suggest that homologous recombination is highly disturbed in these meiotic
cells, leaving aberrant recombination events to repair the meiotic double
strand breaks. The “brca2” meiotic phenotype was eliminated in spo11
mutant plants. Our experiments point to an essential role of Brca2 at meiosis
in Arabidopsis. We also propose a role for Rad51 in the dmc1 context. We are
now examining other genetic functions that might be required for the “brca2”
phenotype.
(1) Siaud N et al (2004) EMBO J inpress
(2) Couteau F et al (1999) Plant Cell 11, 1623-1634
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin
T09-002
Arabidopsis Mutants Enhanced in RNA Silencing
Konstantina Boutsika(1), Francesco Di Serio(2), Eugene Glazov(3), Ueli Klahre(4),
Frederick Meins(1)
1-Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel,
Switzerland
2-Present Address: Istituto di Virologia Vegetale, CNR, Via Amendola 165/A 70126, Bari, Italy
3-Present Address: The Institute for Molecular Bioscience, Queensland Bioscience Precinct. The
University of Queensland, Brisbane QLD 4072, Australia
4-Present Address: Heidelberg Institut fur Pflanzenwissenschaften Im Neuenheimer Feld 360,
D- 69120 Heidelberg, Germany
5-Friedrich Miescher Institute for Biomedical Research, CH-4058 Basel, Switzerland
In spite of extensive effort, previous genetic and molecular studies had identified
only five plant genes required for RNA silencing. To identify additional
silencing-related genes, we screened M2 populations of Arabidopsis ecotype
“Columbia 0” for EMS mutants with enhanced RNA silencing (ESI) of a green
fluorescent protein (GFP) reporter gene. Fifteen recessive mutants showing
high GFP expression in cotyledons, but no GFP expression in true leaves fell
into 5 complementation groups (ESI1-ESI5).
The esi1 and esi2 mutants studied in detail showed some variability in
growth rate, but were otherwise normal in appearance. Nuclear run-on transcription
experiments and the presence of GFP siRNAs verified that esi1 and
esi2 mutants chosen for study exhibit RNA silencing and suggested that both
mutants act upstream of siRNA production. We did not detect marked effects
of esi1 and esi2 on either susceptibility to virus infection or virus-induced
gene silencing (VIGS) as judged from inoculations of mutants, the high-GFP
expressing wild-type line, and a silent GFP reporter line with Cucumber
mosaic virus (CMV) and with RNA representing the genome of a recombinant
Turnip crinkle virus (TCV) with the coat protein ORF replaced by a GFP- phytoene
desaturase transcriptional fusion (TCV GFP-PDS DCP). The CMV 2b
protein has been shown to suppress RNA silencing and is believed to act in
the cell nucleus. Our most interesting finding was that GFP silencing was not
suppressed in esi2 mutants infected with CMV. These results, and the fact a
GFP reporter gene is constitutively silenced in esi2, lead us to propose that
ESI2 is part of host silencing-suppression pathway, which is activated by the
CMV 2b protein.
This work was supported in part by the Novartis Research Foundation and by
Grant No. BBW 00.0224-2 from the Swiss Office for Education and Science
as part of the European Union Gene Silencing in Transgenic Plants Project No.
QLK3-2000-00078.
T09 Genetic Mechanisms (Transcriptional and Chromatin Regulation)