Supplemental Data. Kurzbauer et al. (2012). Plant Cell 10.1105/tpc.111.098459 1 Supplementary Figure 1. Molecular characterisation of the Arabidopsis rad51 null allele. atr rad51 double mutants, with both alleles homozygous for the respective mutation, were grown for two further generations and then analysed by PCR genotyping and DNA gel blot analysis, confirming the presence of both mutant alleles. In rad51-1 (Li et al. 2004) mutants only mRNA corresponding to the 5’ part of the gene, the region upstream of the inserted T-DNA sequence, was detected. To rule out that a hypomorphic, truncated RAD51 protein is made from this truncated mRNA, we generated a new polyclonal antibody against RAD51 (-RAD51*), with the potential to detect the N-terminal part of the Arabidopsis RAD51 protein. The new antibody was successfully tested against different truncated versions of Arabidopsis RAD51 generated in E. coli, including one corresponding to a putative truncated protein in the rad51-1 mutant (RAD51 N-term). Using this antibody, no RAD51 signal could be detected in meiotic spreads of rad51-1 mutant plants. We therefore concluded that the rad51-1 mutant allele represents a true null allele. (A) Top: Schematic representation of the genomic DNA and the transcript of the Arabidopsis RAD51 gene (RAD51; At5g20850). The insertion site of the integrated T- DNA in the rad51 mutant line (rad51-1; GABI_134A01) is indicated as triangle. Black boxes represent exons, primer for genotyping PCR (red), RT-PCR (blue) and DNA gel blotting (green) are depicted as arrows and the BsrGI restriction sites (“B”) for DNA gel blotting are shown. Bottom: Results of genotyping PCR and RT-PCR using the primer shown on the left. PCR on the wild-type allele of RAD51 yields an approximately 950 base pairs (bp) product and on the mutant allele an approximately
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Supplemental Data. Kurzbauer et al. (2012). Plant Cell 10.1105/tpc.111.098459
1
Supplementary Figure 1. Molecular characterisation of the Arabidopsis rad51
null allele.
atr rad51 double mutants, with both alleles homozygous for the respective mutation,
were grown for two further generations and then analysed by PCR genotyping and
DNA gel blot analysis, confirming the presence of both mutant alleles. In rad51-1 (Li
et al. 2004) mutants only mRNA corresponding to the 5’ part of the gene, the region
upstream of the inserted T-DNA sequence, was detected. To rule out that a
hypomorphic, truncated RAD51 protein is made from this truncated mRNA, we
generated a new polyclonal antibody against RAD51 ( -RAD51*), with the potential
to detect the N-terminal part of the Arabidopsis RAD51 protein. The new antibody
was successfully tested against different truncated versions of Arabidopsis RAD51
generated in E. coli, including one corresponding to a putative truncated protein in
the rad51-1 mutant (RAD51 N-term). Using this antibody, no RAD51 signal could be
detected in meiotic spreads of rad51-1 mutant plants. We therefore concluded that
the rad51-1 mutant allele represents a true null allele.
(A) Top: Schematic representation of the genomic DNA and the transcript of the
Arabidopsis RAD51 gene (RAD51; At5g20850). The insertion site of the integrated T-
DNA in the rad51 mutant line (rad51-1; GABI_134A01) is indicated as triangle. Black
boxes represent exons, primer for genotyping PCR (red), RT-PCR (blue) and DNA
gel blotting (green) are depicted as arrows and the BsrGI restriction sites (“B”) for
DNA gel blotting are shown. Bottom: Results of genotyping PCR and RT-PCR using
the primer shown on the left. PCR on the wild-type allele of RAD51 yields an
approximately 950 base pairs (bp) product and on the mutant allele an approximately
Supplemental Data. Kurzbauer et al. (2012). Plant Cell 10.1105/tpc.111.098459
2
800 bp product. PCR on cDNA yields a product using primer upstream of the
insertion site (I) in wild-type and mutants but primer spanning the insertion site (II)
only give a product with wild-type and never with mutant cDNA. DNA gel blotting was
performed to non-ambiguously demonstrate the exclusive presence of the rad51
mutant allele in atr rad51 F3 plants, defined as homozygous for both mutant alleles
by PCR. In total, 77 plants were tested, 14 individuals are shown (1-14) next to wild-
type (wt) and a rad51 single mutant (rad51). The lower band represents the 1000bp
product of the restriction digest of genomic DNA with BsrGI in wild-type, the upper
band also contains the 2091bp insertion present in rad51 mutants.
(B) Immunoblot and immune-histochemical experiments performed with a newly
generated antibody against Arabidopsis RAD51 ( -RAD51*). This antibody detects
the N-terminus of Arabidopsis RAD51, gives numerous foci on spread nuclei of wild-
type PMCs and does not yield any signal on spread nuclei of mutant rad51 PMCs.
(Size bar: 10 m)
Supplemental Data. Kurzbauer et al. (2012). Plant Cell 10.1105/tpc.111.098459
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Supplementary Figure 2. A mutation in ATR suppresses the severe meiotic
defects of rad51 mutants.
(A) Meiotic spreads of pollen mother cells (PMCs) stained with 4',6-diamidino-2-
phenylindole (DAPI). About 36% of atr rad51 double mutant cells undergo meiosis
without DNA fragmentation (see as well Figures 1 and 4). The displayed metaphase
II cell shows DNA fragmentation. dmc1 mutants form univalents and DNA repair is
not compromised (4% seed formation, n=289 siliques). The same phenotype is
observed in atr dmc1 double mutants (1% seed formation, n=114 siliques). A
mutation in ATRIP only partially alleviates the rad51 phenotype (4% seed formation,
n=48 siliques) and most meiocytes display DNA repair defects.
(B) Images of nuclear spreads of PMCs at early to mid-zygotene stage with RAD51
(red) and ASY1 (green) detected by immuno-fluorescence. Bars represent mean
RAD51 foci numbers per meiotic nucleus. RAD51 foci numbers are moderately but
significantly decreased in atr but not in dmc1 mutants. (Error bars represent standard
deviation; * p<0.05; Wilcoxon-Mann-Whitney test; Size bars: 10 m)
Supplemental Data. Kurzbauer et al. (2012). Plant Cell 10.1105/tpc.111.098459
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Supplementary Figure 3. Synaptonemal complex formation in atr rad51.
Staining for the synaptonemal complex protein ZYP1 showed normal progression of
synapsis in atr rad51 mutant meiocytes. (Size bar: 10 m)
Supplemental Data. Kurzbauer et al. (2012). Plant Cell 10.1105/tpc.111.098459
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Supplementary Figure 4. Antibodies and statistics.
(A) Immunoblot analysis to confirm the specificity of Arabidopsis -RAD51 and -
DMC1 antibodies that have been used for immune-histochemistry. The -DMC1
antibody detects the N-terminus of recombinant Arabidopsis DMC1 and does not
detect recombinant full-length Arabidopsis RAD51. The -RAD51 antibody detects
full-length recombinant RAD51, only shows a very weak residual binding to the
recombinant DMC1 C-terminus protein fragment and does not detect the RAD51 N-
terminus (data not shown).
(B) Monte-Carlo simulations for two further individually scored cells to determine the
expected random frequency of co-localisation of RAD51 and DMC1 foci. In all cases,
the numbers for the observed co-localising RAD51/DMC1 foci (R/D foci) were
significantly lower than the simulated numbers of randomly co-localising events.
Supplemental Data. Kurzbauer et al. (2012). Plant Cell 10.1105/tpc.111.098459
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Supplementary Figure 5. RAD51, DMC1 and H2Ax localisation in meiocytes.
Additional images of nuclear spreads of wild-type leptotene PMCs with RAD51 (red),
DMC1 (green) and H2Ax (blue) detected by immunofluorescence (please refer to
Figure 3 for picture details). (Size bar: 10 m)
Supplemental Data. Kurzbauer et al. (2012). Plant Cell 10.1105/tpc.111.098459
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Supplementary Table 1 – Inter-foci distances
The differences between observed inter-foci distances of different foci classes were
evaluated by two different methods (see below) and were found to not significantly
differ from each other (RD –RAD51 and DMC1 doublet; DD – DMC1 doublet; RR –
RAD51 doublet).
A) Fisher's Exact Test with two distance classes between foci
nm RR RD DD
0-150 7 12 4
150-450 65 89 50
Total 72 101 54
Fisher's Exact Test
p-value
RR vs RD 0,8064
DD vs RD 0,5804
RR vs DD 0,7568
B) Mann-Whitney Test for comparison of (non-parametric) distribution
nm RR RD DD
0-50 0 0 0
50-100 0 2 0
100-150 7 10 4
150-200 6 13 13
200-250 19 13 5
250-300 11 10 8
300-350 10 14 8
350-400 11 19 6
400-450 8 20 10
Mann-Whitney Test for comparison of (non-
parametric distribution)
p-value
RR vs RD 0,1978
DD vs RD 0,06191
RR vs DD 0,4227
Supplemental Data. Kurzbauer et al. (2012). Plant Cell 10.1105/tpc.111.098459