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Comparative Transcriptome Analysis of the Necrotrophic
Fungus
Ascochyta rabiei
during Oxidative Stress: Insight
for Fungal Survival in the Host Plant
Kunal Singh, Shadab Nizam, Manisha Sinha, Praveen K. Verma*
Plant Immunity Laboratory, National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, India
Abstract
Localized cell death, known as the hypersensitive response (HR), is an important defense mechanism for neutralizing
phytopathogens. The hallmark of the HR is an oxidative burst produced by the host plant. We aimed to identify genes of the
necrotrophic chickpea blight fungus Ascochyta rabiei that are involved in counteracting oxidative stress. A subtractive cDNA
library was constructed after menadione treatment, which resulted in the isolation of 128 unigenes. A reverse northern blot
was used to compare transcript profiles after H
2
O
2
, menadione and sodium nitroprusside treatments. A total of 70 unigenes
were found to be upregulated by more than two-fold following menadione treatment at different time intervals. A large
number of genes not previously associated with oxidative stress were identified, along with many stress-responsive genes.
Differential expression patterns of several genes were validated by quantitative real-time PCR (qRT-PCR) and northern
blotting. In planta qRT-PCR of several selected genes also showed differential expression patterns during infection and
disease progression. These data shed light on the molecular responses of the phytopathogen A. rabiei to overcome
oxidative and nitrosative stresses and advance the understanding of necrotrophic fungal pathogen survival mechanisms.
Citation: Singh K, Nizam S, Sinha M, Verma PK (2012) Comparative Transcriptome Analysis of the Necrotrophic Fungus Ascochyta rabiei during Oxidative Stress:
Insight for Fungal Survival in the Host Plant. PLoS ONE 7(3): e33128. doi:10.1371/journal.pone.0033128
Editor: Zhengguang Zhang, Nanjing Agricultural University, China
Received June 10, 2011; Accepted February 10, 2012; Published March 12, 2012
Copyright: ß2012 Singh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was partially supported by a research grant from the Department of Biotechnology, Government of India (File No: BT/PR10605/PBD/16/791/
2008) [http://dbtindia.nic.in/dbt_new/appdproj2008-09.pdf]. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript. No additional external funding received for this study.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: praveen_verma@nipgr.res.in
Introduction
The capability of pathogenic fungi to cause disease requires
competence to survive in the host. Pathogen survival in the host is
in turn dependent on evading or suppressing the host’s immune
responses. Early responses towards attempted pathogen attack of
plants and animals are often accompanied by a coordinate
activation of programmed cell death (PCD) and defense
mechanisms [1]. In plants, this response is termed the Hypersen-
sitive Response (HR) and is orchestrated by an oxidative burst,
which induces localized cell death at the infection site [2], [3]. This
oxidative burst consists of a biphasic production of Reactive
Oxygen Species (ROS) at the site of attempted pathogen invasion
[4–5]. The HR is an important element of the defense strategy
that plants employ against biotrophic pathogens, which derive
nutrition from living tissues [6]. In contrast, necrotrophic fungi
obtain nutrients exclusively from dead tissues and produce toxins
as well as cell wall-degrading enzymes that kill host cells prior to
invasion [7]. The HR produced by the host is reported to facilitate
colonization by necrotrophs such as Botrytis cinerea and Sclerotinia
sclerotiorum [8]. Consequently, necrotrophs are able to exploit host
defense mechanisms, enlarge the infection field and colonize host
tissues [8]. The mechanism by which necrotrophs exploit the HR
for growth is largely unknown. To thrive within the oxidative
environment of necrotic tissues, pathogenic fungi have evolved
multiple defense systems, both enzymatic and non-enzymatic [9],
[10]. The necrotroph B. cinerea has been reported to have an array
of enzymes including catalase and superoxide dismutase that
protect it against an environment rich in ROS [9], [11]. Targeted
gene replacement of Magnaporthe oryzae catalase CATB gene led to
compromised pathogen fitness and reduced pathogenicity [12].
Recently the M. oryzae transcription factor MoAtf1 was found to be
necessary for complete virulence and oxidative stress response of
the fungus [13]. An important homolog of the yeast transcription
factor Yap1 from Cochliobolus heterostrophus and Ustilago maydis was
reported to regulate oxidative stress as well as virulence [14–15].
These studies suggest that the genes involved in ROS detoxifica-
tion and oxidative-stress response are vital for fungal survival and
pathogenesis. ROS also plays an important role during mutualistic
interactions between the fungal endophyte Epichloe festucae and its
grass host Lolium perenne [16].
In plants, nitric oxide (NO) is an important signaling molecule
that regulates a number of critical signal transduction pathways,
including the defense response [17]. NO, together with ROS, has
emerged as a major participant in the HR and plant cell death
[18]. Recent work on plant defense mechanisms has indicated that
establishing and initiating the HR requires the correct relative
levels of both NO and ROS to be produced in the host [19], [20].
Crosstalk between ROS and RNS (reactive nitrogen species)
during the HR is required for the host resistance response [4]. The
human pathogenic fungus Cryptococcus neoformans counteracts
cytotoxic nitrosative stress with the help of enzymes, such as
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flavohemoglobin denitrosylase and S-nitrosoglutathione reductase,
which were shown to promote fungal virulence [21]. Similarly, a
gene encoding flavohemoglobin (BcFHG1) was shown to be
essential for NO detoxification and provide protection against
nitrosative conditions in the necrotrophic fungus B. cinerea [22].
Therefore, the relative sensitivity of fungal pathogens to oxidative
and nitrosative stress depends on the effectiveness of their own
ROS and RNS detoxification machinery.
Transcript profiling is an important strategy for studying the
expression of large gene sets modulated against a particular
condition. The DNA array strategy has been applied in fungi to
investigate the transcript pattern at the time of spore germination
[23], appressorium formation [24] and pathogenesis [25].
Furthermore, most studies have focused on various aspects of
development and infection processes [26] although a few reports
have described transcriptional changes due to oxidative stress [27].
The necrotrophic fungus Ascochyta rabiei (Pass.) Lab. (teleo-
morph: Didymella rabiei) causes Ascochyta blight (AB) disease of the
chickpea (Cicer arietinum L.), which is the second most important
food legume worldwide in terms of productivity [28], and results in
severe losses in grain yield. There is a high degree of variation in
resistance among chickpea cultivars, but complete resistance to A.
rabiei has not been observed [29]. A. rabiei causes typical circular
necrotic lesions on all above ground plant parts. The plant dies
when the main stem is girdled at the collar region [30], [31]. The
generation of a rapid oxidative burst accompanied by the HR was
reported to occur in the chickpea-Ascochyta interaction in tolerant
cultivars [32], [33]. ROS accumulation and subsequent HR were
also observed both in moderately resistant and susceptible
chickpea cultivars in the course of Ascochyta infection [34].
In this study, we aimed to identify oxidative stress-induced genes
of the phytopathogenic fungus A. rabiei. To isolate such genes, a
subtractive cDNA library was constructed from menadione-
treated A. rabiei cultures. In addition to well known stress-
responsive genes, a large number of genes not previously
associated with oxidative stress were identified. The expression
patterns of the isolated genes were analyzed in response to both
oxidative and nitrosative stress conditions. In planta expression
studies of several selected genes revealed high expression during
infection. This study provides new insights about survival of A.
rabiei against oxidative and nitrosative stresses.
Materials and Methods
Fungal isolates and stress treatments
Pure cultures of Ascochyta rabiei (Delhi isolate, D-11) were
obtained from IARI, New Delhi. Cultures were grown on Potato
Dextrose Agar (PDA; Difco Laboratories, USA) or in Potato
Dextrose Broth (PDB; Difco) with shaking at 120 rpm for four
days at 22uC in the dark. For exogenous stress treatments, fungal
spore suspensions (1610
3
spores/ml) were grown for four days at
120 rpm and subjected to treatment with menadione (250 mM,
Sigma-Aldrich, USA), hydrogen peroxide (H
2
O
2
; 5 mM, Sigma-
Aldrich) or sodium nitroprusside (SNP; 500 mM, Sigma-Aldrich).
Fungal cultures were harvested at 0.5, 1, and 3 h after treatment
with menadione, and 1 h after H
2
O
2
and SNP treatments. Mock-
treated samples with the respective solvents were used as controls.
One month old chickpea plants (Pusa-362) were inoculated with A.
rabiei spore suspensions (1610
6
spores/ml). Infected stems and
leaves were collected 1, 3 and 6 d post-inoculation (dpi).
Histochemical detection of ROS and microscopy
The chickpea leaves and stem peels were inoculated with WT
and DsRed-expressing A. rabiei [31]. Two days after infection,
tissues were stained with 3, 39-diaminobenzidine (DAB) using the
DAB-Black kit (Invitrogen, Paisley, UK) according to the
Figure 1. Functional cataloging of menadione-responsive genes. Genes were assigned a putative function based on their homology and
classified on the basis of their functions.
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manufacturer’s instructions. Brownish-black colored polymeriza-
tion products resulting from the reaction of DAB with ROS were
visualized using a Zeiss Axio Examiner microscope with
differential interference contrast optics. DsRed fluorescence was
detected with standard filters for rhodamine (excitation 546/12,
beam splitter 560, emission 607/80 nm; Zeiss). Images were
obtained with a CCD camera.
Assessment of optimal menadione dose
Ascochyta spore suspension (1610
3
spores/ml) cultures were
grown with shaking for four days in PDB as described above with
different menadione concentrations (0.1, 0.25, 1 and 20 mM)
added before the cultures were immediately transferred back to
the shaker. After 24 h of treatment, the mycelial ball diameters
were assessed. In order to measure the fungal biomass dry weight,
mycelial balls were filtered through Whatman filter paper No. 1
and dried at 50uC for 72 h in an air incubator. Three independent
experiments were performed each time with three sets of technical
replicates and respective data set means (6SE) were estimated
from the above cultures.
Isolation of RNA and construction of subtracted cDNA
library
To construct the subtracted cDNA library, total RNA was
isolated from A. rabiei using the TRIzolHreagent (Invitrogen,
USA). Poly A
+
RNA was purified using an mRNA isolation kit
(Roche, Germany) according to the manufacturer’s protocol. A
forward Suppression Subtractive Hybridization (SSH) was per-
formed using a PCR-Select
TM
cDNA Subtraction Kit (BD
Biosciences, USA) according to the manufacturer’s protocol.
The mRNA isolated from the 1 h menadione-treated sample was
used as the ‘tester’, where as the mock-treated sample was used as
the ‘driver’ for subtraction. The enriched differentially expressed
cDNAs were cloned into the pDrive U/A Cloning Vector
(Qiagen, Germany). Recombinant plasmids were further se-
quenced using the Big Dye Terminator
TM
kit version 3.0 (Applied
Biosystems, USA) and examined with the 3700 ABI Prizm 96
capillary sequence analyzer. All sequences were screened for
homology in the GenBank database using BLASTx and tBLASTx
(http://www.ncbi.nlm.nih.gov/BLAST/). Sequences were sub-
mitted to GenBank and the assigned accession numbers are listed
in Table S1.
cDNA macroarray and data analysis
Individual clones of the subtracted cDNA library were
amplified, purified, and denatured by adding an equal volume of
0.6 M NaOH. Equal volumes of each denatured PCR product
(about 100 ng) were spotted on Hybond
TM
N membranes
(Amersham, USA) using a 96-well dot-blot apparatus (Bio-Rad,
USA). In addition, PCR products of A. rabiei Elongation factor 1
alpha (ArEf1a) cDNA using primers EF1a-F (59-TCGGTGT-
CAAGCAGCTCATC-39) and EF1a-R (59-AAGCCTCAACG-
CACATGG-39) and the neomycin phosphotransferase (NPTII)
gene from the binary vector pBI121 (Accession No. AF485783.1)
using primers NPTII-F (59-TGCTCGACGTTGTCACTGAAG-
39) and NPTII-R (59-GTCAAGAAGGCGATAGAAGGC-39)
were spotted as an internal and negative control, respectively.
The membranes were neutralized with neutralization buffer
(0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl) for 3 min, washed with
26SSC and immobilized with UV cross-linker (Stratagene, USA).
Probes were prepared for DNA array hybridization by first-strand
reverse transcription (Primescript
TM
RT, BD Biosciences, USA)
with 1 mg mRNA isolated from menadione, H
2
O
2
and SNP-
treated samples and labeled with a
32
P-dCTP (10 mCi ml
21
; 3,000
Ci mmol
21
). Radiolabeled cDNAs were purified on a Sephadex
G-50 column (GE Healthcare, Sweden), suspended in pre-
hybridization buffer (7% SDS, 0.3 M Sodium phosphate buffer
pH 7.4, 1 mM EDTA) and hybridized at 60uC overnight. The
membranes were initially washed three times with washing buffer
(16SSC, 1% SDS, 20 min each at 60uC). Autoradiographs were
scanned with a Fluor-S-Multi-imager (FSMI; Bio-Rad) to acquire
images. Detection and quantification of signals representing the
hybridized cDNAs were performed using Quantity One
TM
software version 4.2.3 (Bio-Rad) and signal intensities were
analyzed by subtracting the background. A total of six replicates
representing three biological replicates were analyzed for all
experiments. ArEf1a cDNA was used as an internal control where
the subtracted density value was used for normalization.
Differential screening and expression pattern data were generated
as the mean (6SD) of expression ratios for all independent
experiments. A paired Student’s t-test on log
2
-transformed data
was applied to determine whether statistical differences between
the expression ratios of each treatment and control pair were
evident. Genes that were significantly different from controls in
any of the treatments were selected and presented. The following
two criteria were chosen to demarcate differentially expressing
genes based on previous reports [35]: (a) a greater than two-fold
induction level; and (b) a P,0.05 level of significance as
determined by a t-test for each experiment, and through analysis
of variance (ANOVA) [36]. ANOVA was performed for each
clone and the significance of differences in expression patterns was
tested with a high stringency threshold Pvalue of 0.01 using InStat
version 3.1 software (GraphPad). Expression profiles of inducible
cDNAs were also analyzed by clustering performed by Self
Organizing Tree Algorithm (SOTA) using average linkage by
TIGR Multiple Experiment Viewer version 3.0 (available at
http://www.tigr.org/software/tm4/menu/TM4).
Northern hybridization
Ten micrograms of total RNA from control and treated samples
were fractionated in a 1.2% agarose gel containing formaldehyde
and transferred onto a positively charged Hybond
TM
N
+
membrane (Amersham) according to Sambrook and Russell
[37]. Equal loading and lane transfer was verified by membrane
staining with methylene blue (0.02%). PCR-amplified individual
cDNA fragments (with primers corresponding to adaptor 1 and
2R, provided in the SSH kit) were purified from agarose gels. In
addition, b-tubulin TUB-F (59-CATCTCCGGCGAGCATGGC-
39) and TUB-R (59-CCAGTTGTTACCAGCACCAG-39) was
amplified and purified from the agarose gel. Probes were labeled
with a
32
P-dCTP using the NEBlotHkit (New England Biolabs,
USA) according to the manufacturer’s instructions and purified as
described above. Hybridization and high stringency washing was
carried out using standard protocols [37]. Development and
scanning of autoradiographs were carried out as previously
described.
Figure 2. Comparison of gene expression after menadione treatment at different time intervals. (a) Heat map of hierarchical clustering
performed for selected genes illustrates differential induction patterns after menadione-treatment for 0.5 h, 1 h and 3 h. (b) The Venn diagram
represents the distribution of transcripts that was significantly induced (.2-fold) at different time intervals.
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Quantitative real-time PCR
Total RNA was extracted as described above and treated with
DNase I (Promega, USA) according to manufacturer’s protocol to
remove any contaminating genomic DNA. First-strand cDNA was
synthesized with 1 mg of total RNA primed with Oligo-dT using
NovaScript III RNase H minus Reverse Transcriptase (Life
Technologies, India) as per the instructions in the manual. The
primers were designed using Primer ExpressH(version 3.0)
software (Applied Biosystems) with the default parameters. The
primers used for quantitative RT-PCR are shown in Table S2.
Real-Time PCR (RT-PCR) was carried out in 96-well plates on a
7900HT Sequence Detection System using Sequence Detection
Systems Software version 2.3 (Applied Biosystems) and Power
SYBR Green PCR Master Mix (Applied Biosystems) in a final
volume of 20 ml. The default cycling program was used with the
following cycling conditions: 2 min at 50uC, 10 min at 95uC, and
40 cycles of 15 s at 95uC and 1 min at 60uC. Each experiment was
performed with three replicates. The primer pair specificity was
visualized by dissociation curve monitoring and agarose gel
electrophoresis. The gene encoding ArEf1awas used as the gene
for calibration in all experiments. Expression ratios were
calculated from cycle threshold values using the 2
2DDCT
method.
Results
Menadione induces oxidative stress in A. rabiei broth
culture
The Diaminobenzidine (DAB) staining and microscopic studies
in the susceptible chickpea cultivar, Pusa 362 showed that
intracellular ROS was generated after A. rabiei infection (Fig.
S1). To isolate oxidative stress induced genes, menadione was used
to generate ROS in broth cultures of A. rabiei. Menadione
produces superoxide anion (O
2
2
), which readily dismutates to
H
2
O
2
or combines with NO to form a strong oxidant peroxynitrite
[38], [39]. Molecular, pharmacological and genetic studies also
support the hypothesis that the primary source of ROS during HR
is O
2
2
generated by plant NADPH oxidase [2]. Since previous
studies showed the extremely toxic effect of menadione on certain
filamentous fungi [40], we, therefore optimized the menadione
concentration in the A. rabiei broth cultures to generate non-lethal
doses of oxidative stress. The measurement of fungal dry weight
and mycelia ball size was carried out after 24 h incubation. Both
parameters suggested that a 250 mM menadione concentration
could induce oxidative stress in the fungus without imposing high
toxic effects (Fig. S2). At this menadione concentration, the
mycelial growth returned to normal levels 48 h after treatment.
Subtractive library identifies many unique and stress
responsive genes
To study the early molecular responses of A. rabiei against
oxidative stress, a forward subtractive cDNA library was
constructed using the Suppression Subtractive Hybridization
(SSH) strategy with the 1 h menadione-treated samples. The
SSH strategy enriches the less abundant transcripts by normalizing
and amplifying the subtracted cDNAs [41]. As a result, 756
recombinant clones were obtained that contained cDNA frag-
ments ranging from 138 to 1024 bp with an average clone size of
,350 bp. Annotation, screening and sequence analysis of these
clones identified 128 independent sequences (unigenes) by
similarity searches against GenBank databases (NCBI). Sequence
analysis revealed that out of the 128 unigenes, 28.13% were
present as a single copy, 13.28% were in duplicates and 58.59%
were present three or more times in the library. Out of the 128
sequences, 13 (10.15%) showed no homology with known
sequences and were classified as ‘no hits’. Seven sequences with
high E-values were also considered as ‘no hits’. Two genes were
also found with Pvalues ,0.05 after conducting a test of
significance (t-test) and were eliminated from analysis. All of the
remaining 106 genes were selected and used for further analysis.
The genes were classified into 10 functional categories based on
their potential cellular function (Table S1; Fig. 1). Major
functional categories corresponded to genes involved in stress
including stress response (7%), protein modification (6%) and
oxidative stress (8%). Other significant categories included protein
transport (4%), protein synthesis (6%), metabolism and homeo-
stasis (9%), miscellaneous (18%), cell signaling (8%), transcription
(3%) and proteins of unknown function (31%). When the roles of
gene products had overlapping functional categories they were
classified according to their most probable role during oxidative
stress. Genes known to be involved in ROS detoxification were
well-represented in the library and included catalase (CAT),
alternative oxidase, superoxide dismutase (SOD) and thioredoxin
(TRX). Other genes with well-established functions during various
stresses such as ubiquitin and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) were categorized under stress responsive
genes. Among the genes from other categories, export control
protein CHS7-like (protein transport), serine/threonine phospha-
tase PP2A (PP2A, signal transduction), myo-inositol phosphate
synthase (metabolism) and opsin (miscellaneous) were previously
reported to be involved in the stress responses. Genes involved in
fatty acid synthesis/metabolism were represented by ATP citrate
synthase, fatty acid synthase subunit beta dehydratase and acyl-
CoA desaturase.
Expression kinetics after menadione-treatment identified
several upregulated genes
A macroarray analysis was performed to determine the early
induction kinetics of all the unigenes. Samples were collected at
0.5, 1, and 3 h after 0.25 mM menadione treatment, as described
in ‘Materials and Methods’. Since there was no sequence available
in the database for any A. rabiei house-keeping genes, we amplified
the cDNA sequences of elongation factor and tubulin using
primers based on their conserved sequences. The constitutive
expression of these genes during oxidative stress was confirmed by
northern blot analysis (Fig. S3). On this basis, EF1a was used as an
internal control during array hybridization experiments. The
densitometry data obtained by macroarray analysis was statisti-
cally analyzed and used for generation of heat-map and cluster
analysis (Fig. 2a). To reduce the noise level, expression ratios
obtained by macroarray were log
2
transformed. Genes were
Figure 3. Clustering analysis of menadione-responsive gene expression profiles. (a) SOTA cluster tree of selected genes illustrates
differential induction patterns after menadione, H
2
O
2
and NO treatments. Accession numbers provided by NCBI are given in the heat map and the
corresponding gene names are listed in Table S1. (b) The 106 genes were grouped into 11 clusters based on their expression profiles. The expression
profile of each individual gene in the cluster is depicted by grey lines, while the mean expression profile is marked in pink for each cluster. The
number of genes in each cluster is given in the left upper corner and the cluster number in the right lower corner. (c) Functional cataloging of the
genes present in different clusters (clusters with n.10 were taken into consideration). (d) The Venn diagram represents the distribution of transcripts
that were significantly induced (.2 fold) in macroarray analysis after menadione, H
2
O
2
and NO treatments.
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considered to be induced when the expression ratio increased by
more than two-fold. Our results revealed that 27 genes were
induced after 0.5 h treatment and increased to 51 genes after 1 h
treatment (Fig. 2b), which substantiates that the library was indeed
enriched with genes induced early on during oxidative stress. The
number of genes with high expression was reduced to 19 at the 3 h
time point and altogether a total of 70 genes were found to be
upregulated. The expression results of fold-induction at different
time-points are summarized in Table S1. A few genes showed
early biphasic induction (Ar93 and Ar96). A set of six genes (Ar16,
Ar27,Ar30,Ar33,Ar57 and Ar90) showed induction 1 h after
menadione treatment, but their expression was increased by two-
fold only at 3 h post-treatment. A total of 36 genes were found to
be not induced or below the cut-off value at all three time points,
indicating that these genes either were induced late after treatment
or to levels below the two-fold cut-off value.
Comparative expression analysis in response to
menadione, H
2
O
2
and SNP revealed that several genes
have similar expression patterns
To achieve a comprehensive overview of the expression pattern
of genes that were co-expressed during menadione, H
2
O
2
, and
SNP treatment, SOTA clustering was performed by analyzing
macroarrays of samples collected 1 h after each treatment (Fig. 3a,
b). SOTA analysis yielded 11 clusters and those with n.10 were
selected for additional studies of the expression patterns of
functionally similar genes (Fig. 3b, c). The details of each cluster
are provided in Table S3. The maximum number of genes were
grouped into cluster 11 (30 genes), which was comprised of genes
having high expression during menadione and H
2
O
2
treatments
and represented all of the functional categories. The second major
group was cluster 6, which contained 19 genes that are involved in
oxidative stress and lipid metabolism. Genes from miscellaneous
and unknown function categories were represented in almost all
the clusters, which may be due to the heterogeneous composition
of these categories and their high representation in the library
(Table S1). In addition, expression was compared on the basis of
induction level (fold-induction), and fifty genes showed greater
than two-fold induction by menadione. Nitrosative stress also
induced the expression of 21 genes to levels above the cut-off value
compared to the control (Fig. 3d).
An ANOVA with a Pvalue cut-off of ,0.01 was performed for
each gene to identify those that showed significant variation in
their expression levels following oxidative and nitrosative stresses.
This analysis revealed that a large majority of genes were
significantly expressed in at least one of the oxidative stress
conditions compared to nitrosative stress (70 genes, 66.03%). The
expression ratios of menadione and SNP treated genes were
compared to determine the preferential expression of the genes.
Among the 66 genes (62.26%) preferentially expressed following
one kind of treatment, 54 genes (50.9%) showed higher expression
levels with menadione while 12 genes (11.32%) were upregulated
by NO. When all the three stress conditions were compared by
ANOVA, a total of 22 genes showed similar expression levels.
These genes are involved either in signaling or had an unknown
function. The putative signaling genes that were co-expressed
included endosomal cargo receptor Erv14 (Ar79), C2 domain
containing protein (Ar77), RAC-alpha serine/threonine-protein
kinase (Ar70), phosphoinositide 3-phosphate phosphatase (Ar20)
and protein kinase C domain containing protein (Ar72), and the
majority exceeded two-fold induction for at least one stress
condition.
qRT-PCR and northern blot analysis validates expression
kinetics of several genes
To validate the reliability of the reverse northern analysis
results, 12 genes were selected from the library and their
expression profiles assessed using real-time quantitative PCR
including CAT, GAPDH, Cytochrome C (CYC), F-box and WD
domain containing protein (FBO), FMN-dependent dehydroge-
nase (FMN), neutral trehalase (TRE), SSC1-like heat-shock
protein (SSC1), ubiquitin-conjugating enzyme E2N, dnaK-type
molecular chaperone BiP (BIP), peptidyl-prolyl cis-trans isomerase
(PPI), mannosylphosphate transferase (MNN4) and NADH-
ubiquinone oxidoreductase (NUO). The selection of these genes
was based both on their putative function inferred from sequence
comparison together with their unique and distinct expression
pattern revealed by macroarray analysis. The samples were
collected at different time points after menadione treatment and
used for qRT-PCR (Fig. S4). The same set of genes was also used
to validate their expression in H
2
O
2
and SNP-treated samples (Fig.
S5). Although the data obtained with real-time PCR were
consistent with those obtained from the array experiments, the
relative expression ratios obtained were often higher. Similar
differences between the two methods were reported previously,
suggesting that the ratios calculated using macroarray are often
underestimated [42]. Real-time PCR allows for the specific
amplification of a gene using gene-specific primers, whereas for
macroarrays, the possibility of cross hybridization among the
homologous genes cannot be ruled out [43]. Furthermore, a few
genes were also randomly selected for their expression analysis by
northern hybridization (Fig. S6). The genes encoding NADH
oxidase (NOX), SOD, GAPDH, histone H1, and a hypothetical
protein (GW996392) were selected. The results obtained by
northern analysis further substantiated the macroarray data.
In planta expression analysis of selected genes showed
high transcript accumulation
To check the relevance of genes isolated in this study to A. rabiei
infection and disease progression, in planta expression analysis of
major cluster 6 was carried out by qRT-PCR using the susceptible
chickpea cultivar, Pusa 362 and samples collected 1, 3 and 6 d
after inoculation (Fig. 4; Table S4). Major cluster 6 consists of
some of the hypothetical proteins and genes from the oxidative
stress category. In addition, other selected genes from the oxidative
stress and stress responsive category together with some genes that
showed high expression under oxidative stress were analyzed for in
planta expression (Fig. 5; Table S4). Two genes of cluster 6, namely
Ar12 and Ar65 encoding acetylglutamate kinase and NADH-
ubiquinone oxidoreductase respectively, showed very high induc-
tion but other genes, such as Ar74,Ar77 and Ar81, showed
repression during infection and disease progression. The majority
Figure 4.
In planta
quantification of expression of genes belonging to cluster 6. Line diagrams representing the expression pattern of
cluster 6 genes from A. rabiei-infected chickpea samples (Pusa-362) are shown as fold-change compared to control. Expression was analyzed at 1, 3
and 6 dpi. Error bars represent 6SE. Ar2, Hypothetical protein SNOG_16463; Ar3, Cytochrome c; Ar11, Cartenoid oxygenase; Ar12, Acetylglutamate
kinase; Ar25, Neutral trehalase; Ar26, Hypothetical protein; Ar66, Hypothetical protein ACLA_073190; Ar69, Hypothetical protein SNOG_10250; Ar74,
Hypothetical protein; Ar77, C2 domain containing protein; Ar81, Alternative oxidase; Ar96, Plasma membrane ATPase; Ar14, ATP-citrate synthase; Ar48,
Hypothetical protein SNOG_00366; Ar65, NADH-ubiquinone oxidoreductase.
doi:10.1371/journal.pone.0033128.g004
Ascochyta Transcriptome under Oxidative Stress
PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e33128
Ascochyta Transcriptome under Oxidative Stress
PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e33128
of genes identified in the stress responsive category showed a
greater than 2-fold induction at various time points after infection
(Ar3,Ar9,Ar13,Ar34,Ar35 and Ar104) while a few others showed
repression (Ar19,Ar57 and Ar71). Five genes, including Ar9,Ar12,
A13,Ar49 and Ar86 showed very high induction (.50 fold).
Discussion
The current study has identified genes that were upregulated
by menadione-treatment, and illustrates the processes affected by
oxidative stress in phytopathogenic fungi. Since isolation of
Ascochyta genes from the infected chickpea plants precisely at the
time of the initial oxidative burst was not realistic, we used liquid
cultures with mild menadione treatment to mimic in planta
oxidative stress. Many genes showed high induction immediately
after menadione treatment. The utility of the SSH strategy is
highlighted by the fact that a large number of genes identified
from the library were not previously characterized or were
categorized as genes having unknown function (31%). The short
length of some clones and the long 39-untranslated region in the
truncated cDNAs could also account for the high number of
sequences with unknown function [36], [41], although the
sequence length did exceed 500 bp in a few cases (Ar106 and
Ar113). A large number of ‘no hits’ (10.93%) found in the library
also demonstrated that subtractive libraries can be used to
identify new genes that are overlooked by large scale EST
projects. Furthermore, with the 70 up-regulated genes represent-
ing 54.69% of the 128 SSH clones, the subtractive approach was
shown to be highly efficient. Since the subtractive library was
generated 1 h after menadione treatment of the fungus, it is
Figure 5.
In planta
expression analysis of stress responsive genes. Line diagrams representing the expression pattern of selected genes from
A. rabiei-infected chickpea samples (Pusa-362) are shown as fold-change compared to control. Expression was analyzed at 1, 3 and 6 dpi. Error bars
represent 6SE. Ar19, FMN dependent dehydrogenase; Ar34, NADH oxidase; Ar35, Catalase; Ar15, Thioredoxin; Ar9, E3 SUMO-protein ligase PIAS1; Ar13,
F-box and WD domain containing protein; Ar46, Ubiquitin-conjugating enzyme E2 N; Ar71, Ubiquitin; Ar57, Mannosylphosphate transferase; Ar104,
Usp domain-containing protein; Ar36, C6 transcription factor; Ar49, Molecular chaperone BiP; Ar50, 41 kDa peptidyl-prolyl cis-trans isomerase; Ar55,
Ribosomal protein S5; Ar86, protein phosphatase PP2A.
doi:10.1371/journal.pone.0033128.g005
Figure 6. Schematic representation of the putative regulatory and functional networks induced during oxidative stress in
Ascochyta
rabiei
.This figure summarizes data obtained in the present work and hypothesized mechanisms. Genes are grouped according to their most
probable localization and function in the cell. Abbreviation for genes: PP2A, Protein phosphatase PP2A; PKC (C1), Protein kinase C (C1); EsdC, GTP-
binding protein EsdC; PKA, RAC-alpha serine/threonine-protein kinase; C6, C6 transcription factor; ZK-TF, Zinc knuckle transcription factor; Rds2, Zn
cluster transcription factor Rds2; HSP78, Heat shock protein 78; AOX, Alternative oxidase; MITPC, Mitochondrial phosphate carrier protein; SOD,
Superoxide dismutase; CAT, Catalase; ERV14, Endosomal cargo receptor Erv14; BIP, Molecular chaperone BIP; CHS7, Export control protein chitin
synthase 7; ACS, ATP-citrate synthase; FAS, Fatty acid synthase; ACD, Acyl-CoA desaturase; COX, Carotenoid oxygenase; ACT, Acetylglutamate kinase;
PIAS, E3 SUMO-protein ligase PIAS1; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; Mnn4, Mannosylphosphate transferase; FBO, F-box and
WD domain containing protein; TRE, Trehalase; RPS5, Ribosomal protein S5; HSP 70, Heat shock protein 70; SSC1, SSC1-like heat-shock protein; PPI,
peptidyl-prolyl cis-trans isomerase; RBOH, Respiratory burst oxidase homolog.
doi:10.1371/journal.pone.0033128.g006
Ascochyta Transcriptome under Oxidative Stress
PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e33128
possible that some genes may be induced at later time points and
would have escaped detection.
Analyses of macroarray results suggest that menadione
treatment induces many genes potentially involved in protecting
cells from oxidative stress and ROS detoxification. Among these
SOD, CAT and TRX (thioredoxin) are known to be involved
directly in ROS detoxification. Thioredoxins are small, thermo-
stable proteins identified as donors of hydrogen to ribonucleotide
reductase [44]. The Cryptococcus neoformans thioredoxin was
reported to regulate oxidative and nitrosative stress [45]. The
upregulation of A. rabiei thioredoxin by menadione, SNP and
H
2
O
2
treatments suggests it may play a similar role. Additionally,
the up-regulation of many genes involved in lipid metabolism,
protein synthesis, protein folding and modification is suggestive of
their involvement in fungus survival during the HR. For example,
the role of heat-shock proteins as stress regulators during oxidative
stress is reflected by the up-regulation of HSP70 (HscA), PPI and
30 kDa HSP. All of these chaperones have well-established roles
during different stresses in other organisms [46], [47], and
therefore the isolation and up-regulation observed here also
suggests their importance in phytopathogenic fungi. A schematic
model based on our work that illustrates the regulatory and
functional networks activated under oxidative stress is depicted in
Figure 6.
Out of the four major clusters identified by SOTA clustering
analysis (Fig. 3), we selected cluster 6 for in planta analysis since
this cluster included many genes of unknown function together
with a few genes that participate in the oxidative stress response.
The majority of the genes grouped in cluster 6 showed induction
under oxidative stress. In planta expression analysis was carried
out using infected samples collected 1, 3, and 6 dpi. Previous A.
rabiei studies have revealed that the initiation of spore germina-
tion, germ tube elongation and penetration occurred around
1 dpi, followed by colonization after 2–3 dpi [30], [31]. Visual
symptoms of the disease were observed after 5–6 dpi. Expression
analysis of cluster 6 revealed a set of genes having high induction
while some showed repression. Although genes of this cluster
were induced during oxidative stress, many of them, including
thioredoxin (Ar15) and alternative oxidase (Ar81), showed
repression during infection. Interestingly, the majority of genes
involved in stress responses were induced during infection with a
few exceptions. A few genes, including superoxide dismutase
(Ar94), glyceraldehyde 3-phosphate dehydrogenase (Ar61) and
Niemann-Pick C1 protein precursor (Ar62) showed prominant
multiple peaks in qRT-PCR and were not considered for in planta
expression analysis..
Among the isolated genes, those encoding acetylglutamate
kinase, E3 SUMO-protein ligase PIAS1, the molecular chaperone
BiP, F-box and WD protein (FBO), and protein phosphatase 2A
(PP2A) showed very high induction. F-box domain containing
proteins are considered to be scavenger proteins in the cell [48]
and are reported to have important roles in various phytopatho-
genic fungi, including M. oryzae and Fusarium oxysporum [49]. PP2A
is a critical regulator of many cellular activities [50], [51], and in
phytopathogenic fungi is necessary for vitality and pathogenicity as
in the biotroph U. maydis [52] and the necrotroph S. sclerotiorum
[53]. The very high induction of PP2A (.200-fold) observed in
infected chickpea samples predicts its importance for A. rabiei
during infection. The acetylglutamate kinase of the crucifer
anthracnose fungus Colletotrichum higginsianum has been reported
to be a pathogenicity factor [54]. The gene encoding neutral
trehalase (Ar25) showed no significant change in expression during
infection, which supports the finding that the M. oryzae trehalase,
TRE1 did not play a significant role in pathogenesis [55].
Previously, the expression patterns of many genes in response to
oxidative stress were studied for the filamentous fungi Aspergillus
nidulans and M. oryzae using microarray analysis [27], [40].
Comparison of the results for certain A. nidulans genes with those
in the present study yielded varying results with both studies
showing menadione dependent expression of alternative oxidase
(AOX), while MnSOD had no significant expression in A. nidulans.
The difference between the two studies may be due to the very
high concentration of ROS generators used in Po´csi et al. [40] to
provide a ‘minimum lethal dose’.
When comparing different treatment time points by macroarray
analysis, a large number of genes isolated from the library were
found to be induced immediately after the 0.5 h of menadione-
treatment. These genes primarily coded for stress-responsive genes
together with a few genes from other categories. Furthermore,
many genes of the library were upregulated by RNS along with
ROS treatment. These genes are likely part of the cell machinery
that acts in co-ordination to regulate responses to stress. ANOVA
analyses suggest that signaling between oxidative and nitrosative
stress overlaps in A. rabiei since many signaling genes were found to
be co-expressed. Therefore, our results support the theory that the
signal transduction pathways initiated by these two types of stress
consistently overlap [56]. Overall, this study suggests that fungi
employ various pathways and regulatory networks of genes to
resist various types of stress imparted by ROS and RNS.
In conclusion, we report here the detailed investigation of gene
expression under oxidative and nitrosative stress conditions in the
phytopathogenic fungi, Ascochyta rabiei. The high expression of
many genes is likely indicative of their role during infection,
although further research using gene knock-out and infection
studies on chickpea plants will provide additional evidence that
some of these identified genes are required for coping with host-
induced oxidative stress and cell death. The data presented here
also provide a portfolio of candidate genes for further studies on
their role in oxidative and nitrosative stress tolerance.
Supporting Information
Figure S1 DAB staining to detect ROS production
during Ascochyta-chickpea interaction. (I) A light micro-
graph of an A. rabiei germinating spore infecting chickpea tissue (II)
merged micrograph of DsRed-expressing A. rabiei infecting
chickpea stem peel. DAB precipitates are visible as black
precipitates in the vicinity of the spores (I) or hypha (II).
Bars = 10 mm.
(TIF)
Figure S2 Measurement of fungal biomass (dry weight)
and mycelia ball size. The measurement of fungal biomass
(dry weight) and mycelia ball size carried out after incubation for
24 h with different menadione concentrations.
(TIF)
Figure S3 RNA gel-blot analysis of actin and b-tubulin.
(TIF)
Figure S4 Real-time quantitative PCR validation of
macroarray analysis after menadione- treatment for
different time intervals. Bar diagrams representing the
expression pattern of 12 genes are shown as the fold-change
compared to their controls. The solid black bars represent the
qRT PCR results. Error bars represent 6SE.
(TIF)
Figure S5 Validation of macroarrays by real-time
quantitative PCR after menadione, H
2
O
2
and NO
treatments. Bar diagrams representing the expression pattern
Ascochyta Transcriptome under Oxidative Stress
PLoS ONE | www.plosone.org 11 March 2012 | Volume 7 | Issue 3 | e33128
of twelve genes are shown as the fold-change compared with their
controls. The solid black bars represent the qRT PCR results.
Error bars represent 6SE.
(TIF)
Figure S6 RNA gel-blot analysis of selected genes from
the library.
(TIF)
Table S1 Genes differentially expressed in response to
menadione, H
2
O
2
and NO.
(DOC)
Table S2 List of primers for real-time quantitative
PCR.
(DOC)
Table S3 Group of clusters after SOTA analysis.
(DOC)
Table S4 Relative expression of selected genes in planta
determined by qRT-PCR.
(DOC)
Acknowledgments
We are grateful to the Director, National Institute of Plant Genome
Research for providing facilities and help during this study. The Ascochyta
rabiei, (Delhi isolate, D11) was provided by Dr. S.C. Dube, Division of Plant
Pathology, Indian Agricultural Research Institute, New Delhi. The authors
thank Prof. Sushil Kumar for critically reading the manuscript. KS and SN
as well as MS acknowledge the Council of Scientific and Industrial
Research and University Grant Commission for fellowships, respectively.
Author Contributions
Conceived and designed the experiments: PKV. Performed the experi-
ments: KS SN MS. Analyzed the data: KS SN PKV. Contributed
reagents/materials/analysis tools: PKV. Wrote the paper: KS PKV.
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