Hypovirus Infection Research Articles

Changes in VOCs from a chestnut blight fungus Cryphonectria parasitica by a hypovirus infection: mycoviral infection alters fungal smell for insect vectors

BackgroundThe chestnut blight fungus, Cryphonectria parasitica, and its Cryphonectria hypovirus 1 (CHV1) comprise a useful model system to study fungus–virus interactions. CHV1 infection results in various phenotypic changes in the fungal host, including hypovirulence and other associated symptoms. Many studies have investigated the effects of hypoviral infection and how this infection affects physiological and biochemical states: however, no studies have examined volatile organic compounds (VOCs).ResultsThis study characterized the global profile of VOCs released from C. parasitica, and by comparing CHV1-free with CHV1-infected isogenic strains, proved that CHV1 infection significantly affects the composition and levels of VOCs. We demonstrated that these VOC alterations were caused by viral regulation of the expression of fungal genes encoding enzymes responsible for the production of VOCs. The changed VOC profile resulting from CHV1 infection was more attractive to insects than the VOC profile from the virus-free strain, suggesting differences in the efficacy of fungal dissemination by the insect vector.ConclusionsWe characterized VOCs from C. parasitica and demonstrated that mycovirus (CHV1) infection affects fungal VOCs. We provided evidences that these alterations are caused by the modulation of the corresponding gene expression by CHV1 and resulted in changes in attractiveness to insects, suggesting the improved efficacy of hypovirulent C. parasitica for insect-borne dissemination.Graphical

Hypovirus infection induces proliferation and perturbs functions of mitochondria in the chestnut blight fungus.

The chestnut blight fungus, Cryphonectria parasitica, and hypovirus have been used as a model to probe the mechanism of virulence and regulation of traits important to the host fungus. Previous studies have indicated that mitochondria could be the primary target of the hypovirus. In this study, we report a comprehensive and comparative study comprising mitochondrion quantification, reactive oxygen species (ROS) and respiratory efficiency, and quantitative mitochondrial proteomics of the wild-type and virus-infected strains of the chestnut blight fungus. Our data show that hypovirus infection increases the total number of mitochondria, lowers the general ROS level, and increases mitochondrial respiratory efficiency. Quantification of mitochondrial proteomes revealed that a set of proteins functioning in energy metabolism and mitochondrial morphogenesis, as well as virulence, were regulated by the virus. In addition, two viral proteins, p29 and p48, were found to co-fractionate with the mitochondrial membrane and matrix. These results suggest that hypovirus perturbs the host mitochondrial functions to result in hypovirulence.

Distinct Roles of Two DNA Methyltransferases from Cryphonectria parasitica in Fungal Virulence, Responses to Hypovirus Infection, and Viral Clearance.

ABSTRACTTwo DNA methyltransferase (DNMTase) genes from Cryphonectria parasitica have been previously identified as CpDmt1 and CpDmt2, which are orthologous to rid and dim-2 of Neurospora crassa, respectively. While global changes in DNA methylation have been associated with fungal sectorization and CpDmt1 but not CpDmt2 has been implicated in the sporadic sectorization, the present study continues to investigate the biological functions of both DNMTase genes. Transcription of both DNMTases is regulated in response to infection with the Cryphonectria hypovirus 1 (CHV1-EP713). CpDmt1 is upregulated and CpDmt2 is downregulated by CHV1 infection. Conidium production and response to heat stress are affected only by mutation of CpDmt1, not by CpDmt2 mutation. Significant changes in virulence are observed in opposite directions; i.e., the CpDmt1-null mutant is hypervirulent, while the CpDmt2-null mutant is hypovirulent. Compared to the CHV1-infected wild type, CHV1-transferred single and double mutants show severe growth retardation: the colony size is less than 10% that of the parental virus-free null mutants, and their titers of transferred CHV1 are higher than that of the wild type, implying that no defect in viral replication occurs. However, as cultivation proceeds, spontaneous viral clearance is observed in hypovirus-infected colonies of the null mutants, which has never been reported in this fungus-virus interaction. This study demonstrates that both DNMTases are significant factors in fungal development and virulence. Each fungal DNMTase affects fungal biology in both common and separate ways. In addition, both genes are essential to the antiviral responses, including viral clearance which depends on their mutations.

Functional analysis of an essential Ran-binding protein gene, CpRbp1, from the chestnut blight fungus Cryphonectria parasitica using heterokaryon rescue

A Ran binding protein (RanBP) homolog, CpRbp1, from Cryphonectria parasitica, has been identified as a protein that is affected by hypovirus infection or tannic acid supplementation. In this study, functional analyses of CpRbp1 were performed by constructing a knockout mutant and analyzing the resulting heterokaryon. Transformation-mediated gene replacement resulted in two putative CpRbp1-null mutants and genotype analyses identified these two mutants as heterokaryotic transformants consisting of two types of nuclei, one with the wild-type CpRbp1 allele and another with the CpRbp1-null mutant allele. Although stable mycelial growth of the heterokaryotic transformant was observed on selective medium containing hygromycin B, neither germination nor growth of the resulting conidia, which were single-cell monokaryotic progeny, was observed on the medium. In trans complementation of heterokaryons using a full-length wild-type allele of the CpRbp1 gene resulted in complemented transformants. These transformants sporulated single-cell monokaryotic conidia that were able to grow on media selective for replacing and/or complementing markers. These results clearly indicate that CpRbp1 is an essential gene, and heterokaryons allowed the fungus to maintain lethal CpRbp1-null mutant nuclei. Moreover, in trans complementation of heterokaryons using chimeric structures of the CpRbp1 gene allowed for analysis of its functional domains, which was previously hampered due to the lethality of the gene. In addition, in trans complementation using heterologous RanBP genes from Aspergillus nidulans was successful, suggesting that the function of RanBP is conserved during evolution. Furthermore, in trans complementation allowed for functional analyses of lethal orthologs. This study demonstrates that our fungal heterokaryon system can be applied effectively to determine whether a gene of interest is essential, perform functional analyses of a lethal gene, and analyze corresponding heterologous genes.

Spatial and temporal dynamics of the fungal community of chestnut blight cankers on American chestnut (Castanea dentata) in Michigan and Wisconsin

Abstract Chestnut blight cankers caused by the fungal pathogen Cryphonectria parasitica on infected American chestnut trees can be invaded by hypoviruses that infect C. parasitica mycelia. Hypoviruses lower pathogen virulence (i.e. hypovirulence) and decrease the probability of stem girdling. Cankers also are susceptible to invasion by non-C. parasitica fungi; however, the influence of invading fungi on disease severity is unknown. Fungi may antagonize C. parasitica growth and combine with hypovirulence to further reduce the probability of stem girdling. We conducted a survey to investigate how spatial and temporal dynamics of the fungal community within cankers correlate with canker severity and the likelihood of girdling. Cankers from six American chestnut populations were monitored from 2012 to 2016. We found that the spatial distribution of the fungal community within a canker resembled a mosaic that increased in fungal diversity over time. Cankers with increased fungal diversity were not associated with higher stem survivorship over time, compared to cankers with abundant hypovirulence in recovering chestnut populations. Fungal communities also were unstable, and cankers on surviving trees containing hypovirulence consistently were invaded by non-C. parasitica fungi. Our results suggest that canker communities are not stable, and volatility of the fungal community indicates that cankers can change quickly from less severe to more severe cankers through loss of hypovirulence. Non-C. parasitica fungi may facilitate canker expansion through greater inhibition of hypovirulent C. parasitica relative to virulent C. parasitica, which may permit virulent C. parasitica to escape hypovirus infection and resume rapid canker expansion.

Transcriptional and Small RNA Responses of the White Mold Fungus Sclerotinia sclerotiorum to Infection by a Virulence-Attenuating Hypovirus.

Mycoviruses belonging to the family Hypoviridae cause persistent infection of many different host fungi. We previously determined that the white mold fungus, Sclerotinia sclerotiorum, infected with Sclerotinia sclerotiorum hypovirus 2-L (SsHV2-L) exhibits reduced virulence, delayed/reduced sclerotial formation, and enhanced production of aerial mycelia. To gain better insight into the cellular basis for these changes, we characterized changes in mRNA and small RNA (sRNA) accumulation in S. sclerotiorum to infection by SsHV2-L. A total of 958 mRNAs and 835 sRNA-producing loci were altered after infection by SsHV2-L, among which >100 mRNAs were predicted to encode proteins involved in the metabolism and trafficking of carbohydrates and lipids. Both S. sclerotiorum endogenous and virus-derived sRNAs were predominantly 22 nt in length suggesting one dicer-like enzyme cleaves both. Novel classes of endogenous small RNAs were predicted, including phasiRNAs and tRNA-derived small RNAs. Moreover, S. sclerotiorum phasiRNAs, which were derived from noncoding RNAs and have the potential to regulate mRNA abundance in trans, showed differential accumulation due to virus infection. tRNA fragments did not accumulate differentially after hypovirus infection. Hence, in-depth analysis showed that infection of S. sclerotiorum by a hypovirulence-inducing hypovirus produced selective, large-scale reprogramming of mRNA and sRNA production.

Comparative Methylome Analysis Reveals Perturbation of Host Epigenome in Chestnut Blight Fungus by a Hypovirus.

In eukaryotic genomes, DNA methylation is an important type of epigenetic modification that plays crucial roles in many biological processes. To investigate the impact of a hypovirus infection on the methylome of Cryphonectria parasitica, the chestnut blight fungus, whole-genome bisulfite sequencing (WGBS) was employed to generate single-base resolution methylomes of the fungus with/without hypovirus infection. The results showed that hypovirus infection alters methylation in all three contexts (CG, CHG, and CHH), especially in gene promoters. A total of 600 differentially methylated regions (DMRs) were identified, of which 144 could be annotated to functional genes. RNA-seq analysis revealed that DNA methylation in promoter is negatively correlated with gene expression. Among DMRs, four genes were shown to be involved in conidiation, orange pigment production, and virulence. Taken together, our DNA methylomes analysis provide valuable insights into the understanding of the relationship between DNA methylation and hypovirus infection, as well as phenotypic traits in C. parasitica.

Transcriptome-Based Discovery of Fusarium graminearum Stress Responses to FgHV1 Infection.

Fusarium graminearum hypovirus 1 (FgHV1), which is phylogenetically related to Cryphonectria hypovirus 1 (CHV1), is a virus in the family Hypoviridae that infects the plant pathogenic fungus F. graminearum. Although hypovirus FgHV1 infection does not attenuate the virulence of the host (hypovirulence), it results in defects in mycelial growth and spore production. We now report that the vertical transmission rate of FgHV1 through asexual spores reached 100%. Using RNA deep sequencing, we performed genome-wide expression analysis to reveal phenotype-related genes with expression changes in response to FgHV1 infection. A total of 378 genes were differentially expressed, suggesting that hypovirus infection causes a significant alteration of fungal gene expression. Nearly two times as many genes were up-regulated as were down-regulated. A differentially expressed gene enrichment analysis identified a number of important pathways. Metabolic processes, the ubiquitination system, and especially cellular redox regulation were the most affected categories in F. graminearum challenged with FgHV1. The p20, encoded by FgHV1 could induce H2O2 accumulation and hypersensitive response in Nicotiana benthamiana leaves. Moreover, hypovirus FgHV1 may regulate transcription factors and trigger the RNA silencing pathway in F. graminearum.

A Comparison of the Growth and Asexual Reproduction by Cryphonectria parasitica Isolates Infected with Hypoviruses CHV3-County Line, CHV1-Euro7, and CHV1-Ep713

The impact of chestnut blight, caused by the fungal pathogen Cryphonectria parasitica, has diminished in Europe due to a natural biological control caused by hypovirus infection. Hypovirulence-mediated biological control has been far less successful in North America meriting further evaluation of field isolates that have the ability to produce non-lethal cankers, generate hypovirulent inoculum, and exhibit a greater ecological fitness in forest systems. In this study, Cryphonectria hypoviruses (CHV) CHV3-County Line, CHV1-Euro7, and CHV1-Ep713 were evaluated in five different isolates of C. parasitica. One hundred and eighty cankers representing each treatment combination were initiated on American chestnut sprouts in the Monongahela National Forest, West Virginia, USA. The size of cankers, the persistence of hypovirulent (HV) isolates, stroma production, and hypovirus transmission to conidia were assessed four and 12 months after canker expansion. CHV3-County Line infected isolates produced significantly smaller cankers than the isolates infected with either CHV1-type. With regard to CHV1-Euro7 isolates, the fungal genome appeared to contribute to the differences in canker size. After four months, HV isolates harboring either CHV1-type (30%) were retrieved at a significantly higher rate than isolates containing CHV3-County Line (14%). After 12 months, the HV recovery was similar among the three hypoviruses indicating smaller cankers will maintain their HV status after one year. Very few stroma were produced after one year in the field from HV isolates. In vitro, CHV3-County Line (49%) had a significantly lower rate of hypovirus transmission to conidia when compared to CHV1-Euro7 (87%) and CHV1-Ep713 (80%). Significant differences existed among the five different isolates indicating HV transmission is dependent on the fungal genome. This research provided additional evidence that each hypovirus interacts with its host differently and certain isolate/hypovirus combinations have better biological control potential than others.

Modeling and Analysis of American Chestnut Populations Subject to Various Stages of Infection

AbstractAmerican chestnuts, Castanea dentata, were once a dominant tree in eastern deciduous forests of the United States before the chestnut blight fungus, Cryphonectria parasitica, was introduced unintentionally in the early 1900s in New York. This fungus rapidly devastated American chestnut populations until a hypovirus infection of the fungus began to reduce pathogen virulence on chestnut trees. The subsequent reappearance of large reproducing chestnut trees, associated with a large proportion of blight-infected isolates being parasitized by this hypovirus, is currently taken to indicate recovery of American chestnut populations. We explore, using previously-established matrix population models, the dynamics of healthy, fungus-infected, and hypovirus-infected American chestnut populations to test the efficacy of this recovery. Our main result is that populations transitioning from being fungus-infected to hypovirus-infected are predicted to show large transient amplifications as a result of demographi...

CpBir1 is required for conidiation, virulence and anti-apoptotic effects and influences hypovirus transmission in Cryphonectria parasitica

Inhibitors of apoptosis proteins (IAPs) are critically important in the regulation of unicellular yeast and metazoan apoptosis. All IAPs contain one to three baculovirus IAP repeat (BIR) domains, which are essential for the anti-apoptotic activity of the IAPs. A homolog of IAPs, CpBir1, which bears two BIR domains, was recently identified from the chestnut blight fungus Cryphonectria parasitica genome. CpIAP was deleted by gene replacement, and the phenotypes of ΔIAP were characterized. CpBir1 was significantly down-regulated by hypovirus infection but up-regulated by H2O2. Similar to Saccharomyces cerevisiae Bir1p, the Cpbir1 mutant was sensitive to H2O2, and constitutive overexpression of CpBir1 increased resistance to H2O2. The Cpbir1 mutant also showed defects in aerial hyphal formation, colony growth, mycelial morphology, conidiogenesis, pigmentation, resistance to stress conditions and virulence. Genetic complementation with native Cpbir1 fully recovered all these defective phenotypes. The CpBir1-eGFP fusion protein was localized to the nucleus in juvenile cultures, while it was found in the cytoplasm in old cultures, suggesting that the localization pattern of CpBir1 may correlate with the process of anti-apoptosis. Increased accumulation of reactive oxygen species (ROS) in the Cpbir1 deletion mutant further supports the anti-apoptotic function of CpBir1. Among five selected vegetative compatible (vc) types of C. parasitica, Cpbir1 deletion was found to block virus from transferring between Cpbir1 mutants. However, hypovirus infected Cpbir1 mutants showed a similar ability to transmit virus to other virus-free isolates compared with the infected wild-type strain. In summary, Cpbir1 encodes an IAP CpBir1 that is down-regulated by hypovirus infection and required for conidiation, virulence and anti-apoptosis, as well as affects hypovirus transmission in chestnut blight fungus C. parasitica.

Comparative vesicle proteomics reveals selective regulation of protein expression in chestnut blight fungus by a hypovirus

The chestnut blight fungus (Cryphonectria parasitica) and hypovirus constitute a model system to study fungal pathogenesis and mycovirus-host interaction. Knowledge in this field has been gained largely from investigations at gene transcription level so far. Here we report a systematic analysis of the vesicle proteins of the host fungus with/without hypovirus infection. Thirty-three differentially expressed protein spots were identified in the purified vesicle protein samples by two-dimensional electrophoresis and mass spectrometry. Down-regulated proteins were mostly cargo proteins involved in primary metabolism and energy generation and up-regulated proteins were mostly vesicle associated proteins and ABC transporter. A virus-encoded protein p48 was found to have four forms with different molecular mass in vesicles from the virus-infected strain. While a few of the randomly selected differentially expressed proteins were in accordance with their transcription profiles, majority were not in agreement with their mRNA accumulation patterns, suggesting that an extensive post-transcriptional regulation may have occurred in the host fungus upon a hypovirus infection.

Comparative proteomic analysis of chestnut blight fungus,Cryphonectria parasitica, under tannic-acid-inducing and hypovirus-regulating conditions

Chestnut blight fungus, Cryphonectria parasitica , and its hypovirus present a useful model system for investigating the mechanisms of hypoviral infection. To identify gene products associated with fungal pathogenicity and hypoviral regulation, we attempted a proteomic analysis of the virus-free EP155/2 strain and its isogenic virus-infected UEP1 strain in response to tannic acid (TA), which is abundant in the bark of chestnut trees. In this study, pretreatment of mycelia grown on TA-supplemented media was developed for proteomic analysis. Approximately 704 proteins from the mycelia of the EP155/2 strain were reproducibly present in 3 independent extractions. Among these, 111 and 79 spots were found to be responsive to hypovirus infection and TA supplementation, respectively. The TA-grown UEP1 strain yielded 28 spots showing an expression pattern different from that of untreated UEP1. Thirty protein spots showing considerable differences in spot density were selected for further analysis. Hybrid tandem LC-MS/MS spectrometry of the 30 selected protein spots revealed that 29 were identified while 1 was unidentified. Among the identified 29 proteins, 15 were metabolic enzymes; 5 were stress-related, of which 4 were heat-shock proteins and 1 was glutathione S-transferase; 5 were signaling and cellular process-related proteins; 2 were structural proteins; and 2 matched proteins of hypothetical genes.

Structure of Oxalacetate Acetylhydrolase, a Virulence Factor of the Chestnut Blight Fungus

Oxalacetate acetylhydrolase (OAH), a member of the phosphoenolpyruvate mutase/isocitrate lyase superfamily, catalyzes the hydrolysis of oxalacetate to oxalic acid and acetate. This study shows that knock-out of the oah gene in Cryphonectria parasitica, the chestnut blight fungus, reduces the ability of the fungus to form cankers on chestnut trees, suggesting that OAH plays a key role in virulence. OAH was produced in Escherichia coli and purified, and its catalytic rates were determined. Oxalacetate is the main OAH substrate, but the enzyme also acts as a lyase of (2R,3S)-dimethyl malate with approximately 1000-fold lower efficacy. The crystal structure of OAH was determined alone, in complex with a mechanism-based inhibitor, 3,3-difluorooxalacetate (DFOA), and in complex with the reaction product, oxalate, to a resolution limit of 1.30, 1.55, and 1.65 A, respectively. OAH assembles into a dimer of dimers with each subunit exhibiting an (alpha/beta)(8) barrel fold and each pair swapping the 8th alpha-helix. An active site "gating loop" exhibits conformational disorder in the ligand-free structure. To obtain the structures of the OAH.ligand complexes, the ligand-free OAH crystals were soaked briefly with DFOA or oxalacetate. DFOA binding leads to ordering of the gating loop in a conformation that sequesters the ligand from the solvent. DFOA binds in a gem-diol form analogous to the oxalacetate intermediate/transition state. Oxalate binds in a planar conformation, but the gating loop is largely disordered. Comparison between the OAH structure and that of the closely related enzyme, 2,3-dimethylmalate lyase, suggests potential determinants of substrate preference.

Major impacts on the primary metabolism of the plant pathogen Cryphonectria parasitica by the virulence-attenuating virus CHV1-EP713

Cryphonectria parasitica, the chestnut blight fungus, can be infected by virulence-attenuating mycoviruses of the family Hypoviridae. Previous studies have led to the hypothesis that the hypovirus-infected phenotype is partly due to metabolic changes induced by the viral infection. To investigate this, we measured the metabolic rate and respiration of C. parasitica colonies grown on solid medium. These experiments supported historical observations of other fungal species done in liquid cultures that the metabolic rate steadily declines with age and differentiation of the mycelium. Hypovirus infection increased metabolic rate in the youngest mycelium, but a subsequent decline was also observed as the mycelium aged. By measuring both CO(2) production and O(2) consumption, we also observed that changes occur in carbohydrate metabolism as a result of ageing in both infected and uninfected mycelium. Mycelium on the periphery of the colony exploited fermentation pathways extensively, before transitioning to aerobic carbohydrate metabolism and finally lipid metabolism in the interior regions, despite abundant remaining glucose. However, the hypovirus affected the extent of these changes, with infected mycelium apparently unable to utilize lipid-related metabolic pathways, leading to an increased depletion of glucose. Finally, we used metabolic profi fi ling to determine the changes in accumulation of primary metabolites in wild-type and hypovirus-infected mycelium and found that approximately one-third of the 164 detected metabolites were affected. These results are consistent with those expected from the physiological measurements, with significant alterations noted for compounds related to lipid and carbohydrate metabolism. Additionally, we observed an increase in the accumulation of the polyamine spermidine in the presence of hypovirus. Polyamines have been implicated in antiviral responses of mammalian systems; therefore this may suggest a novel antiviral response mechanism in fungi.

Hypovirus-Responsive Transcription Factor Gene pro1 of the Chestnut Blight Fungus Cryphonectria parasitica Is Required for Female Fertility, Asexual Spore Development, and Stable Maintenance of Hypovirus Infection

We report characterization of the gene encoding putative transcription factor PRO1, identified in transcriptional profiling studies as being downregulated in the chestnut blight fungus Cryphonectria parasitica in response to infection by virulence-attenuating hypoviruses. Sequence analysis confirmed that pro1 encodes a Zn(II)(2)Cys(6) binuclear cluster DNA binding protein with significant sequence similarity to the pro1 gene product that controls fruiting body development in Sordaria macrospora. Targeted disruption of the C. parasitica pro1 gene resulted in two phenotypic changes that also accompany hypovirus infection, a significant reduction in asexual sporulation that could be reversed by exposure to high light intensity, and loss of female fertility. The pro1 disruption mutant, however, retained full virulence. Although hypovirus CHV1-EP713 infection was established in the pro1 disruption mutant, infected colonies continually produced virus-free sectors, suggesting that PRO1 is required for stable maintenance of hypovirus infection. These results complement the recent characterization of the hypovirus-responsive homologue of the Saccharomyces cerevisiae Ste12 C(2)H(2) zinc finger transcription factor gene, cpst12, which was shown to be required for C. parasitica female fertility and virulence.

Two‐dimensional fractal growth properties of the filamentous fungus Cryphonectria parasitica: the effects of hypovirus infection

Whole-colony two-dimensional fractal growth patterns produced by hypovirus-infected Cryphonectria parasitica (EP155/CHV1-EP713) were measured and compared with those produced by the isogenic virus-free strain (EP155) on solid medium. We have quantified statistically significant differences in the rates of expansion and spatial dynamics of colony growth between the two strains and concluded that fractal dimension is affected by the presence of the hypovirus. Therefore, fractal dimension measurement is an effective quantitative tool for testing the effects of mycovirus infection on fungal growth parameters.

Comparative Analysis of Alterations in Host Phenotype and Transcript Accumulation following Hypovirus and Mycoreovirus Infections of the Chestnut Blight Fungus Cryphonectria parasitica

Infection of the chestnut blight fungus, Cryphonectria parasitica, by hypovirus CHV1-EP713 or by reovirus MyRV1-Cp9B21 or MyRV2-CpC18 results in reduced fungal virulence (hypovirulence). However, additional phenotypic changes caused by the two groups of mycoviruses are quite different. We now report that the loss of female fertility and the resulting absence of virus transmission through sexual spores observed after hypovirus infection was not observed for reovirus-infected C. parasitica. Consistent with this result, expression of two genes involved in sexual reproduction, the pheromone precursor gene, Mf2/1, and the yeast STE12-like transcriptional factor gene, cpst12, was less reduced in reovirus-infected strains than in the hypovirus CHV1-EP713-infected strain. Analysis with a custom microarray cDNA chip containing expressed sequence tag clones representing approximately 2,200 unique C. parasitica genes identified 140 and 128 host genes that were responsive to MyRV1-Cp9B21 or MyRV2-CpC18 infection, respectively. Comparison of these virus-responsive genes revealed an overlap of 85 genes, even though the nucleotide sequence identity for the two reoviruses is less than 50%. Significantly, 84 of the 85 genes were altered in the same direction. Further comparison revealed that 51% and 48% of the MyRV1-Cp9B21- and MyRV2-CpC18-responsive genes were also responsive to CHV1-EP713 infection. Finally, similar to results reported for CHV1-EP713 infection, a high percentage (59% and 66%) of the reovirus-responsive genes were also differentially expressed following disruption of the cellular G-protein signal transduction pathway. These data support the hypothesis that hypovirus and reovirus infections perturb common and specific C. parasitica regulatory pathways to cause hypovirulence and distinct sets of phenotypic changes.

Ste12 Transcription Factor Homologue CpST12 Is Down-Regulated by HypovirusInfection and Required for Virulence and Female Fertility of the Chestnut Blight Fungus Cryphonectria parasitica

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.

A gene encoding phosphatidyl inositol-specific phospholipase C from Cryphonectria parasitica modulates the lac1 expression

Hypovirus infection of the chestnut blight fungus Cryphonectria parasitica is known to downregulate the fungal laccase1 ( lac1), the modulation of which is tightly governed by the inositol triphosphate (IP 3) and calcium second messenger system in a virus-free strain. We cloned the gene cplc1 encoding a phosphatidyl inositol-specific phospholipase C (PLC), to investigate the regulation of lac1 expression and to better characterize fungal gene regulation by hypovirus. Sequence analysis of the cplc1 gene indicated that the protein product contained both the X and Y domains, which are the two conserved regions found in all known PLCs, with a 133 amino acid extension between the 2nd β-strand and the α-helix in the X domain. In addition, the gene organization appeared to be highly similar to that of a δ-type PLC. Disruption of the cplc1 gene resulted in slow growth and produced colonies characterized by little aerial mycelia and deep orange in color. Accordingly, reduced virulence of the cplc1-null mutant as compared to the wild-type was observed, which can be ascribed to the growth defect. However, other PLC-associated characteristics including temperature sensitivity and osmosensitivity did not differ from the wild-type strain. Northern blot analysis revealed no accumulation of the lac1 gene transcript due to the disruption of the cplc1 gene. Functional complementation of the cplc1-null mutant with the PLC1 gene from Saccharomyces cerevisiae restored lac1 expression, which suggests that the cloned gene encodes PLC activity. The present study indicates that the cplc1 gene is required for normal mycelial growth rate and colony morphology, and that it regulates the lac1 expression, which is also modulated by the hypovirus. Although several PLC genes have been identified in various simple eukaryotic organisms, the deletion analysis of the cplc1 gene in this study appears to be the first report on the functional analysis of PLC in filamentous fungi.