Download The Arabidopsis RAD51 paralogs RAD51B, RAD51D and XRCC2

Research
The Arabidopsis RAD51 paralogs RAD51B, RAD51D and XRCC2
play partially redundant roles in somatic DNA repair and gene
regulation
Yingxiang Wang1, Rong Xiao2*, Haifeng Wang1,3*, Zhihao Cheng1, Wuxing Li2, Genfeng Zhu1, Ying Wang1 and
Hong Ma1,2,3
1
State Key Laboratory of Genetic Engineering and Institute of Genetics, Institute of Plant Biology, Center for Evolutionary Biology, School of Life Sciences, Fudan University, Shanghai
200433, China; 2Department of Biology, Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA 16802, USA; 3Institutes of Biomedical Sciences, Fudan
University, Shanghai 200032, China
Summary
Author for correspondence:
Hong Ma
Tel: +86 21 55665569
Email: [email protected]
Received: 12 May 2013
Accepted: 14 August 2013
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doi: 10.1111/nph.12498
Key words: Arabidopsis, DNA repair, gene
regulation, homologous recombination,
molecular evolution, RAD51 paralogs.
The eukaryotic RAD51 gene family has seven ancient paralogs conserved between plants
and animals. Among these, RAD51, DMC1, RAD51C and XRCC3 are important for homologous recombination and/or DNA repair, whereas single mutants in RAD51B, RAD51D or
XRCC2 show normal meiosis, and the lineages they represent diverged from each other
evolutionarily later than the other four paralogs, suggesting possible functional redundancy.
The function of Arabidopsis RAD51B, RAD51D and XRCC2 genes in mitotic DNA repair
and meiosis was analyzed using molecular genetic, cytological and transcriptomic approaches.
The relevant double and triple mutants displayed normal vegetative and reproductive
growth. However, the triple mutant showed greater sensitivity than single or double mutants
to DNA damage by bleomycin. RNA-Seq transcriptome analysis supported the idea that the
triple mutant showed DNA damage similar to that caused by bleomycin. On bleomycin treatment, many genes were altered in the wild-type but not in the triple mutant, suggesting that
the RAD51 paralogs have roles in the regulation of gene transcription, providing an explanation for the hypersensitive phenotype of the triple mutant to bleomycin.
Our results provide strong evidence that Arabidopsis XRCC2, RAD51B and RAD51D have
complex functions in somatic DNA repair and gene regulation, arguing for further studies of
these ancient genes that have been maintained in both plants and animals during their long
evolutionary history.
Introduction
Genome stability is important for cellular homeostasis and an
organism must be able to repair DNA damage. Among a variety
of DNA damage, double-strand DNA breaks (DSBs) are caused
by ionizing radiation, genotoxic chemicals or errors in DNA replication (Kuzminov, 2001; Tonami et al., 2005). Failure to correctly
repair DSBs can cause genome instability, mutations, cell cycle
arrest and even cell death (Glazer & Glazunov, 1999; Mills et al.,
2003; Dudasova et al., 2004; Sasaki et al., 2004). DSBs are known
to be repaired by two major pathways: homologous recombination
(HR) and non-homologous end-joining (NHEJ). The NHEJ
pathway involves the rejoining of two broken DNA ends without
a template of similar sequence, often resulting in deletions or insertions. By contrast, HR is a relatively accurate pathway that
depends on the homologous DNA sequence, thereby retaining the
*These authors contributed equally to this work.
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correct genetic information in the repair process (West et al.,
2004; Bleuyard et al., 2006). In addition to its function in somatic
DNA repair, HR is also required for normal meiosis to maintain
the association of homologous chromosomes and contributes to
the redistribution of genetic diversity among progeny.
The genes involved in HR were first identified in budding
yeast and mainly belong to the RAD52 epistasis group, including
RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55,
RAD57, RAD59, MRE11 and XRS2 (Paques & Haber, 1999;
Krogh & Symington, 2004). Further identification of their
homologs in animals and plants suggests that the HR repair pathway is highly conserved (Krogh & Symington, 2004; Bleuyard
et al., 2006). Among them, members of the RAD51 family,
including DMC1, RAD51 and five RAD51 paralogs (RAD51B,
RAD51C, RAD51D, XRCC2 and XRCC3) have crucial roles in
HR or DNA repair in mammals. Mutations in several of these
genes lead not only to elevated sensitivity to DNA damaging
agents, but also to embryonic lethality (Tsuzuki et al., 1996; Shu
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et al., 1999; Deans et al., 2000; Pittman & Schimenti, 2000),
suggesting that they are important for DNA repair during the
mitotic cell cycle.
Homologs of DMC1 and RAD51 have been studied in many
eukaryotes, including fungi, invertebrate animals and plants
(Bishop et al., 1992; Habu et al., 1996; Klimyuk & Jones, 1997;
Couteau et al., 1999). In Arabidopsis thaliana, they function in
DNA repair via HR in somatic or meiotic cells (Couteau et al.,
1999; Bleuyard & White, 2004; Li et al., 2004, 2005; Abe et al.,
2005; Bleuyard et al., 2005; Osakabe et al., 2005). For simplicity,
unless otherwise noted, the RAD51 paralogous genes and
mutants refer to those of Arabidopsis. Both rad51c and xrcc3
knockout mutants are hypersensitive to DNA damaging agents
and sterile with striking meiotic chromosome fragmentation, suggesting that RAD51C and XRCC3 are involved in mitotic DNA
repair by somatic and meiotic recombination (Bleuyard &
White, 2004; Abe et al., 2005; Bleuyard et al., 2005; Li et al.,
2005). By contrast, the rad51b, rad51d and xrcc2 mutants show
normal fertility without detectable meiotic defects, but are sensitive to various DNA damaging agents (Bleuyard et al., 2005),
and RAD51B and XRCC2 seem to have a role in the suppression
of meiotic recombination (Ines et al., 2013), suggesting that
RAD51B, RAD51D and XRCC2 are involved in somatic and meiotic HR. Furthermore, the Arabidopsis RAD51B, RAD51C and
RAD51 proteins also interact in yeast two-hybrid systems, similar
to their mammalian counterparts, suggesting that they have
conserved functions (Osakabe et al., 2005).
In mammals, the embryonic lethality of mutations in RAD51
and its paralogs makes it difficult to analyze their function
in vivo. By contrast, none of the Arabidopsis RAD51 paralogs is
required for survival in individual single mutants. It has been
reported that the RAD51B, RAD51D and XRCC2 homologs
form the three groups that occupy the last three branches in the
RAD51 family tree (Lin et al., 2006). Therefore, these genes
might have overlapping/redundant functions in plant mitotic cell
cycle or meiotic cells. It is also possible that RAD51B, RAD51D
and XRCC2 are not required for normal meiosis, even when their
functions are lost simultaneously. Nevertheless, the Arabidopsis
RAD51B, RAD51D and XRCC2 proteins might form a protein
complex that interacts with RAD51C in the process of DNA
repair by HR. To date, the genetic relationship among RAD51B,
RAD51D and XRCC2 in Arabidopsis has not been studied. The
study of their genetic interactions should provide clues to the
understanding of the function and relationship of these genes
during their long evolutionary history.
Materials and Methods
Plant materials and growth conditions
The xrcc2 (SALK_029106) and rad51b (SALK_024755) T-DNA
insertional lines have been characterized previously by Bleuyard
et al. (2005). rad51d (also named ssn1) was obtained from Professor Xinnian Dong’s laboratory (Durrant et al., 2007). All plants
were grown in growth chambers under a 16-h light : 8-h dark
photoperiod at 22°C : 18°C, unless otherwise indicated.
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Characterization of the double and triple mutants
F1 double heterozygous plants were generated by crosses between
xrcc2, rad51b and rad51d homozygous single mutant plants. The
triple heterozygous F1 plants were generated by crossing the
rad51b xrcc2 double homozygous mutant with rad51d. The F2
or F3 progeny plants were genotyped with each of the genespecific primers for RAD51B and XRCC2, combining with the
T-DNA left board primer (Supporting Information Table S1).
To genotype rad51d, PCR products were digested with SphI to
produce two fragments of 233 and 86 bp for the wild-type and
one fragment of 319 bp for the mutant allele.
Light microscopy
Photographs of plants were taken with a Sony digital camera
DSC-707 (Tokyo, Japan). The viability of mature pollen grains
was examined after staining with Alexander’s solution (Alexander,
1969). Mitosis was examined using root tips of 1-wk-old seedlings, as described previously (Li et al., 2004). Male meiosis was
examined using chromosome spreading with 4′,6-diamidino2-phenylindole (DAPI) staining, as described previously (Ross
et al., 1996). Both pollen and meiotic cells were photographed
using a Nikon dissecting microscope (Tokyo, Japan) with an
Optronics digital camera (Goleta, CA, USA).
Treatment with DNA damaging agents
The eight genotypes of Col (wild-type), rad51b, rad51d (ssn1),
xrcc2, rad51b rad51d, rad51d xrcc2, rad51b xrcc2 and rad51b
rad51d xrcc2 were treated with either of two types of DNA damaging agent: the cross-linking agents cisplatin (Sigma P4394),
methyl methanesulfonate (MMS; Sigma M4016) and mitomycin-C (MMC; Sigma M4287); the DSB-inducing agent bleomycin (Sigma B5507). Seeds were surface sterilized with 10%
NaClO for 5 min and 75% ethanol for 5 min, and then sown on
Murashige and Skoog (MS) plates containing different concentrations of MMC, bleomycin, cisplatin or MMS, as indicated in
the text. The plates were placed at 4°C for 3 d, and then transferred to a growth chamber. The resistance or sensitivity was estimated by the average fresh weight of four plants after growth for
3 wk.
The comet assay
Fourteen-day-old plants grown on half-strength MS plates under
normal conditions were incubated in 2 lg l 1 of bleomycin for
6 h and then harvested in liquid nitrogen. Comet assay for DNA
damage was performed according to a previously described protocol (Menke et al., 2001; Zhu et al., 2006) with minor modifications. Comet slides were prepared and subjected to
electrophoresis on ice for 2 min (2 V cm 1, 11 mA). Images of
comets were captured under a Zeiss Axio Imager A2 fluorescence
microscope with a high-resolution microscopy camera AxioCam
MRc Rev. 3 FireWire (D). The comet data analysis was performed using CometScore software (http://autocomet.com). The
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DNA fragments in comet tails (% tail-DNA) were used to estimate the extent of DNA damage.
RNA extraction and real-time PCR
Three-week-old young plants grown without or with DNA damaging agent at a concentration of 23.5 lg ml 1 (in the same manner as for the phenotypic analyses) were collected and quickly
frozen in liquid nitrogen. Total RNA was extracted using an
RNeasy Plant Kit (Qiagen, Valencia, CA, USA) and its concentration was estimated on an Agilent 2100 Bioanalyzer (Agilent
Technologies, Waldbronn, Germany). Approximately 1 lg of
total RNA was used to synthesize cDNA according to the manufacturer’s instruction (Promega, Madison, WI, USA). Real-time
PCR was performed as described previously (Yang et al., 2011) in
triplicate for each sample, using gene-specific primers (Table S1).
RNA-seq library preparation and sequencing
The ribosomal RNA was then removed by two purification steps
with a PolyATtract® mRNA Isolation System (Promega) and a
Poly (A) PuristTM Kit (Ambion, Austin, TX, USA), respectively.
The removal of ribosomal RNAs was confirmed on an Agilent
2100 Bioanalyzer. Approximately 0.8 lg of mRNA was fragmented by RNase III at 37°C for 10 min and ligated with adaptor Mix A for reverse transcription. The 100–200 nucleotides of
the first-strand cDNA were recovered by separation in 6% TBE
(Tris-borate-EDTA)-Urea Gel (Invitrogen, Carlsbad, CA, USA).
The fractionated cDNAs were amplified with 11–15 cycles of
PCR and then purified to yield the SOLiD Fragment Library;
600 pg of the library was used for emulsion PCR; 50-base
sequence reads were obtained using a SOLiD sequencer (ABI,
Foster City, CA, USA).
Estimation of expression level and differential gene
expression
Reads from each sample were aligned to The Arabidopsis Information Resource (TAIR) 10 Arabidopsis reference genome
(http://www.arabidopsis.org) using SOLiDTM BioScopeTM Software 1.3 (https://products.appliedbiosystems.com), a SOLiD
data analysis package for transcriptome sequencing and other
sequencing technologies. Afterwards, the aligned reads matching
the annotated genes were used to estimate gene expression levels
and to identify differentially expressed genes between treatments
by Cufflinks (v1.2; Trapnell et al., 2010). To reduce the falsepositive rate, a threshold for differential expression was set at a P
value of 0.05 or less in the Cufflinks output, with a further
requirement of a minimal gene expression level of at least 1.0
FPKM (fragments per kilobase of transcripts per millions of
mapped reads).
Gene ontology (GO) enrichment analysis
We used the GO terms defined by the TAIR 10 GO Slim database (ftp.arabidopsis.org:/Ontologies/Gene_Ontology) for GO
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enrichment analysis employing the online tools AgriGO with
Fisher’s exact test and false discovery rate (FDR) correction
(http://bioinfo.cau.edu.cn/agriGO/analysis.php). Transcription
factor (TF) family annotations were downloaded from the PlantTFDB v2.0 database, containing 2023 TFs in 58 families for
Arabidopsis thaliana (http://planttfdb.cbi.edu.cn/index.php;
Zhang et al., 2011). The heat map of the expressed TFs was
implemented by the pheatmap (Pretty Heatmaps) function in the
pheatmap package (R version, 2.15, pheatmap version, 0.6.1; R
Core Team, Vienna, Austria).
Results
Expression patterns of the three RAD51 paralogs
Previous studies have shown that RAD51B is expressed widely
and at a higher level in the floral buds than in other tissues
(Osakabe et al., 2005). Similarly, RAD51D is expressed widely,
but at very low levels (Durrant et al., 2007). The expression of
XRCC2 has not been reported. To further examine the expression
of these three Arabidopsis RAD51 paralogs (referred to hereafter
as the RAD51 paralogs for convenience), we searched our microarray data and male meiocyte transcriptome by RNA-Seq (Zhang
et al., 2005; Yang et al., 2011). Consistent with previous reports,
our microarray data showed that RAD51B and RAD51D were
widely expressed with relatively low levels, as was XRCC2
(Fig. S1a). Unlike DMC1, RAD51 and RAD51C, which showed
the highest expression in stage-6 anthers containing meiocytes,
RAD51B and RAD51D were expressed at lower levels in stage-6
anthers than in other tissues (Fig. S1a). Their low-level expression
was further confirmed by our male meiocyte transcriptome data
(Yang et al., 2011; Fig. S1b).
Mutants defective in the RAD51 paralogs showed normal
vegetative and reproductive growth
Previous studies have shown that the rad51b, rad51d and xrcc2
single mutants display normal vegetative and reproductive
growth (Bleuyard et al., 2005; Durrant et al., 2007). To test
whether these genes were redundant for normal development, we
generated the relevant double and triple mutants and found that
they all exhibited normal development of the vegetative and floral
organs and produced seedpods with normal seed numbers
(Fig. 1). Furthermore, pollen grains from all single and multiple
mutants were viable and indistinguishable from those of the
wild-type (Fig. 1). Because mutations in RAD51 paralogs in
mouse and chicken cause severe mitotic defects that are accompanied by chromosome fragmentation (Takata et al., 2000, 2001),
it is possible that there are minor defects in mitotic cell division
not observed from gross examination of the vegetative and reproductive growth of the Arabidopsis mutants. Mitotic chromosomes were further examined using DAPI staining of cells from
1-wk-old root tips of the eight genotypes. The results showed
that mitotic chromosome features at metaphase, anaphase and
telophase were indistinguishable among the eight phenotypes:
wild-type (70 cells), rad51b (54 cells), rad51d (35 cells), xrcc2
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Fig. 1 Phenotypes of wild-type (WT) and the
Arabidopsis rad51b, rad51d, xrcc2 single,
double and triple mutants. The left columns
show the flower phenotype for the eight
genotypes (as indicated on the left), the
middle columns display the features of
siliques and the right columns present pollen
grains stained with Alexander’s solution of
the eight genotypes. No obvious differences
were found among the eight genotypes.
Fig. 2 Mitotic chromosomes in 1-wk-old
Arabidopsis root tips of wild-type (WT) and
the rad51b, rad51d, xrcc2 single, double and
triple mutants. Mitotic chromosomes were
visualized with 4′,6-diamidino2-phenylindole (DAPI) at metaphase,
anaphase and telophase stages during the
mitotic cell cycle.
(60 cells), rad51b rad51d (27 cells), rad51b xrcc2 (52 cells),
rad51d xrcc2 (59 cells) and rad51b rad51d xrcc2 (74 cells; Fig. 2).
Taken together, these results suggest that these three genes combined are not required for plant vegetative and reproductive
development.
The single, double or triple mutants in the RAD51 paralogs
appeared normal in male meiosis
In Arabidopsis, both RAD51C and XRCC3 are required for
meiosis and DNA repair (Bleuyard & White, 2004; Abe et al.,
2005; Li et al., 2005). Even though the mutants in the three
RAD51 paralogs showed normal pollen phenotypes, we could
not rule out the possibility that minor male meiotic defects
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might still be present, but not sufficiently severe to affect fertility, as was the case for the mus81 mutant (Berchowitz et al.,
2007). We found that male meiosis in all mutant genotypes
showed normal chromosome behavior, including typical diakinesis with five bivalents (Fig. 3). In addition, similar to the
wild-type, the bivalents in all mutants were well aligned at the
equatorial plane at metaphase I (Fig. 3) and then segregated to
form two groups of chromosomes at anaphase I. The two
groups of chromosomes further underwent decondensation and
recondensation, and were again aligned at two division planes
at metaphase II (Fig. 3). After anaphase II and telophase II,
sister chromatids were separated to form four nuclei, which
were packed into four microspores at the end of male meiosis
(Fig. 3). We examined a total of > 300 meiocytes for the triple
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Fig. 3 The Arabidopsis male meiosis in the
wild-type (WT) and rad51b, rad51d, xrcc2
single, double and triple mutants.
Chromosome features were stained by
4′,6-diamidino-2-phenylindole (DAPI) in the
WT and mutants at diakinesis, metaphase I,
metaphase II and tetrad stages. No obvious
differences were found among the eight
genotypes at each stage.
mutant, but found no obvious meiotic defects. Therefore, we
conclude that these three genes are not essential for male meiosis, because mutations in the three genes together did not
result in detectable abnormality.
RAD51B, RAD51D and XRCC2 were partially redundant
for somatic DSB repair
Mutations in different RAD51 paralogs caused sensitivity to
DNA damaging agents, such as c-irradiation, MMC, cisplatin
and bleomycin, in both animal cells and Arabidopsis (Liu et al.,
1998; Takata et al., 2001; Abe et al., 2005; Osakabe et al., 2005),
suggesting that the functions of the RAD51 paralogs in DNA
damage repair are conserved between vertebrates and plants. To
investigate the relationship between RAD51B, RAD51D and
XRCC2 in DNA damage repair, we first examined whether the
double and triple mutant plants conferred greater sensitivity than
the single mutants to MMC, a DNA cross-linking agent (Warren
et al., 1998). As shown in Fig. 4(c,d), the growth of the eight
genotypes was indistinguishable at a low dose of MMC
(30 lg ml 1). At 60 lg ml 1or higher concentrations, the growth
of all mutants was inhibited significantly compared with that of
the wild-type, to similar extents among the single, double and
triple mutants (Fig. 4c,d). In a parallel experiment, the single,
double and triple mutants exhibited slight or mild sensitivity in a
similar manner to other DNA cross-linking agents, such as MMS
and cisplatin (data not shown). These results indicate that these
three genes play similar, but not redundant, roles in the repair of
damage caused by DNA cross-linking agents.
Previous studies have demonstrated that RAD51B, RAD51D
and XRCC2 have a role in DSB repair (Bleuyard et al., 2005;
Osakabe et al., 2005; Durrant et al., 2007), but their genetic relationship in DSB repair is still lacking. Thus, we evaluated the
sensitivity of the single, double and triple mutants to bleomycin,
which causes DSBs in DNA (Favaudon, 1982). As shown in
Fig. 4(e,f), all mutants grew normally on medium supplemented
with 7.05 lg ml 1 of bleomycin. At 14.1 lg ml 1, all single,
double and triple mutants were more severely affected than the
wild-type, with the triple mutants being slightly more sensitive
than the double mutants (Fig. 4e,f). The dose of 23.5 lg ml 1
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bleomycin strongly inhibited the growth of all plants, with more
dramatic effects on mutant genotypes, especially for the triple
mutants, with significant differences compared with all other
genotypes (Fig. 4e,f). The hypersensitivity of the triple mutant to
the high dose of bleomycin was different from its response to the
cross-linking agents, such as MMC, suggesting that the three
RAD51 paralogs have partially redundant functions or are
involved in different pathways for the repair of DNA damage
induced by bleomycin.
To further evaluate the levels of DNA damage in the double
and triple mutants exposed to bleomycin, the comet assay experiment, which reveals damaged DNA in a tail resembling that of a
comet after electrophoresis, was performed to estimate the
amount of DNA damage in 2-wk-old seedlings of the eight genotypes induced by bleomycin. The results showed that, under normal growth conditions, wild-type and triple mutant plants
showed no significant difference in the levels of DNA damage
(Fig. 5a,b). By contrast, with 2 lg ml 1 bleomycin induction for
6 h, the accumulation of DNA in the comet tail in the xrcc2 single mutants and all double and triple mutants was significantly
higher than that in rad51b, rad51d and the wild-type (Fig. 5a,b).
It is especially worth noting that the triple mutants showed the
highest level of DNA damage with 92.22% of the nuclear DNA
in the tail, consistent with the observation that the triple mutant
displayed the highest sensitivity to bleomycin. These results further support the proposal that these three genes have partially
redundant roles in DNA repair.
Expression of DNA repair genes was induced in the triple
mutant or by bleomycin
Previous studies have revealed that mutants, such as rad51 and
rad51d, which are defective in mitotic HR or DNA repair, are
also affected in gene expression (Durrant et al., 2007; Tuteja
et al., 2009; Wang et al., 2010; Liu & Gong, 2011). To investigate whether the hypersensitive phenotype of the triple mutant to
bleomycin is related to the expression of HR or other
DNA repair genes, we examined several representative genes in
3-wk-old seedlings with or without bleomycin treatment. In
mammalian cells, RAD51B, RAD51D and XRCC2 form a
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(a)
(b)
(c)
(d)
(e)
(f)
Fig. 4 Mitomycin-C (MMC) and bleomycin sensitivity assay of wild-type (WT) and rad51b, rad51d, xrcc2 single, double and triple mutants. The
Arabidopsis plants were grown on half-strength Murashige and Skoog (MS) medium without or with different doses of methanesulfonate (MMS) or
bleomycin for 3 wk, with 16 plants for each genotype in each replicate. The growth phenotypes were for normal conditions (a) for the eight genotypes
arranged as indicated in (b), for 60 and 120 lg ml 1 of MMC (c) and for 14.1 and 23.5 lg ml 1 of bleomycin (e); genotypes for plants shown in (c) and
(e) are also the same as indicated in (b). Fresh weight was determined for groups of four plants, and the average weight for 16 plants was calculated.
Shown are the average fresh weights from three independent tests for each genotype under a series of MMC treatments (d) and bleomycin treatments (f).
Bars show standard error (**, P < 0.01 for comparison between each mutant and WT, as well as between the single or double mutants and the triple
mutant at 23.5 lg ml 1 in (d) and (f)). CK, control.
complex with RAD51C (the BCDX2 complex), and RAD51C
with XRCC3 (the CX3 complex; Wiese et al., 2002); furthermore,
some of the plant members can also interact physically in a yeast
two-hybrid assay (Osakabe et al., 2005). Therefore, we first examined the expression of RAD51 paralogs, including RAD51 and
RAD51C. As shown in Fig. 6, RAD51 and RAD51C expression
showed no significant difference between the wild-type and triple
mutants with or without bleomycin treatment (Fig. 6). By contrast, the expression level of GAMMA RESPONSE1 (GR1) was
increased dramatically by either the triple mutations or bleomycin
induction (Fig. 6). We then examined the expression of BRCA1,
which is important for HR and DNA repair in plants (Bundock &
Hooykaas, 2002; Block-Schmidt et al., 2011), and found that
BRCA1 expression in the wild-type and triple mutants resembled
that of RAD51 under normal conditions (Fig. 6). By contrast,
BRCA1 expression was sharply increased in the triple mutant compared with the wild-type under bleomycin treatment (Fig. 6). It is
possible that DNA damage induced by bleomycin was repaired less
effectively in the triple mutant, as suggested by its greater sensitivity to bleomycin relative to the single or double mutants, thereby
triggering the elevated expression of some of the DNA repair
genes.
RAD51B, RAD51D and XRCC2 affected normal gene
expression
To identify additional genes with altered expression in the triple
mutant, RNA-Seq was performed using mRNA isolated from
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wild-type and triple mutant seedlings by the SOLiD 3 platform,
as described previously (Yang et al., 2011). We obtained a total
of c. 378 million single-end reads of 50 bp (Table S2). Approximately 65.9% of reads were mapped to the Arabidopsis reference
genome (TAIR 10), representing c. 70.8% of the annotated genes
in TAIR 10 (Table S3) and providing high-quality data to
explore the transcriptome.
We first identified genes with altered expression in the triple
mutant compared with the wild-type (Fig. 7a), and found that
2111 genes were differentially expressed (FPKM ≥ 1 and
P ≤ 0.05), including 1450 up-regulated (Table S4) and 661
down-regulated (Table S5) genes, although many of these genes
might not be regulated directly by the RAD51 paralogs. The GO
categorization for the 1450 up-regulated genes showed that
molecular functions of DNA repair, transcriptional regulator
activity, DNA binding, enzyme activity and developmental regulation were over-represented (P < 10 4, Fig. 7b). Specifically,
among these were over 45 genes with known functions in somatic
DNA repair or meiotic recombination, such as COM1/GR1,
MND1 and RPA1A, as well as RAD3, RAD54, MutS homolog 2,
4, 7 and DNA damage repair 1 (DRT101; Table S6), suggesting
that the triple mutant had more DNA damage even without
bleomycin treatment, although this was not obvious using the
comet assay.
To investigate possible effects of the DNA damaging agent
bleomycin on gene expression in the wild-type, we compared the
wild-type transcriptome with or without bleomycin treatment,
and found that 4311 genes were differentially expressed
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(a)
Fig. 6 Analysis of the expression of RAD51, RAD51C, GR1 and BRCA1
genes in wild-type (Wt) and triple mutants using quantitative real-time
PCR. The 3-wk bleomycin-treated and untreated Arabidopsis seedlings in
wild-type and mutants were the same as in Fig. 4. Data are means SD of
three technical replicates (**, P < 0.01 for comparison with wild-type).
Similar patterns were obtained from two biological replicates. CK, control.
(a)
(c)
(b)
(b)
Fig. 5 Evaluation of DNA damage in wild-type (WT) and rad51b, rad51d,
xrcc2 single, double and triple mutants by the comet assay. Samples for
comet assay were prepared from untreated and bleomycin-treated
14-d-old Arabidopsis WT and mutant seedlings, as described in the
Materials and Methods section. (a) Representative comet images are
shown for the untreated WT and the triple mutant, as well as the eight
treated genotypes. The red color shows the DNA stained by propidium
iodide (PI). (b) The fraction of DNA from untreated and treated samples
was separated in the comet tails by electrophoresis, which is defined as an
indicator for double-strand DNA break (DSB) repair, and was quantified
using a computerized CCD camera digital image analysis system (Tritek
CometScore). The values shown are averages from two to four technical
replicates, each with three slides, and from each slide 25 nuclei were
scored. (*, P < 0.05; **, P < 0.01; ***, P < 0.001, for comparison between
each mutant and WT; ▲▲▲, P < 0.001 for comparison between the double
mutants and the triple mutant).
(FPKM ≥ 1and P ≤ 0.05), with 2835 genes up-regulated
(Table S7) and 1476 genes down-regulated (Table S8) in bleomycin-treated seedlings. The GO categorization indicated that
DNA repair (53 genes; Table S6), response to stimulus, immune
response, DNA binding and several kinds of enzymes were most
enriched in the up-regulated genes (Fig. 7b). In the down-regulated genes, the most enriched categories were the same as those
in the up-regulated genes of the triple mutant, such as transcriptional regulation, DNA binding and enzyme activity (Table S9),
but the specific genes did not overlap between these sets.
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Fig. 7 Comparisons of the differentially expressed genes affected by the
triple mutations and/or bleomycin treatment in Arabidopsis. (a) A
comparison of the differentially expressed genes caused by the triple
mutations and bleomycin treatment (in the wild-type (Wt)). (b) Gene
ontology (GO) annotation of three sets of up-regulated genes from (a).
DEF_707, DEF_739 and DEF_2047 represent the differentially expressed
up-regulated genes in the triple mutant, in both triple mutant and
bleomycin-induced wild-type and in the bleomycin-induced wild-type,
respectively. (c) A comparison of the differentially expressed genes caused
by the triple mutations and bleomycin treatment in the triple mutant. CK,
control.
The fact that several known genes involved in DNA repair
were induced in both the triple mutant and by bleomycin treatment suggested that the respective sets of differentially expressed
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genes might be similar when compared with the untreated
wild-type. To test this possibility, we compared the two sets of
differentially expressed genes, and found that the number of
differentially expressed genes in the bleomycin-treated wild-type
was more than double the number of genes differentially
expressed in the triple mutant, suggesting that bleomycin has
more severe effects than the three mutations. In addition, among
the up-regulated genes in the triple mutant, 739 genes (> 50%)
were also found in the up-regulated genes caused by bleomycin
treatment (Fig. 7a, Table S10), suggesting that a large number of
genes were induced in the triple mutant probably as a result of
the accumulation of DNA damage when the repair functions
were reduced. Furthermore, functional annotation of these genes
showed that the main molecular functions were related to DNA
repair, chromatin structure and stimulus response (Fig. 7b),
including 30 genes related to DNA repair as mentioned already,
such as COM/GR1, MND1, RPA1A, DRT101 and MSH7. These
results support the idea that bleomycin and mutations in the
three RAD51 paralogs both cause the accumulation of DNA
damage. Moreover, transcriptional regulation was also most
enriched in a set of 704 genes induced in the triple mutant, but
not by bleomycin (Fig. 7b), whereas the categories of stimulus
and immune response, enzyme activity and cell death were most
enriched in the 2047 genes induced by bleomycin, but unaffected
in the triple mutant (Fig. 7b), indicating that bleomycin treatment and the triple mutations also induced distinct changes in
gene expression.
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Although the expression of similar numbers of genes was
affected by bleomycin in both the wild-type and the triple
mutant, the fact that the triple mutant was hypersensitive to bleomycin suggested that distinct sets of genes might be affected in
these two genotypes. Indeed, 1428 of the genes up-regulated in
the bleomycin-treated wild-type were not induced in the triple
mutant by bleomycin, and 1001 of the genes induced in the triple mutant were not up-regulated in the treated wild-type
(Fig. 8a, Table S18). Likewise, 624 of the genes that were downregulated in the bleomycin-treated wild-type were not repressed
by bleomycin in the triple mutant, whereas 1224 of the genes
(a)
(b)
(c)
(d)
The triple mutant exhibited a strong transcriptomic
response to bleomycin
Although the triple mutant showed altered expression relative
to the wild-type for many genes, it still responded to bleomycin in gene regulation. We therefore compared the transcriptomes between bleomycin-treated and untreated triple mutant
seedlings, and found 2408 and 2076 genes to be up-regulated
and down-regulated, respectively, in treated triple mutant seedlings (Fig. 7c, Tables S11, S12). GO annotation showed that
most enriched categories in the up-regulated genes were the
same as those in the bleomycin-treated wild-type (Table S13),
whereas transcriptional regulation, DNA binding and oxidoreductase activity were enriched in down-regulated genes
(Table S14).
We also found that bleomycin affected very different sets of
genes relative to the triple mutations, as supported by the relatively large number of genes showing opposite effects for the triple mutations and bleomycin: 270 were expressed at higher levels
in the wild-type than in the triple mutant, but were repressed by
bleomycin, whereas 352 showed lower levels of expression in the
wild-type than in the triple mutant, but were induced by bleomycin (Fig. 7c, Tables S15, S16). GO annotation analysis revealed
that TF and regulation activity, DNA binding and enzyme inhibitor activity were enriched in the 352 genes induced by bleomycin in the triple mutant (Table S17), suggesting that the
up-regulatory genes are important for the response to stress by
the triple mutant.
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(e)
(f)
Fig. 8 Additional comparisons of the differentially expressed genes caused
by mutations in the RAD51 paralogs and/or bleomycin in Arabidopsis. (a)
A comparison of the genes differentially expressed by bleomycin in either
the wild-type (Wt) or triple mutant. (b) A comparison of the genes
differentially expressed by the triple mutations in either the absence or
presence of bleomycin. (c) Hierarchical clustering of the 84 differentially
expressed genes encoding transcription factors in the four samples. (d–f)
Heat-map of bHLH, ERF and MYB genes with putative or known functions
in development or responses to stress. Red represents genes with a higher
expression level in the mutants, and blue indicates reduced expression. CK,
control.
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300 Research
repressed by bleomycin in the triple mutant did not decrease in
the bleomycin-treated wild-type (Fig. 8a). It is possible that the
genes affected by bleomycin in the wild-type, but not in the triple
mutant, require the function of the RAD51 paralogs for increased
expression by bleomycin. Among the 1001 genes specifically
induced by bleomycin in the triple mutant are those related to
defense and immune responses, apoptosis, cell death and enzyme
activities (Table S19). These genes might be sensitized by the
defects of the RAD51 paralogs and might be more easily induced
by bleomycin in the mutant compared with the wild-type.
To further investigate whether the hypersensitivity of the triple
mutant to bleomycin is related to the expression of specific genes,
we compared the expression of genes with known DNA repair
functions, and found that most genes examined showed dramatically lower levels of expression in the bleomycin-treated triple
mutant than in the bleomycin-treated wild-type (Table S6), similar to the above real-time PCR results (Fig. 6). The low-level
expression of these DNA repair genes, in addition to the triple
mutations, suggested that the DNA repair capacity was low and
DNA damage probably accumulated abnormally in the triple
mutant when treated with bleomycin.
To identify additional differentially affected genes, we compared the transcriptomes between the triple mutant and wildtype, both treated with bleomycin, and found 562 differentially
expressed genes, with 238 up-regulated and 324 down-regulated
in the triple mutant compared with the wild-type (FPKM ≥ 1
and P ≤ 0.05; Fig. 8b). Most of these genes (428) did not overlap
with those differentially expressed between the triple mutant and
wild-type when both were untreated with bleomycin (Fig. 8b),
suggesting an interaction between the triple mutations and bleomycin. In other words, these 428 genes required the presence of
bleomycin for differential expression by the triple mutations,
with 189 and 239 genes up-regulated and down-regulated,
respectively (Fig. 8b, Tables S20, S21). GO analysis of the 428
genes revealed gene function categories for metabolism, signaling,
stresses, catalytic activity and transcriptional regulation (Fig. S2).
A group for response to stimulus included 20 crucial genes for
disease response, such as PR1, PRB1 and JAZ4 (Table S22), consistent with the previous finding that RAD51 and RAD51D regulate directly defense-related genes (Durrant et al., 2007; Wang
et al., 2010).
To further examine the genes affected by the triple mutations
in the presence of bleomycin, we focused on 84 TF genes belonging to 10 families (21 up-regulated and 63 down-regulated), and
divided into five groups by hierarchical clustering, with additional expression data from the triple mutant and wild-type without bleomycin (Fig. 8c). The expression patterns of these genes
suggested that transcriptional regulation was dramatically altered
in the triple mutant, even when its morphology was normal. The
expression of group I genes was induced by bleomycin in the
wild-type, but generally similar in the triple mutant with or without bleomycin, indicating that their induction by bleomycin was
dependent on the RAD51B–RAD51D–XRCC2 functions
(Fig. 8c). By contrast, the expression of group II genes was
induced by bleomycin in the triple mutant, but not in the wildtype, suggesting that these triple mutations might have sensitized
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these genes, making them more easily induced by bleomycin
(Fig. 8c). Expression of the group III genes was reduced dramatically in the wild-type by bleomycin, but the difference in the triple mutant, with or without bleomycin treatment, was not
dramatic (Fig. 8c), again suggesting a role of the RAD51 paralogs
in the altered expression caused by bleomycin. The group IV
genes were negatively regulated by the RAD51 paralogs, but bleomycin canceled the effect of the triple mutations (Fig. 8c). The
group V genes were slightly repressed by bleomycin in the wildtype, and even more repressed by bleomycin in the triple mutant,
suggesting an additive effect (Fig. 8c). Moreover, TF genes of the
same family also showed distinct expression patterns in the four
samples. For example, among five bHLH genes examined here,
the expression pattern of At5G51780 and At1G74500 was similar
to that of the group I genes, At5G15160 was similar to the group
III genes, whereas At3G59060 and At5G56960 were similar to
the group V genes (Fig. 8d). Similar phenomena were also found
for MYB and ERF genes (Fig. 8e,f). The variety of expression patterns of the TF genes caused by the triple mutation or bleomycin
treatment suggest that plants use many different regulators to
achieve a comprehensive response to DNA damage from different causes.
Discussion
The role of RAD51B, RAD51D and XRCC2 in somatic DNA
repair
Similar to vertebrates, the Arabidopsis genome also contains
seven RAD51 homologs, which can be divided into two ancient
groups, the RADa and RADb subfamilies (Lin et al., 2006). The
RADa subfamily includes both RAD51 and DMC1, whereas the
RADb subfamily includes RAD51B, RAD51C, RAD51D, XRCC2
and XRCC3, which are also known as the five Arabidopsis
RAD51 paralogs. Among the seven genes, RAD51, DMC1,
RAD51C and XRCC3 have non-redundant roles in meiotic HR
and are required for normal fertility (Li & Ma, 2006). Except for
the meiosis-specific DMC1, the other three genes also function in
somatic DNA repair (Bray & West, 2005). By contrast, single
mutants defective in any of the RAD51B, RAD51D and XRCC2
genes exhibit increased sensitivity to DNA damaging agents, suggesting that they have a role in HR and/or DNA repair, but they
show normal vegetative growth and fertility. The normal morphological phenotypes of these mutants are in dramatic contrast
with the cellular phenotype and embryo lethality caused by the
mutations in their corresponding homologs in humans and mice
(Silva et al., 2010). In addition, yeast two-hybrid and immunoprecipitation studies have shown that animal XRCC2, RAD51B
and RAD51D form a complex with RAD51C (the BCDX2 complex) and function as a complex in homologous recombinational
DNA repair (Dosanjh et al., 1998; Liu et al., 1998, 2002; Schild
et al., 2000; Masson et al., 2001; Wiese et al., 2002). This complex may facilitate the formation of RAD51 foci important for
HR (Takata et al., 2000, 2001). Indeed, the survival of cell lines
carrying mutations in the RAD51 paralogs is reduced significantly in response to c-irradiation treatment (Takata et al., 2000,
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2001). Recent studies have shown that the BCDX2 and CX3
complexes act upstream and downstream, respectively, of RAD51
recruitment on DNA damage in human cells (Chun et al., 2013).
A recent study has reported that Arabidopsis single mutants in
the three genes do not affect the formation of radiation-induced
RAD51 foci (Ines et al., 2013). Our study has demonstrated that
the triple mutant exhibits greater sensitivity to bleomycin than
the single and double mutants (Fig. 4), indicating that their functions are partially redundant. For example, they might interact
with proteins in parallel pathways that have overlapping functions in somatic DNA repair. This idea is supported by recent
high-throughput proteomic analyses of protein complexes containing mouse RAD51C, RAD51D and XRCC2, which identified > 100 candidates for interaction with each protein (Rajesh
et al., 2009). More than 60% of these proteins were involved in
DNA/RNA modification or metabolism, including DNA mismatch repair protein MSH2, DNA replication-licensing factor
MCM2, SFPQ and NONO. Further studies have demonstrated
that SFPQ–NONO form a heteromeric complex to repair DSB
by rejoining DSB ends (Rajesh et al., 2009, 2010). In addition,
plant cells may require different recombination factors in different DNA repair pathways; for example, mutations in the plant
MRE11 and COM1 homologs do not affect either synthesisdependent strand annealing (SDSA) or single-strand annealing
(SSA), whereas mutations in RAD51, RAD51C and XRCC3, as
well as RAD54, affect SDSA but not SSA (Roth et al., 2012). The
idea that the three RAD51 paralogs together are involved in
multiple pathways is supported by our transcriptomic results,
which show that the expression of genes coding for factors
involved in both SDSA and SSA pathways is affected in the triple
mutant. It is also quite striking that the Arabidopsis RAD51B,
RAD51D and XRCC2 genes have partially redundant functions
because they diverged at least 1 billion yr ago.
Regulation of transcriptome by RAD51B, RAD51D and
XRCC2
Previous studies have shown that RAD51D and RAD51 regulate
pathogen-related genes on salicylic acid induction (Durrant et al.,
2007; Wang et al., 2010), suggesting that other RAD51 paralogs
might also have a role in transcriptional regulation. Our transcriptomic analysis detected 2111 differentially expressed genes
in the triple mutant compared with the wild-type (Fig. 7a). In
addition to several pathogen-related genes, other genes important
for DNA repair, abiotic stress and transcriptional regulation were
also enriched, suggesting that RAD51 paralogs have additional
roles in gene regulation. It is possible that some of these genes
might be regulated directly by the RAD51 paralogs, but many
could be affected by the accumulation of DNA damage caused
by the mutations in the RAD51 paralogs. However, this regulation does not seem to be vital for plant development under normal conditions, as the triple mutant showed normal vegetative
and reproductive growth (Figs 1, 2). Genetic studies support a
major function of these genes in somatic DNA repair. The normal development of the mutants suggests that little DNA damage, including DSBs, occurs under normal conditions, as
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Research 301
supported by the comet assay (Fig. 5), consistent with the similar
levels of RAD51 expression between the wild-type and triple
mutant (Fig. 6) and the indistinguishable patterns of radiationinduced RAD51 foci between each single mutant and the wildtype (Ines et al., 2013), as well as the normal growth of the rad51
mutant (Li et al., 2004).
Bleomycin causes DNA DSBs (Favaudon, 1982) and inhibits
the growth of both animal and plant cells, probably as a result of
the accumulation of unrepaired DSBs. However, the molecular
basis of the bleomycin-induced growth phenotype in plants is
not clear. We found that of the > 4000 differentially expressed
genes in the wild-type treated by bleomycin, over 58% of genes
were also found to be altered in the same direction in the triple
mutant, including 30 DNA repair genes (Table S6), suggesting
that bleomycin and the triple mutations have similar effects on a
large number of genes, including those for DNA repair.
Although this result further supports the hypothesis that the
main functions of the three RAD51 paralogs are in DNA repair,
the fact that both bleomycin and the triple mutations also have
specific sets of differentially expressed genes suggests that each
also has a distinct role in gene regulation. The existence of genes
that are specifically altered in the wild-type by bleomycin
suggests that bleomycin might cause more severe damage or
different type (s) of damage than that found in the triple mutant.
However, the presence of genes specifically induced/repressed by
the triple mutations raises the possibility that some genes are
regulated by the RAD51 paralogs, but not by DNA damage,
perhaps in a manner similar to the regulation of pathogen-related
genes by RAD51 and RAD51D. The RAD51 paralogs are
thought to be ATPases that associate with RAD51 and chromatin (Li & Ma, 2006; Lin et al., 2006); it is possible that, in addition to DNA repair, they affect chromatin structure and gene
expression.
Although the triple mutant was clearly hypersensitive to bleomycin and probably deficient in DNA repair, > 4000 genes were
still differentially expressed in the triple mutant in response to
bleomycin (Fig. 8a), consistent with the above discussion that
there is a substantial difference between the effects of mutations
and bleomycin treatment. Over 60% of the genes altered in the
triple mutant by bleomycin overlapped with those in the wildtype treated with bleomycin, indicating that these genes
responded to bleomycin independent of the functions of the
RAD51 paralogs. Nevertheless, the hypersensitivity of the triple
mutant to bleomycin (Fig. 8a) can be explained by the observation that nearly one-half of the bleomycin-induced genes in the
wild-type were not induced in the triple mutant, including some
genes crucial for the repair of damage caused by bleomycin, such
as COM1/GR1, MND1, RPA1A, RAD54, DRT101, ERCC1 and
MSH2/7. We noted that a number of genes encoding TFs
(groups I and III, Fig. 8c) were induced/repressed by bleomycin
in the wild-type but not in the triple mutant; some of these TFs
might be responsible for bleomycin-induced alteration in gene
expression in the wild-type, and the failure of the triple mutant
to regulate the expression of these TF genes provides an explanation
for the lack of differential expression of many of the bleomycininduced genes.
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302 Research
The other 40% of differentially regulated genes caused by bleomycin only in the mutant but not in the wild-type could be the
result of regulatory pathways affected by the RAD51 paralogs. In
particular, a number of TF genes (groups II and IV, Fig. 8c) were
induced/repressed by bleomycin in the mutant but not in the
wild-type; these could provide the needed regulatory function for
mutant-specific changes in gene expression caused by bleomycin.
Some of the genes might be important for DNA repair, and lowlevel accumulation of DNA damage in the triple mutant might
have sensitized the plant cells for response to bleomycin. Another
perspective is that the combination of the triple mutations and
bleomycin might have a much greater effect (possible synergism)
than either alone. In any case, the comparison of transcriptomes
in both the triple mutant and wild-type, with or without bleomycin treatment, has revealed that there is an interaction between
the triple mutations and bleomycin, suggestive of a complex function for the three RAD51 paralogs that was previously unappreciated. Our results support multiple hypotheses and highlight the
importance of further studies with regard to the functions of these
ancient genes that have been maintained in both animals and
plants over the long history of eukaryotic evolution.
Acknowledgements
We thank X. N. Dong (Duke University, Durham, NC, USA)
for providing the rad51d (ssn1) mutant, A. W. Dong’s laboratory
(Fudan University, Shanghai, China) for comet assay assistance
and the Ohio State University Arabidopsis Stock Center for
providing the SALK lines. This work was supported by grants
from the Ministry of Sciences and Technology of China
(2011CB944603), the National Natural Science Foundation of
China (91131007), Rijk Zwaan, Fudan University (to H.M.)
and Zhuoxue Plan of Fudan University, and the Shanghai Committee of Science and Technology Fund for 2013 Qimingxing
Project (13QA1400200) (to Y.W.).
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Supporting Information
Additional supporting information may be found in the online
version of this article.
Fig. S1 Expression patterns of members of the RAD51 family.
Fig. S2 Gene ontology (GO) annotation of 562 differentially
expressed genes.
Table S1 List of primers used in this study
Table S2 Summary of mapped reads
Table S3 Summary of transcript types detected by short reads
Table S4 List of up-regulated genes in the triple mutant and
wild-type
Table S5 List of down-regulated genes in the triple mutant and
wild-type
Table S6 List of differentially expressed genes related to DNA
repair or meiotic recombination
Table S7 List of the bleomycin-induced genes in the wild-type
Table S8 List of the bleomycin-repressed genes in the wild-type
Table S9 Gene ontology (GO) annotation of the bleomycinrepressed genes in the wild-type
Table S10 List of the 739 overlapping genes
Table S11 List of the bleomycin-induced genes in the triple
mutant
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Table S12 List of the bleomycin-repressed genes in the triple
mutant
Table S18 List of the 1001 genes induced in the triple mutant
that were not up-regulated in the bleomycin-treated wild-type
Table S13 Gene ontology (GO) annotation of the bleomycininduced genes in the triple mutant
Table S19 Gene ontology (GO) annotation of the 1001 genes
Table S14 Gene ontology (GO) annotation of the bleomycinrepressed genes in the triple mutant
Table S15 List of the 352 overlapping genes in the up-regulated
set
Table S20 List of the 189 up-regulated genes in the wild-type
and triple mutant, both treated with bleomycin
Table S21 List of the 238 down-regulated genes in the wild-type
and triple mutant, both treated with bleomycin
Table S22 List of the 20 crucial pathogen-related genes
Table S16 List of the 270 overlapping genes in the down-regulated set
Table S17 Gene ontology (GO) annotation of the 352 overlapping genes in the up-regulated set
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