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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{triangledown}

Fuyou Deng, Todd D. Allen,{dagger} 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
 
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
 
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{alpha} 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{alpha}- 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
 
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 ({alpha}) 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 {Delta}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
 
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.


Figure 1
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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.

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 {Delta}cpst12-E1 and {Delta}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).


Figure 2
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  		FIG. 2. Genomic organization of C. parasitica cpst12 gene and map of the cpst12 gene disruption construct. (A) A 4.5-kb genomic DNA containing the cpst12 coding region composed of four exons is indicated by the boxes. A cpst12 gene replacement construct was generated by insertion of the 2.8-kb hygromycin cassette using a PCR-based strategy (see Materials and Methods). (B) Southern analysis of wild-type EP155, cpst12 disruptant strains {Delta}cpst-E1 (EP155/{Delta}cpst-E1), {Delta}cpst-E7 (EP155/{Delta}cpst-E7), and ectopic transformant A8. All DNA samples were digested with HindIII and XhoI (left) or independently with ClaI (right). Fragment sizes are indicated in the figure margins. The blot was hybridized with the PCR fragments shown in panel A. (C) Real time RT-PCR analysis of cpst12 gene expression in wild-type EP155, cpst12 disruptant strains EP155/{Delta}cpst-E1 and EP155/{Delta}cpst-E7 and hypovirus CHV1-infected strain EP713.

The growth characteristics and colony morphology of the cpst12 disruptants, {Delta}cpst12-E1 and {Delta}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.


Figure 3
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  		FIG. 3. (A) Phenotypes of wild-type strain EP155, CHV1-infected strain EP713 and two cpst12 disruptant strains, {Delta}cpst-E1 and {Delta}cpst-E7. Photograph was taken on day 7 of culture on PDA. (B) Cultures of wild-type EP155 and cpst12 disruptant {Delta}cpst-E1 (EP155/{Delta}cpst-E1) to show the pycnidia and conidial spore production after 2 weeks. Compared to wild-type EP155, the cpst12 disruptant strain {Delta}cpst-E1 produced more pycnidia, which sporulate abundantly on PDA.


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  		TABLE 1. Virulence and sporulation of disruptant strains compared to the wild-type and hypovirulent strainsa

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.


Figure 4
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  		FIG. 4. (A) Virulence assay on dormant American chestnut stems. Representative cankers formed by wild-type strain EP155, strain EP155-infected with hypovirus CHV1-EP713 (EP155/CHV1-EP713), and two cpst12 disruptants (EP155/{Delta}cpst-E1 and EP155/{Delta}cpst-E7). Photographs of cankers were taken 3 weeks postinoculation. (B) The cpst12 disruptants (EP155/{Delta}cpst-E1,shown) failed to produce the stromal pustules that are formed by wild-type strain EP155 on the bark of inoculated dormant chestnut stems, even after prolonged incubation, indicating that the cpst12 disruptants are unable to erupt through the bark of the stems. (C) The cpst12 disruptant {Delta}cpst-E1 was able to grow on the surface of unscored autoclaved chestnut twigs but failed to produce stromal pustules like the wild-type strain EP155, similar to results observed for inoculated chestnut stems.

The {Delta}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 {Delta}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, {Delta}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/{Delta}cpst-E1/pCPST-C and EP155/{Delta}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/{Delta}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).


Figure 5
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  		FIG. 5. Representative cankers formed by wild-type strain EP155, EP155/CHV1-EP713, cpst12 disruptant EP155/{Delta}cpst-E1, ectopic transformant A8, and the two cpst12 disruptant complemented strains EP155/{Delta}cpst-E1/pCPST-C and EP155/{Delta}cpst-E1/pCPST-G2. Photographs were taken 2 weeks after inoculation.

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 {Delta}cpst-E1 with the wild-type cpst12 cDNA (EP155/{Delta}cpst-E1/pCPST-C and EP155/{Delta}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 {Delta}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 {Delta}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 {Delta}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).


Figure 6
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  		FIG. 6. Similarity in expression profiles between CHV-1-EP713 hypovirus-infected strain EP155 and {Delta}cpst12 disruptant strain {Delta}cpst-E1. (A) Venn diagram illustrating the total number of differentially expressed genes identified in hybridizations between EP155/CHV1-EP713 versus EP155 and EP155/{Delta}cpst-E1 versus EP155 (the present study). A total of 47 genes were found on both lists of differentially expressed clones, and these genes are described in Table 2. When differential expression was defined as genes with 1.5-fold changes relative to the wild type, EP155, a total of 99 hypovirus CHV1-EP713-responsive genes overlapped with the differentially expressed genes of the disruptant. (B) Bar chart illustrating the relative magnitude and direction of change in transcript levels produced by microarray for the genes differentially expressed in both CHV1-EP713-infected and cpst12 disruptant strains. Blue-shaded bars indicate the magnitude of transcript accumulation change after CHV1-EP713 infection (fold change [y axis]), whereas the magnitude of change for the same genes in the {Delta}cpst12 disruptant is indicated in purple. The order of AEST clones from left to right is as presented in Table 2. That is, values for AEST-01-G-08 are shown at the far left of the figure.


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  		TABLE 2. C. parasitica genes responsive to both CHV1-EP713 hypovirus infection and cpst12 gene disruptiona

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 {Delta}cpst12 disruptant strain {Delta}cpst-E1a

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 {Delta}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
 
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{alpha} 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.


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ACKNOWLEDGMENTS
 
This study was supported in part by Public Health Service grant GM55981 to D.L.N.


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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. Back

{triangledown} Published ahead of print on 17 November 2006. Back

{dagger} Present address: Harrisburg Area Community College-Lancaster, 206R, 1641 Old Philadelphia Pike, Lancaster, PA 17602. Back


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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
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