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Eukaryotic Cell, February 2007, p. 235-244, Vol. 6, No. 2
1535-9778/07/$08.00+0 doi:10.1128/EC.00302-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Ste12 Transcription Factor Homologue CpST12 Is Down-Regulated by Hypovirus Infection and Required for Virulence and Female Fertility of the Chestnut Blight Fungus Cryphonectria parasitica
Fuyou Deng,
Todd D. Allen,
and
Donald L. Nuss*
Center for Biosystems Research, University of Maryland Biotechnology
Institute, Shady Grove Campus, 9600 Gudelsky Drive, Rockville, Maryland 20850
Received 19 September 2006/
Accepted 10 November 2006
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ABSTRACT
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A
putative homologue of the Saccharomyces cerevisiae Ste12
transcription factor was identified in a series of expressed sequence
tag-based microarray analyses as being down-regulated in strains of the
chestnut blight fungus, Cryphonectria parasitica, infected by
virulence-attenuating hypoviruses. Cloning of the corresponding gene,
cpst12, confirmed a high level of similarity to Ste12
homologues of other filamentous fungi. Disruption of cpst12
resulted in no alterations in in vitro growth characteristics or colony
morphology and an increase in the production of asexual spores,
indicating that CpST12 is dispensable for vegetative growth and
conidiation on artificial medium. However, the disruption mutants
showed a very substantial reduction in virulence on chestnut tissue and
a complete loss of female fertility, two symptoms normally conferred by
hypovirus infection. Both virulence and female fertility were restored
by complementation with the wild-type cpst12 gene. Analysis of
transcriptional changes caused by cpst12 gene disruption with
a custom C. parastica cDNA microaray chip identified 152
responsive genes. A significant number of these putative
CpST12-regulated genes were also responsive to hypovirus infection.
Thus, cpst12 encodes a cellular transcription factor, CpST12,
that is down-regulated by hypovirus infection and required for female
fertility, virulence and regulated expression of a subset of hypovirus
responsive host
genes.
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INTRODUCTION
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Hypovirus infection of the chestnut blight fungus Cryphonectria
parasitica results in a persistent and stable set of phenotypic
changes that can include reduced pigmentation, suppressed asexual
sporulation, loss of female fertility, and hypovirulence (reduced
virulence) (recently reviewed in references
17 and
32). The pleiotropic
nature of these phenotypic changes suggested that hypoviruses may
perturb one or several cellular regulatory pathways. Consistent with
this view, a series of studies have provided evidence that hypovirus
infection alters G-protein-linked, cyclic AMP-mediated
(9,
10,
21,
36) and
calcium/calmodulin/inositol trisphosphate-dependent
(26,
27) signaling cascades,
as well as a mitogen-activated protein kinase (MAPK) signaling pathway
(35).
The
development of an expressed sequence tag (EST)-based microarray
representing approximately 2,200 C. parasitica genes has
provided the means to monitor transcriptional responses to hypovirus
infection and to the disruption of specific cellular signaling
pathways. Infection by the prototypic hypovirus CHV1-EP713 was shown to
result in the modulation of ca. 13.4% of the C. parasitica
transcriptome, representing a broad spectrum of biological functions,
including stress responses, carbon metabolism, and transcriptional
regulation (1). In similar
analyses of C. parasitica strains deleted for the G
subunit gene cpg-1 and the Gß subunit gene
cpgb-1, respectively, more than 250 C. parasitica
genes that are potentially regulated by G-protein signaling were
identified (19).
Comparisons of transcriptional profiles revealed that more than half
(53.7%) of the CHV1-EP713 responsive genes were also modulated in at
least one of the G-protein subunit-null mutants. Interestingly, an EST
with homology to the S. cerevisiae transcription factor Ste12
was consistently found to be down-regulated in C. parasitica
strains infected by different hypovirus and in the G
- and
Gß-null mutants
(1,
2,
19).
Saccharomyces
cerevisiae transcription factor Ste12 is activated in response to
stimulation by mating pheromones through a G-protein-coupled receptor,
leading to the induction of various genes required for mating (reviewed
in references 20 and
22). Roles in sexual
development and reproduction have been reported for Ste12 homologues in
several filamentous fungi, e.g., Aspergillus nidulans
(43), Candida
albicans (29),
Cryptococcus neoformans
(46), and Neurospora
crassa (28).
Moreover, Ste12 homologues have been reported to play important roles
in the pathogenicity of several animal, e.g., C. albicans
(29), C.
glabrata (6),
Cryptococcus neoformans
(7,
8,
45), Cryptococcus
gattii (37), and
plant pathogenic fungi, e.g., Colletotrichum lagenarium
(42) and Magnaporthe
grisea (33,
34). In C.
parasitica, both hypovirus infection and disruption of the
G-protein signaling pathway result in virulence attenuation and female
sterility. Thus, it was of interest to further characterize the
STE12-related C. parasitica EST identified in
transcriptional profiling analyses in order to confirm the homology of
the corresponding gene and to determine its potential role in virulence
and sexual reproduction. The results provide the first link between
hypovirus-mediated down-regulation of a specific host transcription
factor, reduced virulence, female infertility, and the regulated
expression of a subset of hypovirus-responsive host
genes.
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MATERIALS AND METHODS
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Fungal strains, growth conditions, and phenotypic measurements.
C. parasitica hypovirus-free
strain EP155 (ATCC 38755), isogenic hypovirus CHV1-EP713-infected
strain EP713 (ATCC 52571), and hypovirus-free strain EP146 (ATCC 64671)
were maintained on potato dextrose agar (PDA; Difco, Detroit, MI) at 22
to 24°C with a 12-h light-dark cycle at 1,300 to 1,600 lx.
C. parasitica cultures used for RNA preparations were grown
under similar conditions on PDA overlaid with a cellophane membrane.
Virulence assays were performed with dormant American chestnut tree
stems as previously described
(24), with six duplicate
inoculations per fungal strain. Inoculated stems were kept at room
temperature in a glass tank to maintain moisture. Cankers were measured
1, 2, and 3 weeks after inoculation. Sporulation was measured from
cultures grown on PDA for 14 days. Conidia were washed from the plates
with 0.015% Tween 80 and counted with a hemacytometer. Statistical
analysis for sporulation and virulence assay was performed by using the
Proc GLM procedure of SAS program version 8.0 (SAS Institute, Cary,
NC), and the type I error rate (
) was set at 0.05. Mating was
performed on autoclaved twigs of American chestnut embedded in 2% water
agar as described by Anagnostakis
(4). Each cross was
performed in triplicate, with EP155 (mating type Mat-2) x EP146
(mating type Mat-1) crosses serving as controls. The twigs were
mechanically scored multiple times with a scalpel prior to inoculation
to ensure effective colonization by the strains serving as the female
parent in the crosses.
Cloning and disruption of the C. parasitica cpst12 gene.
A C. parasitica EST cDNA
clone (CEST-47-D-11) identified in microarray analyses as a
hypovirus-responsive, G-protein-regulated gene with homology to STE12,
and the related Magnaporthe grisea transcription factor MST12
(1,
2,
19) was used to screen a
C. parasitica EP155 phage genomic library. The corresponding
gene, cpst12, was sequenced by using the ABI Prism BigDye
terminator ready-reaction cycle sequencing kit (Applied Biosystems).
These sequence data have been submitted to the GenBank database under
accession number DQ458788
[GenBank]
. DNA sequences were
analyzed with the SeqMan (v5.53) module of LaserGene (DNASTAR, Inc.).
Multiple alignments were created by using the CLUSTAL W program
(41).
Disruption of
C. parasitica cpst12 was performed by the PCR-based strategy
described by Davidson et al.
(16), using a gene
cassette conferring hygromycin resistance
(15) flanked by
cpst12-specific sequences. In the first round of PCR, the
5' end (1.6 kb) of cpst12 was amplified using
chromosomal DNA with primers MstFor-1
(5'-CCCTTCTTTCTGAGTGACCAGGTA-3')
and MstRev-H
(5'-CACTGGAACAACTGGCATGAATTCGCCGCCTGGTTGTTGATACATAA TA-3').
The 3' end (1.5 kb) of cpst12 was amplified
by using the primers MstFor-H
(5'-GGTAATCCTTCTTTCTAGAGGATCCTCCAATACGACTCGTCGCAGTCGC-3')
and MstRev-1 (5'-GAAAGCCTGTCATCCGTCCA-3).
The hygromycin resistance cassette, driven by an Aspergillus
nidulans trpC promoter and terminator, was PCR amplified from
plasmid pCPXHY3 (38) by
using the primers HygBTrpCFor
(5'-GAATTCATGCCAGTTGTTCCAGTG-3')
and HygBTrpCRev
(5'-GGATCCTCTAGAAAGAAGGATTACC-3').
The portion of the primers MstRev-H and MstFor-H corresponding to the
trpC promoter and terminator sequences flanking the hygromycin
resistance gene are underlined.
The amplified products were gel
purified by using a QIAquick gel extraction kit (QIAGEN) according to
the manufacturer's instructions. Flanking primers MstFor-1 and MstRev-2
(5'-CATCATGACGGCGGTGTTC-3') were
then used to stitch the three independent PCR products together in a
single 6.0-kb disruption cassette. The disruption cassette was gel
purified and used to transform spheroplasts of C. parasitica
strain EP155 according to the method of Churchill et al.
(12). Hygromycin (40
µg/µl) was added to the regeneration medium to provide
for selection of transformants. PCR analysis was used to screen
hygromycin-resistant, single-spored transformants. Putative
cpst12 knockout strains were further confirmed by Southern
blot and real-time PCR
analyses.
Functional complementation of the cpst12 mutant.
A cDNA copy of the cpst12
gene was amplified using primer set CST-F
(5'-GCAGTGTGGTACCACTATTATGTATCAACAACCAG-3')
and CST-R
(5'-CATTCTCGGTACCGTTATTACATCATGACGGC-3'),
with each primer containing a KpnI recognition sequence (indicated in
boldface). The KpnI-digested cpst12 cDNA fragment was first
inserted into the KpnI site of plasmid pCPXHY-1
(14), liberated from the
vector by KpnI digestion, filled in with T4 DNA polymerase (BioLabs),
and inserted into the HpaI site of plamid pCPXNBn1 to generate the
complementation vector pCPST, which contains the benomyl resistance
cassette and the C. parasitica glyceraldehyde-3-phosphate
dehydrogenase promoter to drive expression of the inserted
cpst12 cDNA
(39). The resulting pCPST
vector was used to transform cpst12 disruptant
cpst12-E1.
Southern blot analysis.
Genomic DNA
(10 µg) was digested with restriction enzymes HindIII and XhoI
or independently with ClaI and separated by electrophoresis in a 1%
agarose gel. After electrophoresis, the gel was denatured in 50 mM NaOH
and 150 mM NaCl for 15 min and soaked in 10x SSC for 10 min,
before being blotted onto Hybond-N+ membrane (Amersham
Biosciences). Probe preparation and hybridization were performed by
using the AlkPhos direct labeling reagents (Amersham Biosciences)
according to the manufacturer's
instructions.
Real-time reverse transcription-PCR (RT-PCR).
Real-time PCR analysis of transcript
accumulation was performed by using TaqMan reagents (Applied
Biosystems, Foster City, CA) and a GeneAmp 5700 PCR apparatus as
described previously
(36). Analyses were
performed at least twice, in triplicate for each transcript, from at
least two independent RNA preparations, with primers and probes
specific for 18S rRNA and the target genes. Calculations of transcript
accumulation values in the mutants relative to those in strain EP155
were performed by using the comparative CT method
as described previously
(36) using the 18S rRNA
values to normalize for variations in template
concentration.
Microarray fluorescent probe generation, hybridization, and scanning.
Fluorescence-labeled cDNA probes were
prepared from total RNA (25 µg per probe) by the direct
incorporation of Cy3- or Cy5-dUTP using a CyScribe first-strand cDNA
labeling kit (Amersham Pharmacia) primed with oligo(dT) according to
the manufacturer's instructions. Unincorporated nucleotides were
removed with a Microcon-30 spin column, and probes were processed
according to the method of Allen et al.
(1). Prehybridization,
hybridization, and posthybridization wash steps were performed as
suggested by the manufacturer of the GAPS II slides (Corning). Each
hybridized chip was scanned in both the Cy3 and Cy5 channels with an
Affymetrix 418 scanner as described by Allen et al.
(1).
Microarray data analysis and management.
Integrated pixel intensity values for
each spot were calculated by using TIGR Spotfinder software (The
Institute for Genomic Research, Rockville, MD) and saved in
tab-delimited format for analysis in Mathematica 5.1 (Wolfram Research,
Inc., Champaign, IL). All hybridization data among two sets of dye-swap
experiments (representing a total of four datasets) were processed
according to the following pipeline steps. (Step 1) Each hybridization
data set was individually processed by using a locally weighted linear
regression (Lowess) algorithm (smoothing factor of 0.15) to remove
systemic dye bias present in each group of spots within a single
metarow, metacolumn block of the microarray chip. (Step 2)
After the intraslide normalization routine described above, all
Lowess-normalized datasets were loaded simultaneously to rescale each
spot through the use of a Z-transformation. Specifically, the global
log2(cy3/cy5) mean and standard deviations were
calculated across all four datasets simultaneously and then used to
rescale each spot in all four normalized datasets by using the
following equation: the rescaled log2(cy3/cy5) value of an
individual spot = [the spot's own log2(cy3/cy5)
value the global mean of all log2(cy3/cy5)
values]/by the global standard deviation of all
log2(cy3/cy5) values. These data have been submitted to the
Gene Expression Omnibus (GEO) at the National Center for Biotechnology
Information under series accession number GSE
6371.
The arrayed EST (AEST) library used to
construct the cDNA microarray chip consisted of 3,864 clones
representing ca. 2,200 unique C. parasitica genes, of which
roughly 1,600 were estimated to be present as a single clone
(1,
18). (Step 3) To reduce
the redundancy in lists of differentially expressed ESTs for
publication purposes, the data from all four normalized, rescaled
datasets were grouped so that each clone on the arrayed chip that is
identical to another clone (as defined by a Blastn comparison cutoff
value of E-80) were considered the same gene. A supplemental file
showing AESTs and their redundant clone groups has been submitted to
GEO. Every log2 ratio of each member of a redundant clone
group was then averaged together to calculate the representative
log2 ratio of the gene represented by multiple, redundant
clones. The representative AEST ID for each redundant clone group used
to report the averaged log2 value for the group was chosen
to be the lowest, canonical member of the group. (Step 4) To identify
differentially expressed genes, the nonredundant list of microarray
values established above were sorted in descending order according to
their log2 values. Any AEST in the sorted list which had a
log2 value equal to or greater than ±1.0 standard
deviations in a minimum of three of four hybridizations were considered
differentially
expressed.
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RESULTS
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Cloning and sequence analysis of cpst12.
The EST (CEST-47-D-11) corresponding to
the hypovirus down-regulated C. parasitica gene with homology
to STE12 was used as a probe to screen a C.
parasitica EP155 phage library. A 4.5-kb region containing the
cpst12 gene was sequenced in four independent genomic clones,
revealing a 2521-bp open reading frame interrupted with three introns.
The presence of introns was confirmed by sequencing DNA fragments
generated from cpst12 mRNA by PCR with primers designed to
amplify the entire coding sequence. This open reading frame was
predicted to encode a 699-amino-acid protein with significant
similarity to Ste12 homologues from other fungi that included a
homeodomain at the N-terminal region (residues 59 to 203) and two
C2/H2-Zn2+ finger motifs at
the C terminus. Phylogenic analysis showed that the characterized
fungal Ste12 homologues formed two groups (Fig.
1), with CpST12 falling within the group that contained the
plant pathogens Colletotrichum lagenarium,
C. lindemuthianum, Fusarium graminearum, and
M. grisea.

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FIG. 1. Phylogram
of the Ste12 homologues from C. parasitica (CpST12) and other
fungi. S. cerevisiae Ste12 (GenBank accession no.
p13574) was used as an outgroup. Notations of
the fungal Ste12 homologues and GenBank accession numbers:
CpST12 (DQ458788, C.
parasitica), MST12(AF432913, M. grisea),
CST1 (AB090340,
Colletotrichum lagenarium), CLSTE12(AJ459778, Colletotrichum
lindemuthianum, Fst12(AF509340, Fusarium graminearum), pp-1
(AY027529, Neurospora crassa), AnST-12
(XM654802, Aspergillus nidulans),
AfST12 (EAL91975,
Aspergillus fumigatus), and stlA
(AF284062, Penicillium marneffei). The
bar represents distances scaled as substitutions per amino acid
residue.
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Targeted disruption of cpst12 and phenotypic characterization.
The
functional role of CpST12 was examined by gene disruption. A PCR-based
strategy was used to generate a disruption fragment consisting of a
hygromycin selectable maker flanked by cpst12 gene specific
sequences (Fig.
2A) that was used to transform C. parasitica strain EP155.
Hygromycin-resistant transformants were screened by PCR, and
cpst12 gene disruption events were confirmed by Southern
hybridization. As indicated in Fig.
2B (left side), the 1.1-kb
fragment detected in HindIII-XhoI-digested EP155 DNA with a probe
specific to the 5'-noncoding region and the first exon of
cpst12 (Fig. 2A)
was replaced with fragments of
3.8 kb and
4.4 kb in
cpst12 disruptants
cpst12-E1 and
cpst12-E7, respectively. In contrast, both a
3.8- and a
1.1-kb fragment was detected in
HindIII-XhoI-digested DNA from a randomly selected transformant, A8,
indicating that the gene disruption cassette integrated ectopically in
the genome of this transformant (Fig.
2B, left). The difference
in the hybridization pattern observed for the two cpst12
disruptants (Fig. 2B,
left) is likely due to rearrangements during integration of the
disruption construct or the introduction of a mutation at one of the
HindIII/XhoI restriction sites during PCR amplification. Consistent
with these possibilities, identical hybridization patterns were
observed for the two disruptants when the same probe was used to
hybridize to ClaI-digested genomic DNA. The ClaI recognition sequence
is present both in the hygromycin cassette and in the cpst12
sequences (Fig. 2A),
yielding a 1.5-kb fragment for the two disruptants in contrast to the
1.9-kb fragment generated for the wild-type EP155 (Fig.
2B, right). In addition to
Southern analysis, real-time RT-PCR was performed to further confirm
the disruption of cpst12 gene in the two disruptants. Compared
to the wild-type isolate EP155, cpst12 transcripts were
undetectable in the two disruptants and, as previously reported
(1,
2,
19), significantly
reduced in the hypovirus-infected isolate EP713 (Fig.
2C).
The growth
characteristics and colony morphology of the cpst12
disruptants,
cpst12-E1 and
cpst12-E7, on PDA medium under standard laboratory
conditions were similar to untransformed strain EP155 (Fig.
3A), with the exception that the disruptants produced more pycnidia and
conidial spores than did the wild type (Fig.
3B). The cultures of the
two disruptants formed pycnidia earlier and produced about four- to
five times more conidial spores (Table
1) than wild-type EP155. These results
suggest that CpST12 is dispensable for vegetative growth and
conidiation on artificial medium.
The cpst12 disruptants
were also inoculated onto American chestnut stems to test whether
disruption of this gene affected virulence. As illustrated in the
representative photographs in Fig.
4A and the quantitative data presented in Table
1, wild-type strain EP155
produced large cankers with densely packed orange spore-containing
stromal pustules protruding through the bark surface. In contrast, the
two cpst12 disruptants were severely reduced in the ability to
expand on chestnut tissue, forming small cankers that contain few,
often no, pustules (Fig.
4B). The cankers formed by
the cpst12 deletion mutants resembled cankers formed by
hypovirus CHV1-EP713-infected strain EP155. Even 6 weeks after
inoculation, very few stromal pustules had erupted through the bark of
the cankers formed by the cpst12 deletion mutants, in striking
contrast to the cankers formed by wild-type EP155.
The
cpst12 mutants also differed from wild-type C.
parasitica in the ability to colonize autoclaved chestnut stems
embedded in agar. As shown in Fig.
4C, when inoculated on the
agar substrate adjacent to the twig, strain EP155 was able to
effectively colonize the twig and form an abundance of stromata that
was seen erupting through the bark surface; protrusion was especially
evident through the bark lenticels. In contrast, the mutant strains
were unable to effectively colonize the bark tissue unless the bark was
mechanically scored prior to inoculation of the agar substrate. Thus,
the
cpst12 deletion mutants have difficulty
penetrating through the bark of dormant chestnut stems from the
underlying infected cambium tissue in the standard virulence assay
(Fig. 4A and B) and also
appear to be unable to effectively colonize autoclaved chestnut twigs
in the absence of mechanical wounding of the bark tissue (Fig.
4C).
To ensure that
the decreased virulence and increased conidiation phenotypes were
caused by the disruption of cpst12, a complementation vector
expressing the cDNA of cpst12 gene and conferring resistance
to benomyl was constructed and was used to transform the
cpst12 disruptant,
cpst-E1.
Benomyl-resistant transformants produced conidial spores at the level
comparable to wild-type EP155 (data not shown) and consistent with the
values reported in Table
1. When inoculated onto
dormant American chestnut stems, the two complemented strains
EP155/
cpst-E1/pCPST-C and
EP155/
cpst-E1/pCPST-G2, produced cankers comparable
in size to those produced by wild-type strain EP155 and the ectopically
transformed strain A8 and in contrast to the small cankers produced by
the cpst12 disruptant (EP155/
cpst-E1) and
the hypovirus CHV1-EP713-infected strain (EP155/CHV1-EP713) (Fig.
5). Moreover, the two complemented stains were also restored in the ability
to colonize and penetrate chestnut stems (data not
shown).
CpST12 is required for mating in C. parasitica.
The
two disruptant strains, the ectopic transformant A8, and the wild-type
strain EP155 were all tested for the ability to serve as a male or as a
female parent in crosses with strain EP146 of the opposite mating type.
Spermatization of strain EP146 with conidia from either wild-type
strain EP155, the ectopic transformant A8, or the cpst12
disruptant strains resulted in a similar number of perithecia and
viable ascospores (data not shown). In contrast, spermitization of the
disruptant strains with conidia derived from strain EP146 resulted in
the production of no perithecia even after prolonged incubation,
whereas copious numbers of perithecia were produced when the same batch
of EP146 conidia were used to spermatize strain EP155 or the
ectopic transformant A8. As was observed for the
defect in virulence, complementation of the
cpst12 disruptant
cpst-E1 with the wild-type
cpst12 cDNA (EP155/
cpst-E1/pCPST-C and
EP155/
cpst-E1/pCPST-G2) restored female
fertility with production of viable ascospores (data not shown). We
conclude that disruption of cpst12 results in female
sterility.
CpST12-regulated genes.
In an effort to
identify CpST12-regulated genes, we monitored changes in the
transcriptional profiles of approximately 2,200 C. parasitica
genes represented on a custom C. parastica cDNA microarray
(1) in response to
cpst12 disruption. Reciprocal (dye swap) hybridizations were
performed for each of two RNA preparations (a total of four
hybridizations) obtained from parallel cultures of wild-type EP155 and
cpst12 disruptant
cpst12-E1. An EST clone
(gene) was scored as being differentially expressed if the
log2 ratio of the relative probe signal intensities was at
least one standard deviation from the experimental average log2 ratio
in at least three of four hybridizations. Of the 2,200 C.
parasitica genes represented on the microarray, 152 genes were
scored as differentially expressed as a result of cpst12
disruption in disruptant strain
cpst12-E1
(61 up-regulated and 91 down-regulated
[available at
http://www.umbi.umd.edu/
cbr/deng/cpst12.xls]).
Comparison of these 152 differentially expressed genes with the list of
C. parasitica genes previously identified as being responsive
to CHV1-EP713 infection (reference
1 as refined in reference
19) revealed 47 genes
that were responsive both to cpst12 disruption and to
CHV1-EP713 infection, i.e., 31% of the 152 genes differentially
expressed in the
cpst12-E1 disruptant. (Fig.
6A). Inspection of the transcriptional changes for the 47 common genes
revealed that transcript levels were altered in the same direction for
32 genes and in the opposite direction for 15 genes. Moreover, of the
32 genes altered in the same direction, 28 were changed with a similar
magnitude (Fig. 6B). The
list of these common genes is presented in Table
2 under headings of putative biological processes as assigned by Dawe et
al. (18) according to the
classification guidelines outlined by the Gene Ontology Consortium
(http://www.genontology.org)
and as previously reported for CHV1-EP713-responsive genes by Allen et
al. (1).
To confirm
the changes obtained by the microarray analyses, transcript levels were
validated by real-time RT-PCR analyses for 15 randomly selected genes
that were designated as differentially expressed in the cpst12
disruptant strain. Table
3 shows that microarray-predicted changes in transcript accumulation were
confirmed for 14 of the 15 clones, and only one clear false-positive
result was obtained, a rate of <7%. These data provide a high
degree of confidence in the transcriptional changes that were measured
by microarray analysis in the present study.
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TABLE 3. Real-time
RT-PCR validation of microarray measurements for transcriptional
changes in cpst12 disruptant strain
cpst-E1a
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Since
cpst12 transcript levels are reduced by severalfold in
CHV1-EP713-infected strains
(1) rather than eliminated
as is the case in the cpst12 disruptant strains, we further
compared the two gene lists after lowering the fold change cutoff value
from 2 to 1.5 to compile the CHV1-EP713 responsive list. Under these
parameters, 99 of the 152 (65%) of the genes differentially expressed
in the
cpst12-E1 disruptant were also scored as
CHV1-EP713 responsive (Fig.
6A), with transcript
levels changed in the same direction for 72 of those 99
genes.
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DISCUSSION
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The changes in host
phenotypic traits that result from hypovirus (
13-kb RNA
genome) infection are remarkable both in terms of (i) the persistence,
stability, and pleiotropic nature of the changes caused by a specific
hypovirus and (ii) the differences in the magnitude and constellation
of changes caused by different hypoviruses
(3,
32). These phenotypic
changes have been correlated with virus-mediated alterations in several
different cellular signaling pathways
(9,
10,
21,
27,
35,
36). Moreover, different
hypoviruses have been reported to have differential effects on specific
pathways (36). Studies
employing a C. parasitica cDNA microarray have verified
alterations of G-protein-mediated signaling in hypovirus
CHV1-EP713-infected cells
(19) and have identified
hypovirus-specific and hypovirus-common changes in host gene transcript
accumulation (2). The
observation that a homologue of the yeast transcription factor Ste12 is
down-regulated by hypovirus infection and the identification of a
subset of CpST12-regulated, hypovirus-responsive genes, reported here,
provides a link between hypovirus-mediated modifications of cellular
signaling pathways and changes in the host transcriptional profile. The
observation that CpST12 is required for virulence and female fertility
also provides a possible basis for hypovirus-mediated modulation of
these fungal processes.
Ste12 homologues in filamentous fungi are
characterized by a highly conserved homeodomain at the N terminus and
dual C2/H2-Zn2+ finger
domains, not present in the yeast Ste12 homologues, at the C terminus
(33). In this regard,
CpST12 shares a very high level of amino acid identity with all eight
sequenced fungal Ste12 homologues in both the homeodomain (93%) and the
C2/H2-Zn2+ finger domain
(92%). Moreover, a high level of identity extends over the entire
predicted amino acid sequences for CpST12 and the homologues
pp-1 (71.2%) and MST12 (80.9%) from the closely
phylogenetically related
(18) fungi N.
crassa and M. grisae, respectively.
Ste12 plays
essential roles in the S. cerevisiae mating process and in the
filamentous growth phenotype, promoting the transcription of genes
required for mating as a homodimer and of genes required for invasive
growth as a heterodimer with Tec1 (summarized in reference
47). Although less well
characterized, fungal Ste12 homologues have also been shown to mediate
multiple functions. For example, deletion of the N. crassa
homologue pp-1 resulted in a reduced growth rate on synthetic
medium and the inability to form protoperithecia, resulting in female
sterility (28). In
contrast, disruption of the M. grisea homologue mst12
resulted in no effect on vegetative growth or female fertility
(33). CpST12, like
MST12, was found to be completely dispensable for vegetative
growth on synthetic medium. However, unlike MST12, CpST12 was
required for female fertility. No perithecia formed when the
cpst12 deletion mutants were tested as females in crosses,
even when twigs were mechanically scored with a scalpel to ensure
effective colonization by the disruption mutant prior to spermatization
and after prolonged incubation. Interestingly, the cpst12
disruptant strains produced more pycnidia and asexual spores than the
wild-type strain in culture, a phenotypic change not previously
reported for other fungal Ste12 homologue-null mutants. These conidia
were also shown to serve effectively as spermatia in mating assays
(data not shown). These observations further support the view that the
functional role of fungal Ste12 homologues vary according to the
lifestyle that a particular fungus has adopted over evolutionary
time.
The virulence of cpst12 disruptants was
significantly reduced, as previously reported for the Ste12 homologues
in the plant pathogenic fungi C. lagerarium
(42) and M.
grisea (33).
Infection by the later two fungi involves the formation of an elaborate
infection structure (termed an appressorium), penetration, and then
invasive growth. For both fungi, the Ste12 homologue was dispensable
for appressorium formation but was required for penetration and
invasive growth (42,
33). Infection by C.
parasitica does not involve the formation of specialized infection
structures. Colonization of wound sites is followed by the formation of
parallel arrays of hyphae called a mycelial fan that effectively
penetrates host wound periderm and lignified zones deposited by the
host to confine the invading hyphae
(23). The apparent
similarities in roles for Ste12 homologues for invasive pathogenic
growth by plant pathogenic fungi with quite different infection
strategies and plant hosts deserves further comparative
studies.
Cankers formed by cpst12 disruption mutants
resemble those formed by C. parasitica infected by the severe
hypovirus isolate CHV1-EP713 (Fig.
4). Interestingly,
although the cpst12 disruptants produced more pycnidia and
conidia on solid media, the disruptants, similar to CHV1-EP713-infected
C. parasitica strain EP155, formed small cankers with few, if
any, asexual spore-forming structures (stromal pustules) protruding
through the canker surface. These results suggest that the mutant has
difficulty in penetrating bark tissue from underlying cambial tissue.
In this regard, Kazmierczak et al.
(25) have shown that the
hydrophobin, cryparin, is required for stromal pustule eruption.
However, cryparin gene expression was not down-regulated in
cpst12-disruptant strains as measured by real-time RT-PCR
(data not shown). In addition, the aerial hyphae of cpst12
disruptants were not wettable and also distinct from the
C. parasitica cryparin mutants and hydrophobin-null mutants in
other fungi (5,
25,
40,
44). Therefore, the
defect in stromal pustule eruption observed in the cpst12
disruption mutant appears not to be due to reduced cryparin
production.
Microarray analysis with the custom C.
parasitica EST-based microarray platform used previously to
identify hypovirus-responsive and G-protein-regulated genes revealed
152 differentially expressed genes in the cpst12 disruptant
relative to the EP155 control strain, Consistent with the observation
that cpst12 transcript accumulation is down-regulated after
hypovirus infection (1,
2), comparisons of the
list of cpst12-regulated genes with hypovirus
CHV1-EP713-responsive genes revealed significant overlap. This was
particularly evident (99 of 152 genes [65%]) when the stringency for
scoring CHV1-EP713 responsive genes was adjusted from
2- to
1.5-fold change relative to the wild-type EP155 levels to
reflect the fact that hypovirus infection caused a reduction in, and
not a complete loss of, cpst12 transcript accumulation.
Although hypovirus infection clearly alters multiple signaling pathways
and has been reported to down-regulate other putative transcription
factors (1,
2,
19), these results
suggest that a significant portion of the hypovirus-responsive change
in transcriptional profile is mediated through cpst12.
Although it is too early to link hypovirus-mediated reductions in
cpst12 transcript levels and hypovirus-mediated hypovirulence
and female sterility, the fact that cpst12 disruption mutants
are reduced in virulence and are female sterile is consistent with this
possibility and warrants further study of this possible linkage. The
results of the microarray analysis also provide the first glimpse of
hypovirus-responsive genes that are dependent on a specific cellular
transcription factor for regulated expression.
Ste12 is the
direct target of MAPKs for both S. cerevisiae mating and
pseudohyphal growth pathways with Fus3 regulating Ste12 function in
response to mating pheromone and Kss1 in response to pseudohyphal
signals (13,
30,
31). Characterization of
the Fus3/Kss1 and Ste12 homologues in several filamentous fungi suggest
a degree of evolutionary conservation of this interaction. Li et al.
(28) recently reported
that null mutants of the N. crassa Ste12 and Fus3/Kss1
homologues, PP-1 and MAK-2, respectively, exhibited a very similar set
of phenotypic changes; this is consistent with the view that PP-1 and
MAK-2 are part of the same MAPK signaling cascade. In M.
grisea, deletion of the homologues MST12 and
PMK1 also resulted in a similar set of phenotypic changes,
including a defect in invasive pathogenic growth, with the exception
that the pkm1-null mutant, but not the mst12 mutant,
is defective in appressorium formation
(33,
34,
48).
The C.
parasitica Fus3/Kss1 homologue gene, cpmk2, has been
cloned and disrupted
(11). The
cpmk2-null mutant, like the cpst12 disruptant, was
significantly reduced in virulence and was repressed in the expression
of the pheromone precursor gene Mf2/1, suggesting a possible
defect in female fertility, although the results of mating experiments
were not reported for this mutant. However, unlike the observations
reported for the N. crassa and M. grisea, Fus3/Kss1
and Ste12 homologues, the cpmk2 and cpst12 disruption
mutants exhibited a quite different set of phenotypic traits.
Disruption of cpmk2 resulted in reduced vegetative growth on
solid media, loss of conidiation, and loss of orange pigment production
(11), traits not affected
by cpst12 disruption. Thus, the regulatory interaction between
CpMK2 and CpST12 is less clear than for the Fus3/Kss1 and Ste12
homologues in other closely related filamentous
fungi.
Interestingly, the cpmk2 deletion mutant more
closely resembled the G
subunit cpg-1 deletion mutant
(11). Moreover, both
cpmk2 (data not shown) and cpst12
(19) transcript levels
are reduced in cpg-1 and cpgb-1 (Gß subunit)
deletion mutants (the present study and data not shown). In this
regard, 41 cpg-1-regulated and 41 cpgb-1-regulated
genes (19) were also
found in the list of cpst12-regulated genes (the present study
and data not shown). These observations suggest a level of coordination
between the G-protein and MAPK signaling pathways in the regulation of
a hypovirus-responsive cellular transcription factor that is required
for fungal virulence and sexual reproduction. It is anticipated that
further characterization of hypovirus-responsive cellular transcription
factors such as cpst12 will provide useful new perspectives of
the signaling pathways that regulate important fungal physiological
processes and the mechanisms that underlie hypovirus-mediated
modulation of fungal virulence and development.
 |
ACKNOWLEDGMENTS
|
|---|
This study was supported in
part by Public Health Service grant GM55981 to
D.L.N.
 |
FOOTNOTES
|
|---|
* Corresponding author. Mailing address: Center for Biosystems Research, University of Maryland Biotechnology Institute, Shady Grove Campus, 9600 Gudelsky
Dr., Rockville, MD 20850. Phone: (240) 314-6218. Fax: (240) 314-6255. E-mail:
nuss{at}umbi.umd.edu. 
Published ahead of print on 17 November 2006. 
Present
address: Harrisburg Area Community College-Lancaster, 206R, 1641 Old
Philadelphia Pike, Lancaster, PA 17602. 
 |
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