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Cryphonectria parasitica is the causal agent of chestnut blight, a fungal disease that almost entirely eliminated mature American chestnut from North America over a 50-year period. Here, we formally report the genome of C. parasitica EP155 using a Sanger shotgun sequencing approach. After finishing and integration with simple-sequence repeat markers, the assembly was 43.8 Mb in 26 scaffolds (L 50 = 5; N 50 = 4.0Mb). Eight chromosomes are predicted: five scaffolds have two telomeres and six scaffolds have one telomere sequence. In total, 11,609 gene models were predicted, of which 85% show similarities to other proteins. This genome resource has already increased the utility of a fundamental plant pathogen experimental system through new understanding of the fungal vegetative incompatibility system, with significant implications for enhancing mycovirus-based biological control.

Horizontal transmission of virulence attenuating hypoviruses of Cryphonectria parasitica is restricted by an allorecognition system termed vegetative incompatibility (vic). A super donor formulation of two engineered C. parasitica strains (SD328/SD82) with gene disruptions at four of six vic loci transmitted hypovirus to strains in the laboratory independent of vic genotype. We now report the transmission of hypovirus by the SD328/82 formulation to a diverse, natural C. parasitica population infecting American chestnut in a forest setting. Hypovirulent (HV) isolates were recovered from 94% of cankers treated with the hypovirus-infected SD328/82 formulation compared to 51% of cankers treated with a hypovirus-infected EU5/6 formulation (strains having the same vic genotypes as SD strains but lacking vic gene disruptions). Overall, the SD328/82 formulation transmitted hypovirus into more divergent vic genotypes compared to the EU5/6 formulation. These results demonstrate the SD328/82 formulation can serve as an enhanced hypovirus vector for highly divergent C. parasitica populations., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Chestnut blight is a devastating disease of Castanea spp. Mycoviruses that reduce virulence (hypovirulence) of the causative agent, Cryphonectria parasitica, can be used to manage chestnut blight. However, vegetative incompatibility (vic) barriers that restrict anastomosis-mediated virus transmission hamper hypovirulence efficacy. In order to effectively determine the vegetative incompatibility genetic structure of C. parasitica field populations, we have designed PCR primer sets that selectively amplify and distinguish alleles for each of the six known diallelic C. parasitica vic genetic loci. PCR assay results were validated using a panel of 64 European tester strains with genetically determined vic genotypes. Analysis of 116 C. parasitica isolates collected from five locations in the eastern United States revealed 39 unique vic genotypes and generally good agreement between PCR and tester strain coculturing assays in terms of vic diversity and genotyping. However, incongruences were observed for isolates from multiple locations and suggested that the coculturing assay can overestimate diversity at the six known vic loci. The availability of molecular tools for rapid and precise vic genotyping significantly improves the ability to predict and evaluate the efficacy of hypovirulence and related management strategies., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

An inducible RNA-silencing pathway, involving a single Dicer protein, DCL2, and a single Argonaute protein, AGL2, was recently shown to serve as an effective antiviral defense response in the chestnut blight fungus Cryphonectria parasitica. Eukaryotic RNA-dependent RNA polymerases (RdRPs) are frequently involved in transcriptional and posttranscriptional gene silencing and antiviral defense. We report here the identification and characterization of four RdRP genes (rdr1-4) in the C. parasitica genome. Sequence relationships with other eukaryotic RdRPs indicated that RDR1 and RDR2 were closely related to QDE-1, an RdRP involved in RNA silencing ("quelling") in Neurospora crassa, whereas RDR3 was more closely related to the meiotic silencing gene SAD-1 in N. crassa. The RdRP domain of RDR4, related to N. crassa RRP-3 of unknown function, was truncated and showed evidence of alternative splicing. Similar to reports for dcl2 and agl2, the expression levels for rdr3 and rdr4 increased after hypovirus CHV-1/EP713 infection, while expression levels of rdr1 and rdr2 were unchanged. The virus-responsive induction patterns for rdr3 and rdr4 were altered in the Δdcl2 and Δagl2 strains, suggesting some level of interaction between rdr3 and rdr4 and the dcl2/agl2 silencing pathway. Single rdr gene knockouts Δrdr1-4, double knockouts Δrdr1/2, Δrdr2/3, Δrdr1/3, and a triple knockout, Δrdr1/2/3, were generated and evaluated for effects on fungal phenotype, the antiviral defense response, viral RNA recombination activity and transposon expression. None of the single or multiple rdr knockout strains displayed any phenotypic differences from the parental strains with or without viral infection or any significant changes in viral RNA accumulation or recombination activity or transposon RNA accumulation, indicating no detectable contribution by the C. parasitica rdr genes to these processes.

Vegetative incompatibility (vic), a form of nonself allorecognition, operates widely in filamentous fungi and restricts transmission of virulence-attenuating hypoviruses in the chestnut blight fungus Cryphonectria parasitica. We report here the use of a polymorphism-based comparative genomics approach to complete the molecular identification of the genetically defined C. parasitica vic loci with the identification of vic1 and vic3. The vic1 locus in the C. parasitica reference strain EP155 consists of a polymorphic HET-domain-containing 771-aa ORF designated vic1a-2, which shares 91% identity with the corresponding vic1a-1 allele, and a small (172 aa) idiomorphic DUF1909-domain-containing ORF designated vic1b-2 that is absent at the vic1-1 locus. Gene disruption of either vic1a-2 or vic1b-2 in strain EP155 eliminated restrictions on virus transmission when paired with a vic1 heteroallelic strain; however, only disruption of vic1a-2 abolished the incompatible programmed cell death (PCD) reaction. The vic3 locus of strain EP155 contains two polymorphic ORFs of 599 aa (vic3a-1) and 102 aa (vic3b-1) that shared 46 and 85% aa identity with the corresponding vic3a-2 and vic3b-2 alleles, respectively. Disruption of either vic3a-1 or vic3b-1 resulted in increased virus transmission. However, elimination of PCD required disruption of both vic3a and vic3b. Additional allelic heterogeneity included a sequence inversion and a 8.5-kb insertion containing a LTR retrotransposon sequence and an adjacent HET-domain gene at the vic1 locus and a 7.7-kb sequence deletion associated with a nonfunctional, pseudo vic locus. Combined gene disruption studies formally confirmed restriction of mycovirus transmission by five C. parasitica vic loci and suggested dedicated roles in allorecognition. The relevance of these results to the acquisition and maintenance of vic genes and the potential for manipulation of vic alleles for enhanced mycovirus transmission are discussed., (Copyright © 2014 by the Genetics Society of America.)

Reverse-genetics analysis has played a significant role in advancing fungal biology, but is limited by the number of available selectable marker genes (SMGs). The Cre-loxP recombination system has been adapted for use in filamentous fungi to overcome this limitation. Expression of the Cre recombinase results in excision of an integrated SMG that is flanked by loxP sites, allowing a subsequent round of transformation with the same SMG. However, current protocols for regulated expression or presentation of Cre require multiple time-consuming steps. During efforts to disrupt four different RNA-dependent RNA polymerase genes in a single strain of the chestnut blight fungus Cryphonectria parasitica, we tested whether Cre could successfully excise loxP-flanked SMGs when provided in trans via anastomosis. Stable Cre-producing donor strains were constructed by transformation of wild-type C. parasitica strain EP155 with the Cre-coding domain under the control of a constitutive promoter. Excision of multiple loxP-flanked SMGs was efficiently achieved by simply pairing the Cre-donor strain and the loxP-flanked SMGs-transformed recipient strain and recovering mycelia from the margin of the recipient colony near the anastomosis zone. This method was shown to be as efficient as and much less time consuming than excision by transformation-mediated expression of Cre. It also allows unlimited recycling of loxP-flanked SMGs and the generation of disruption mutant strains that are free of any foreign gene. The successful application of this method to Metarhizium robertsii suggests potential use for optimizing reverse-genetics analysis in a broad range of filamentous fungi., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The idea that viruses can be used to control fungal diseases has been a driving force in mycovirus research since the earliest days. Viruses in the family Hypoviridae associated with reduced virulence (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica, have held a prominent place in this research. This has been due in part to the severity of the chestnut blight epidemics in North America and Europe and early reports of hypovirulence-mediated mitigation of disease in European forests and successful application for control of chestnut blight in chestnut orchards. A more recent contributing factor has been the development of a hypovirus/C. parasitica experimental system that has overcome many of the challenges associated with mycovirus research, stemming primarily from the exclusive intracellular lifestyle shared by all mycoviruses. This chapter will focus on hypovirus molecular biology with an emphasis on the development of the hypovirus/C. parasitica experimental system and its contributions to fundamental and practical advances in mycovirology and the broader understanding of virus-host interactions and fungal pathogenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Hypoviruses Cryphonectria hypovirus 1 (CHV-1)/EP713, CHV-1/Euro7, and CHV-1/EP721, which infect the chestnut blight fungus Cryphonectria parasitica, differ in their degrees of virulence attenuation (hypovirulence), symptom expression, and viral RNA accumulation, even though they share between 90% and 99% amino acid sequence identity. In this report we examine whether this variability is influenced by interactions with the C. parasitica Dicer gene dcl2-dependent RNA-silencing antiviral defense response. The mild symptoms exhibited by strains infected with CHV-1/Euro7 and CHV-1/EP721 relative to those with severe hypovirus CHV-1/EP713 did not correlate with a higher induction of the RNA-silencing pathway. Rather, dcl2 transcripts accumulated to a higher level (∼8-fold) following infection by CHV-1/EP713 than following infection by CHV-1/Euro7 (1.2-fold) or CHV-1/EP721 (1.4-fold). The differences in dcl2 transcript accumulation in response to CHV-1/EP713 and CHV-1/EP721 were unrelated to the suppressor of RNA silencing, p29, encoded by the two viruses. Moreover, the coding strand viral RNA levels increased by 33-, 32-, and 16-fold for CHV-1/EP713, CHV-1/Euro7, and CHV-1/EP721, respectively, in Δdcl2 mutant strains. This indicates that a very robust antiviral RNA-silencing response was induced against all three viruses, even though significant differences in the levels of dcl2 transcript accumulation were observed. Unexpectedly, the severe debilitation previously reported for CHV-1/EP713-infected Δdcl2 mutant strains, and observed here for the CHV-1/Euro7-infected Δdcl2 mutant strains, was not observed with infection by CHV-1/EP721. By constructing chimeric viruses containing portions of CHV-1/EP713 and CHV-1/EP721, it was possible to map the region that is associated with the severe debilitation of the Δdcl2 mutant hosts to a 4.1-kb coding domain located in the central part of the CHV-1/EP713 genome.

Genetic nonself recognition systems such as vegetative incompatibility operate in many filamentous fungi to regulate hyphal fusion between genetically dissimilar individuals and to restrict the spread of virulence-attenuating mycoviruses that have potential for biological control of pathogenic fungi. We report here the use of a comparative genomics approach to identify seven candidate polymorphic genes associated with four vegetative incompatibility (vic) loci of the chestnut blight fungus Cryphonectria parasitica. Disruption of candidate alleles in one of two strains that were heteroallelic at vic2, vic6, or vic7 resulted in enhanced virus transmission, but did not prevent barrage formation associated with mycelial incompatibility. Detailed characterization of the vic6 locus revealed the involvement of nonallelic interactions between two tightly linked genes in barrage formation, heterokaryon formation, and asymmetric, gene-specific influences on virus transmission. The combined results establish molecular identities of genes associated with four C. parasitica vic loci and provide insights into how these recognition factors interact to trigger incompatibility and restrict virus transmission.

Cyclophilins are peptidyl-prolyl cis-trans isomerases that are highly conserved throughout eukaryotes and are the cellular target of the immunosuppressive drug cyclosporin A (CsA). We cloned cyp1, a cyclophilin A-encoding gene in the phytopathogenic fungus Cryphonectria parasitica, and showed that this gene was downregulated following infection by a virulence-attenuating hypovirus. The function of cyp1 was further investigated by construction of a cyp1 deletion mutant. Although the wild-type C. parasitica strain EP155 was sensitive to CsA, the Δcyp1 strain was highly tolerant to CsA, indicating that CYP1 was the target of CsA. Deletion of cyp1 resulted in reduced virulence when inoculated to chestnut stems. Transcriptional analysis revealed that deletion of cyp1 also reduced transcript levels for genes encoding key components of the heterotrimeric guanosine triphosphate-binding protein signalling pathway that are essential for sensing environmental cues and are involved in C. parasitica development and virulence., (© 2010 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2010 BSPP AND BLACKWELL PUBLISHING LTD.)

In contrast to viruses of plants and animals, viruses of fungi, mycoviruses, uniformly lack an extracellular phase to their replication cycle. The persistent, intracellular nature of the mycovirus life cycle presents technical challenges to experimental design. However, these properties, coupled with the relative simplicity and evolutionary position of the fungal host, also provide opportunities for examining fundamental aspects of virus-host interactions from a perspective that is quite different from that pertaining for most plant and animal virus infections. This chapter presents support for this view by describing recent advances in the understanding of antiviral defense responses against one group of mycoviruses for which many of the technical experimental challenges have been overcome, the hypoviruses responsible for hypovirulence of the chestnut blight fungus Cryphonectria parasitica. The findings reveal new insights into the induction and suppression of RNA silencing as an antiviral defense response and an unexpected role for RNA silencing in viral RNA recombination., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Dicer gene dcl2, required for the RNA silencing antiviral defense response in the chestnut blight fungus Cryphonectria parasitica, is inducible upon mycovirus infection and promotes viral RNA recombination. We now report that the antiviral defense response requires only one of the four C. parasitica Argonaute-like protein genes, agl2. The agl2 gene is required for the virus-induced increase in dcl2 transcript accumulation. Agl2 and dcl2 transcripts accumulated to much higher levels in response to hairpin RNA production or infection by a mutant CHV1-EP713 hypovirus lacking the suppressor of RNA silencing p29 than to wild-type CHV1-EP713. Similar results were obtained for an agl2-promoter/EGFP-reporter construct, indicating that p29-mediated repression of agl2 transcript accumulation is promoter-dependent. Significantly, the agl2 deletion mutant exhibited stable maintenance of non-viral sequences in recombinant hypovirus RNA virus vectors and the absence of hypovirus-defective interfering (DI) RNA production. These results establish a key role for an Argonaute gene in the induction of an RNA silencing antiviral defense response and the promotion of viral RNA recombination. They also provide evidence for a mechanism by which a virus-encoded RNA silencing suppressor represses the transcriptional induction of an RNA silencing component.

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.

Defective interfering (DI) RNAs, helper virus-dependent deletion mutant RNAs derived from the parental viral genomic RNA during replication, have been described for most RNA virus taxonomic groups. We now report that DI RNA production in the chestnut blight fungus, Cryphonectria parasitica, persistently infected by virulence-attenuating positive sense RNA hypoviruses, depends on one of two host dicer genes, dcl-2. We further report that nonviral sequences that are rapidly deleted from recombinant hypovirus RNA virus vectors in wild-type and dicer gene dcl-1 deletion mutant strains are stably maintained and expressed in the Deltadcl-2 mutant strain. These results establish a requirement for dcl-2, the C. parasitica dicer gene responsible for antiviral defense and generation of virus-derived small interfering RNAs, in DI RNA production and recombinant virus vector RNA instability.

The prototypic hypovirus CHV1-EP713, responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica, encodes two papain-like proteases, p29 and p48. Protein p29 has been shown to be dispensable for hypovirus RNA replication and to act as a suppressor of RNA silencing. Here we describe a role for p48 in hypovirus RNA propagation. CHV1-EP713 infectious cDNA clones in which the p48 coding region was deleted, Delta p48, were unable to establish infection in C. parasitica when introduced as a DNA form by transformation or as a coding strand transcript by electroporation. However, the Delta p48 mutant virus RNA was rescued when p48 was provided in trans. Surprisingly, the Delta p48 mutant viruses retained replication competence in the apparent absence of p48 following transmission to wild-type C. parasitica and successive subculturing. The replicating Delta p48 mutant virus was reduced in RNA accumulation by 60% both in the absence and presence of p48 provided in trans and was transmitted through asexual spores (conidia) at a rate 3 to 8% of that for full-length CHV1-EP713. Complementary analysis of strains expressing p48 or containing the replicating Delta p48 mutant virus showed that like p29, p48 contributes to virus-mediated suppression of host pigmentation and conidiation, although to a lesser extent, and is dispensable for hypovirus-mediated hypovirulence. The combined results suggest that papain-like protease p48 plays an essential role in the initiation but not the maintenance of virus RNA propagation and also contributes to the regulation of viral RNA accumulation and vertical transmission.

Cryphonectria parasitica is the causal fungal agent responsible for the chestnut blight disease. We report the generation of 14,263 expressed sequence tags representing 6318 unisequences for the fungus. Functional annotation of these unisequences revealed different gene expression patterns for wild-type and hypovirus-infected cultures at the sporulation stage and allowed the reconstruction of key C. parasitica signal transduction pathways conserved in the Sorbidaryomycetes. A list of homologous genes implicated in pathogenicity, sporulation, and virus replication in other fungi were also identified. A large-scale gene comparison indicated that C. parasitica is most closely related to the plant pathogen Fusarium graminearum but more closely related to the non-pathogen Neurospora crassa than to the rice pathogen Magnaporthe grisea. This transcriptional information lays a new and solid ground for further investigation of both the biology of the fungus and interaction between a hypovirus and the host fungus.

The chestnut blight fungus, Cryphonectria parasitica, and associated virulence-attenuating hypoviruses have emerged as an important model system for studying molecular mechanisms underlying fungal-plant pathogenic interactions. As more gene sequence information becomes available as a result of C. parasitica express sequence tags (ESTs) and ongoing whole genome sequencing projects, the development of an efficient gene disruption system has become an urgent need for functional genomics studies of this important forestry pathogen. Here, we report the cloning of the C. parasitica gene cpku80 that encodes a key component of the nonhomologous end joining DNA repair pathway and the construction of a corresponding deletion mutant strain. The cpku80 mutant was indistinguishable from the parental wild-type strain EP155 in colony morphology, ability to support hypovirus replication, conidiation and virulence. As predicted, the Deltacpku80 strain did exhibit an increased sensitivity to the mutagen methyl methanesulfonate. A test with three selected genes resulted in a gene disruption efficiency of about 80% for the Deltacpku80 strain, a significant increase over the 2-5% levels of homologous recombination generally observed for the wild-type strain EP155. This efficient homologous recombination system provides a powerful tool for large-scale analysis of gene functions in C. 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.

The role of RNA silencing as an antiviral defense mechanism in fungi was examined by testing the effect of dicer gene disruptions on mycovirus infection of the chestnut blight fungus Cryphonectria parasitica. C. parasitica dicer-like genes dcl-1 and dcl-2 were cloned and shown to share a high level of predicted amino acid sequence identity with the corresponding dicer-like genes from Neurospora crassa [Ncdcl-1 (50.5%); Ncdcl-2 (38.0%)] and Magnaporthe oryzae [MDL-1 (45.6%); MDL-2 (38.0%)], respectively. Disruption of dcl-1 and dcl-2 resulted in no observable phenotypic changes relative to wild-type C. parasitica. Infection of Deltadcl-1 strains with hypovirus CHV1-EP713 or reovirus MyRV1-Cp9B21 resulted in phenotypic changes that were indistinguishable from that exhibited by wild-type strain C. parasitica EP155 infected with these same viruses. In stark contrast, the Deltadcl-2 and Deltadcl-1/Deltadcl-2 mutant strains were highly susceptible to mycovirus infection, with CHV1-EP713-infected mutant strains becoming severely debilitated. Increased viral RNA levels were observed in the Deltadcl-2 mutant strains for a hypovirus CHV1-EP713 mutant lacking the suppressor of RNA silencing p29 and for wild-type reovirus MyRV1-Cp9B21. Complementation of the Deltadcl-2 strain with the wild-type dcl-2 gene resulted in reversion to the wild-type response to virus infection. These results provide direct evidence that a fungal dicer-like gene functions to regulate virus infection.

Cryphonectria parasitica strain EP721 is infected with a strain of hypovirus CHV1, CHV1-EP721, and exhibits typical hypovirulence-associated traits such as reduced pigmentation and reduced asexual sporulation. However, the accumulation of the viral double-stranded RNA (dsRNA) in this hypovirus-infected C. parasitica strain is atypically low. We now report the complete nucleotide sequence and construction of a full-length infectious cDNA clone for hypovirus CHV1-EP721. The genome sequence of CHV1-EP721 was determined to be 12,724 bp in length and to share extensive homology with two other hypovirus strains, CHV1-Euro7 and CHV1-EP713, with an average of 99% and 90% identities at the nucleotide level and 99% and 92% identities at the amino acid level, respectively. CHV1-EP721 was successfully introduced into virus-free fungal host strain EP721(-v) by transfection with transcripts derived from a full-length viral cDNA. The transfected strain had a phenotype indistinguishable from that of EP721, and the accumulation of CHV1-EP721 dsRNA in the transfectant was lower than those transfected by CHV1-Euro7 and CHV1-EP713 transcripts. Through the construction of chimeric viruses by domain swapping using infectious cDNA clones of CHV1-EP721, CHV1-EP713, and CHV1-Euro7 hypoviruses, the determinant for the low level of viral dsRNA accumulation in CHV1-EP721 was mapped to the second of two CHV1-EP721 open reading frames (ORFs), ORF B. Further refined swapping of domains within ORF B identified a 2.5-kb coding region between p48 and the polymerase domain of CHV1-EP721 as being responsible for the low viral dsRNA accumulation. Evidence is also provided that low rates of hypovirus transmission through conidial spores correlates with low viral dsRNA accumulation.

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.

Infection of the chestnut blight fungus, Cryphonectria parasitica, by the prototypic hypovirus Cryphonectria hypovirus 1-EP713 (CHV1-EP713) or by the type member, Mycoreovirus 1-Cp9B21 (MyRV1-Cp9B21), of a novel genus (Mycoreovirus) of the family Reoviridae results in hypovirulence, but with a different spectrum of phenotypic changes. The former virus depresses pigmentation and conidiation dramatically, whilst the latter virus has little effect on these processes. This study showed that double infection by the two viruses resulted in a phenotype similar to that of CHV1-EP713 singly infected colonies, but with further decreased levels of host conidiation and vegetative growth and increased levels of MyRV1-Cp9B21 genomic dsRNA accumulation (twofold) and vertical transmission (sixfold). In contrast, CHV1-EP713 RNA accumulation was not altered by MyRV1-Cp9B21 infection. It was also found that the papain-like cysteine protease p29, encoded by CHV1-EP713 ORF A, contributes to the phenotypic alterations and transactivation of MyRV1-Cp9B21 replication and transmission. Chromosomally expressed p29 was able to increase MyRV1-Cp9B21 vertical transmission by more than twofold and genomic RNA accumulation by 80 %. Transactivation was abolished by Cys-->Gly mutations at p29 residues 70 and 72 located within the previously identified symptom-determinant domain required for suppression of host pigmentation and sporulation and p29-mediated in trans enhancement of homologous Deltap29 mutant virus RNA replication. Transactivation was not altered by Ser substitutions at the p29 protease catalytic residue Cys(162). These results indicated a link between p29-mediated enhancement of heterologous virus accumulation and transmission and p29-mediated host symptom expression. The role of p29 as a suppressor of RNA silencing is discussed.

Virulence-attenuating hypoviruses of the species Cryphonectria hypovirus 1 (CHV1) encode a papain-like protease, p29, that shares similarities with the potyvirus-encoded suppressor of RNA silencing HC-Pro. We now report that hypovirus CHV1-EP713-encoded p29 can suppress RNA silencing in the natural host, the chestnut blight fungus Cryphonectria parasitica. Hairpin RNA-triggered silencing was suppressed in C. parasitica strains expressing p29, and transformation of a transgenic green fluorescent protein (GFP)-silenced strain with p29 resulted in an increased number of transformants with elevated GFP expression levels. The CHV1-EP713 p29 protein was also shown to suppress both virus-induced and agroinfiltration-induced RNA silencing and systemic spread of silencing in GFP-expressing transgenic Nicotiana benthamiana line 16c plants. The demonstration that a mycovirus encodes a suppressor of RNA silencing provides circumstantial evidence that RNA silencing in fungi may serve as an antiviral defense mechanism. The observation that a phylogenetically conserved protein of related plant and fungal viruses functions as a suppressor of RNA silencing in both fungi and plants indicates a level of conservation of the mechanisms underlying RNA silencing in these two groups of organisms.

Whereas most mycoviruses lead 'secret lives', some reduce the ability of their fungal hosts to cause disease in plants. This property, known as hypovirulence, has attracted attention owing to the importance of fungal diseases in agriculture and the limited strategies that are available for the control of these diseases. Using one pathogen to control another is appealing, both intellectually and ecologically. The recent development of an infectious cDNA-based reverse genetics system for members of the Hypoviridae mycovirus family has enabled the analysis of basic aspects of this fascinating virus-fungus-plant interaction, including virus-host interactions, the mechanisms underlying fungal pathogenesis, fungal signalling pathways and the evolution of RNA silencing. Such systems also provide a means for engineering mycoviruses for enhanced biocontrol potential.

Using an established spotted cDNA microarray platform, the nature of changes in the transcriptional profiles of 2200 unique genes from the chestnut blight fungus Cryphonectria parasitica in response to the absence of either the Galpha subunit CPG-1 or the Gbeta subunit CPGB-1 has been explored. It is reported that 216 transcripts were altered in accumulation in the Deltacpg-1 strain and 163 in the Deltacpgb-1 strain, with a considerable overlap (100 genes) that were changed in both cases. Of note, these commonly altered transcripts were changed in the same direction in every instance, thus suggesting a considerable redundancy in pathway control or extensive crosstalk. To further knowledge of the potential impact on G-protein-signalling of infection by hypovirus CHV1-EP713, the accumulation of CPG-1 and CPGB-1 was also investigated by Western analysis. It was demonstrated that both signalling components were reduced in abundance to approximately 25 % of wild-type levels, while their transcripts were slightly elevated. Comparison of a list of genes with altered expression in the presence of CHV1-EP713 to the data obtained in the absence of either G-protein subunit showed that more than one-half of all the transcripts changed by hypovirus infection were also changed in at least one G-protein mutant strain, with one-third being changed in both. Significantly, 95 % of the co-changed genes were altered in the same direction. These data provide the first evidence for modulation of Gbeta protein levels as well as the Gbetagamma-signalling pathways by hypovirus infection, and support the hypothesis that modification of G-protein-signalling via both Galpha and Gbetagamma provides for a significant contribution to hypovirus-mediated phenotype.

The phenomenon of transmissible hypovirulence (virulence attenuation) associated with biological control of natural populations of the chestnut blight fungus Cryphonectria parasitica can be experimentally reproduced by infection with hypovirus cDNA clones (viral hypovirulence) or by mutation of mitochondrial DNA (mtDNA) in the absence of virus infection (mitochondrial hypovirulence). We now report the use of an established C. parasitica cDNA microarray to monitor nuclear transcriptional responses to an mtDNA mutation of C. parasitica strain EP155, designated EP155/mit2, which was previously shown to induce elevated alternative oxidase activity and hypovirulence (C. B. Monterio-Vitorello, J. A. Bell, D. W. Fulbright, and H. A. Bertrand, Proc. Natl. Acad. Sci. USA 92:5935-5939, 1995). Approximately 10% of the 2,200 genes represented on the microarray exhibited altered transcript accumulation as a result of the mit2 mtDNA mutation. While genes involved in mitochondrial function were clearly represented in the EP155/mit2-responsive gene list, direct parallels to the well-characterized Saccharomyces cerevisiae retrograde response to mitochondrial dysfunction were not observed. Remarkably, 47% of the genes that were differentially expressed following the infection of strain EP155 by the prototypic hypovirus CHV1-EP713 had similarly changed transcript accumulation in the virus-free EP155/mit2 mutant. These results establish a linkage between viral and mitochondrial hypovirulence and raise questions regarding the relationship between hypovirus infection and mitochondrial dysfunction. The combined set of transcriptional profile data provides a foundation for future studies on mitochondrion-to-nucleus communications in the context of hypovirus infection and senescence associated with mitochondrial dysfunction in filamentous fungi.

We report the use of a cDNA microarray to monitor global transcriptional responses of the chestnut blight fungus, Cryphonectria parasitica, to infection by mild and severe isolates of virulence-attenuating hypoviruses that share 87 to 93% and 90 to 98% identity at the nucleotide and amino acid levels, respectively. Infection by the mild hypovirus isolate CHV1-Euro7 resulted in differential expression of 166 of the ca. 2,200 genes represented on the microarray (90 upregulated and 76 downregulated). This is roughly half the number of genes scored as differentially expressed after infection by the severe isolate, CHV1-EP713 (295 genes; 132 upregulated and 163 downregulated). Comparison of the lists of genes responsive to infection by the two hypovirus isolates revealed 80 virus-common responsive genes. Infection by CHV1-EP713 also caused changes in gene transcript accumulation that were, in general, of greater magnitude than those observed with CHV1-Euro7 infections. Thus, the host transcriptional response to infection by severe hypovirus CHV1-EP713 appears to be considerably more dynamic than the response to infection by the mild isolate CHV1-Euro7. Real-time reverse transcription-PCR was performed on 39 different clones, with false-positive rates of 3 and 8% observed for the microarray-predicted list of genes responsive to CHV1-EP713 and CHV1-Euro7 infections, respectively. This analysis has allowed an initial assignment for ca. 2,200 unique C. parasitica-expressed genes as being unresponsive to hypovirus infection, selectively responsive to a specific hypovirus, or generally responsive to hypovirus infection.

Hypoviruses are a family of cytoplasmically replicating RNA viruses of the chestnut blight fungus Cryphonectria parasitica. Members of this mycovirus family persistently alter virulence (hypovirulence) and related fungal developmental processes, including asexual and sexual sporulation. In order to gain a better understanding of the molecular basis for these changes, we have developed a C. parasitica cDNA microarray to monitor global transcriptional responses to hypovirus infection. In this report, a spotted DNA microarray representing approximately 2,200 C. parasitica genes was used to monitor changes in the transcriptional profile after infection by the prototypic hypovirus CHV1-EP713. Altered transcript abundance was identified for 295 clones (13.4% of the 2,200 unique cDNAs) as a result of CHV1-EP713 infection-132 up-regulated and 163 down-regulated. In comparison, less than 20 specific C. parasitica genes were previously identified by Northern analysis and mRNA differential display as being responsive to hypovirus infection. A 93% validation rate was achieved between real-time reverse transcription-PCR results and microarray predictions. Differentially expressed genes represented a broad spectrum of biological functions, including stress responses, carbon metabolism, and transcriptional regulation. These findings are consistent with the view that infection by a 12.7-kbp hypovirus RNA results in a persistent reprogramming of a significant portion of the C. parasitica transcriptome. The potential impact of microarray studies on current and future efforts to establish links between hypovirus-mediated changes in cellular gene expression and phenotypes is discussed.

The prototypic hypovirus CHV1-EP713 attenuates virulence (hypovirulence) and alters several physiological processes of the chestnut blight fungus Cryphonectria parasitica. The papain-like protease, p29, and the highly basic protein, p40, derived, respectively, from the N-terminal and C-terminal portions of the CHV1-EP713-encoded open reading frame (ORF) A polyprotein, p69, both contribute to reduced pigmentation and sporulation. The p29 coding region was shown to suppress pigmentation and asexual sporulation in the absence of virus infection in transformed C. parasitica, whereas transformants containing the p40-coding domain exhibited a wild-type, untransformed phenotype. Deletion of either p29 or p40 from the viral genome also results in reduced accumulation of viral RNA. We now show that p29, but not p40, functions in trans to enhance genomic RNA accumulation and vertical transmission of p29 deletion mutant viruses. The frequency of virus transmission through conidia was found to decrease with reduced accumulation of viral genomic double-stranded RNA (dsRNA): from almost 100% for wild-type virus to approximately 50% for Deltap29, and 10 to 20% for Deltap69. When expressed from a chromosomally integrated cDNA copy, p29 elevated viral dsRNA accumulation and transmission for Deltap29 mutant virus to the level shown by wild-type virus. Increased viral RNA accumulation levels were also observed for a Deltap69 mutant lacking almost the entire ORF A sequence. Such enhancements were not detected in transgenic fungal colonies expressing p40. Mutation of p29 residues Cys(70) or Cys(72), strictly conserved in hypovirus p29 and potyvirus HC-Pro, resulted in the loss of both p29-mediated suppressive activity in virus-free transgenic C. parasitica and in trans enhancement of RNA accumulation and transmission, suggesting a linkage between these functional activities. These results suggest that p29 is an enhancer of viral dsRNA accumulation and vertical virus transmission through asexual spores.

Biolistic bombardment was used to successfully transform three phytopathogenic fungal species with an infectious cDNA clone of the prototypic hypovirus, CHV1-EP713, a genetic element responsible for the virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica. The fungal species included two strains each of C. parasitica and Valsa ceratosperma, as well as one strain of Phomopsis G-type (teleomorph Diaporthe Nitschke); all are members of the order Diaporthales but classified into three different genera. A subset of transformants for each of the fungal species contained CHV1-EP713 dsRNA derived from chromosomally integrated viral cDNA. As has been reported for CHV1-EP713 infection of the natural host C parasitica, biolistic introduction of CHV1-EP713 into the new fungal hosts V ceratosperma and Phomopsis G-type resulted in altered colony morphology and, more importantly, reduced virulence. These results suggest a potential for hypoviruses as biological control agents in plant-infecting fungal pathogens other than the chestnut blight fungus and closely related species. In addition, the particle delivery technique offers a convenient means of transmitting hypoviruses to potential host fungi that provides new avenues for fundamental mycovirus research and may have practical applications for conferring hypovirulence directly on infected plants in the field.

The papain-like protease p29, derived from the N-terminal portion of the hypovirus CHV1-EP713-encoded open reading frame (ORF) A polyprotein, p69, was previously shown to contribute to reduced pigmentation and sporulation by the infected host, the chestnut blight fungus Cryphonectria parasitica, while being dispensable for virus replication and attenuation of fungal virulence (hypovirulence). We now report that deletion of the C-terminal portion of p69, which encodes the highly basic protein p40, resulted in replication-competent mutant viruses that were, however, significantly reduced in RNA accumulation. While the Delta p40 mutants retained the ability to confer hypovirulence, Delta p40-infected fungal strains produced more asexual spores than strains infected with either wild-type CHV1-EP713 or a Delta p29 mutant virus. As observed for Delta p29-infected colonies, pigment production was significantly increased in Delta p40-infected fungal strains relative to that in CHV1-EP713-infected strains. Virus-mediated suppression of laccase production was not affected by p40 deletion. A gain-of-function analysis was employed to map the p40 symptom determinant to the N-terminal domain, encompassing p69 amino acid residues Thr(288) to Arg(312). Evidence that the gain of function was due to the encoded protein rather than the corresponding RNA sequence element was provided by introducing frameshift mutations on either side of the activity determinant domain. Moreover, restoration of symptoms correlated with increased accumulation of viral RNA. These results suggest that p40 indirectly contributes to virus-mediated suppression of fungal pigmentation and conidiation by providing an accessory function in hypovirus RNA amplification. A possible role for p40 in facilitating ORF B expression and the relationship between hypovirus RNA accumulation and symptom expression are discussed.

Hypoviruses persistently alter multiple phenotypic traits, stably modify gene expression, and attenuate virulence (hypovirulence) of their pathogenic fungal host, the chestnut blight fungus Cryphonectria parasitica. The pleiotropic nature of these changes is consistent with hypovirus-mediated perturbation of one or more cellular signal transduction pathways. We now report that two hypoviruses that differ in the severity of symptom expression differentially perturb specific cellular signaling pathways. The C. parasitica 13-1 gene, originally identified as a hypovirus-inducible and cyclic AMP (cAMP)-regulated gene, was used to design a promoter-GFP reporter construct with which to monitor perturbation of cAMP-mediated signaling. Virus-mediated modulation of calcium/calmodulin/inositol trisphosphate-dependent signaling was monitored by measuring transcript accumulation from the C. parasitica laccase gene, lac-1. Infection by the severe hypovirus strain CHV1-EP713 caused a substantial induction of 13-1 promoter activity and a reduction of total extracellular laccase enzymatic activity (LAC-1 and LAC-3). In contrast, 13-1 promoter activity and total laccase activity were only marginally altered upon infection with the mild hypovirus strain CHV1-Euro7. However, examination of lac-1-specific transcript accumulation under previously defined culture conditions revealed that both CHV1-EP713 and CHV1-Euro7 perturbed calcium/calmodulin/inositol trisphosphate-dependent signaling. CHV1-EP713/CHV1-Euro7 chimeric viruses were used to map viral determinants responsible for modulation of cAMP-dependent signaling to domains within the central portion of the second open reading frame.