Genome Sequence of the Chestnut Blight Fungus Cryphonectria parasitica EP155: A Fundamental Resource for an Archetypical Invasive Plant Pathogen.

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 (L50 = 5; N50 = 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.

Engineering super mycovirus donor strains of chestnut blight fungus by systematic disruption of multilocus vic genes.

Transmission of mycoviruses that attenuate virulence (hypovirulence) of pathogenic fungi is restricted by allorecognition systems operating in their fungal hosts. We report the use of systematic molecular gene disruption and classical genetics for engineering fungal hosts with superior virus transmission capabilities. Four of five diallelic virus-restricting allorecognition [vegetative incompatibility (vic)] loci were disrupted in the chestnut blight fungus Cryphonectria parasitica using an adapted Cre-loxP recombination system that allowed excision and recycling of selectable marker genes (SMGs). SMG-free, quadruple vic mutant strains representing both allelic backgrounds of the remaining vic locus were then produced through mating. In combination, these super donor strains were able to transmit hypoviruses to strains that were heteroallelic at one or all of the virus-restricting vic loci. These results demonstrate the feasibility of modulating allorecognition to engineer pathogenic fungi for more efficient transmission of virulence-attenuating mycoviruses and enhanced biological control potential.

Balancing selection at nonself recognition loci in the chestnut blight fungus, Cryphonectria parasitica, demonstrated by trans-species polymorphisms, positive selection, and even allele frequencies.

Balancing selection has been inferred in diverse organisms for nonself recognition genes, including those involved in immunity, mating compatibility, and vegetative incompatibility. Although selective forces maintaining polymorphisms are known for genes involved in immunity and mating, mechanisms of balancing selection for vegetative incompatibility genes in fungi are being debated. We hypothesized that allorecognition and its consequent inhibition of virus transmission contribute to the maintenance of polymorphisms in vegetative incompatibility loci (vic) in the chestnut blight fungus, Cryphonectria parasitica. Balancing selection was demonstrated at two loci, vic2 and vic6, by trans-species polymorphisms in C. parasitica, C. radicalis, and C. japonica and signatures of positive selection in gene sequences. In addition, more than half (31 of 54) of allele frequency estimates at six vic loci in nine field populations of C. parasitica from Asia and the eastern US were not significantly different from 0.5, as expected at equilibrium for two alleles per locus under balancing selection. At three vic loci, deviations from 0.5 were predicted based on the effects of heteroallelism on virus transmission. Twenty-five of 27 allele frequency estimates were greater than or equal to 0.5 for the allele that confers significantly stronger inhibition of virus transmission at three loci with asymmetric transmission. These results are consistent with the allorecognition hypothesis that vegetative incompatibility genes are under selection because of their role in reducing infection by viruses.

Phytoplasmal infection derails genetically preprogrammed meristem fate and alters plant architecture.

In the life cycle of higher plants, it is the fate of meristem cells that determines the pattern of growth and development, and therefore plant morphotype and fertility. Floral transition, the turning point from vegetative growth to reproductive development, is achieved via genetically programmed sequential changes in meristem fate from vegetative to inflorescence, and to floral, leading to flower formation and eventual seed production. The transition is rarely reversible once initiated. In this communication, we report that a bacterial infection can derail the genetically programmed fate of meristem cells, thereby drastically altering the growth pattern of the host plant. We identified four characteristic symptoms in tomato plants infected with a cell wall-less bacterium, phytoplasma. The symptoms are a manifestation of the pathogen-induced alterations of growth pattern, whereas each symptom corresponds to a distinct phase in the derailment of shoot apical meristem fate. The phases include premature floral meristem termination, suppressed floral meristem initiation, delayed conversion of vegetative meristem to inflorescence meristem, and repetitive initiation and outgrowth of lateral vegetative meristems. We further found that the pathogen-induced alterations of growth pattern were correlated with transcriptional reprogramming of key meristem switching genes. Our findings open an avenue toward understanding pathological alterations in patterns of plant growth and development, thus aiding identification of molecular targets for disease control and symptom alleviation. The findings also provide insights for understanding stem cell pluripotency and raise a tantalizing possibility for using phytoplasma as a tool to dissect the course of normal plant development and to modify plant morphogenesis by manipulating meristem fate.

Mutagenesis of the catalytic and cleavage site residues of the hypovirus papain-like proteases p29 and p48 reveals alternative processing and contributions to optimal viral RNA accumulation.

The positive-stranded RNA genome of the prototypic virulence-attenuating hypovirus CHV-1/EP713 contains two open reading frames (ORF), each encoding an autocatalytic papain-like leader protease. Protease p29, derived from the N-terminal portion of ORF A, functions as a suppressor of RNA silencing, while protease p48, derived from the N-terminal portion of ORF B, is required for viral RNA replication. The catalytic and cleavage site residues required for autoproteolytic processing have been functionally mapped in vitro for both proteases but not confirmed in the infected fungal host. We report here the mutagenesis of the CHV-1/EP713 infectious cDNA clone to define the requirements for p29 and p48 cleavage and the role of autoproteolysis in the context of hypovirus replication. Mutation of the catalytic cysteine and histidine residues for either p29 or p48 was tolerated but reduced viral RNA accumulation to ca. 20 to 50% of the wild-type level. Mutation of the p29 catalytic residues caused an accumulation of unprocessed ORF A product p69. Surprisingly, the release of p48 from the ORF B-encoded polyprotein was not prevented by mutation of the p48 catalytic and cleavage site residues and was independent of p29. The results show that, while dispensable for hypovirus replication, the autocatalytic processing of the leader proteases p29 and p48 contributes to optimal virus RNA accumulation. The role of the predicted catalytic residues in autoproteolytic processing of p29 was confirmed in the infected host, while p48 was found to also undergo alternative processing independent of the encoded papain-like protease activities. Importance: Hypoviruses are positive-strand RNA mycoviruses that attenuate virulence of their pathogenic fungal hosts. The prototypic hypovirus CHV-1/EP713, which infects the chestnut bight fungus Cryphonetria parasitica, encodes two papain-like autocatalytic leader proteases, p29 and p48, that also have important functions in suppressing the RNA silencing antiviral defense response and in viral RNA replication, respectively. The mutational analyses of the CHV-1/EP713 infectious cDNA clone, reported here, define the requirements for p29 and p48 cleavage and the functional importance of autoproteolysis in the context of hypovirus replication and exposed an alternative p48 processing pathway independent of the encoded papain-like protease activities. These findings provide additional insights into hypovirus gene expression, replication, and evolution and inform ongoing efforts to engineer hypoviruses for interrogating and modulating fungal virulence.

Multilocus PCR Assays Elucidate Vegetative Incompatibility Gene Profiles of Cryphonectria parasitica in the United States.

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.

Simple and efficient recycling of fungal selectable marker genes with the Cre-loxP recombination system via anastomosis.

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.

Variations in hypovirus interactions with the fungal-host RNA-silencing antiviral-defense response.

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.

A single Argonaute gene is required for induction of RNA silencing antiviral defense and promotes viral RNA recombination.

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.

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.

Gene identification in black cohosh (Actaea racemosa L.): expressed sequence tag profiling and genetic screening yields candidate genes for production of bioactive secondary metabolites.

Black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa, Nutt., Ranunculaceae) is a popular herb used for relieving menopausal discomforts. A variety of secondary metabolites, including triterpenoids, phenolic dimers, and serotonin derivatives have been associated with its biological activity, but the genes and metabolic pathways as well as the tissue distribution of their production in this plant are unknown. A gene discovery effort was initiated in A. racemosa by partial sequencing of cDNA libraries constructed from young leaf, rhizome, and root tissues. In total, 2,066 expressed sequence tags (ESTs) were assembled into 1,590 unique genes (unigenes). Most of the unigenes were predicted to encode primary metabolism genes, but about 70 were identified as putative secondary metabolism genes. Several of these candidates were analyzed further and full-length cDNA and genomic sequences for a putative 2,3 oxidosqualene cyclase (CAS1) and two BAHD-type acyltransferases (ACT1 and HCT1) were obtained. Homology-based PCR screening for the central gene in plant serotonin biosynthesis, tryptophan decarboxylase (TDC), identified two TDC-related sequences in A. racemosa. CAS1, ACT1, and HCT1 were expressed in most plant tissues, whereas expression of TDC genes was detected only sporadically in immature flower heads and some very young leaf tissues. The cDNA libraries described and assorted genes identified provide initial insight into gene content and diversity in black cohosh, and provide tools and resources for detailed investigations of secondary metabolite genes and enzymes in this important medicinal plant.

A host dicer is required for defective viral RNA production and recombinant virus vector RNA instability for a positive sense RNA virus.

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.

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.

Hypovirus papain-like protease p48 is required for initiation but not for maintenance of virus RNA propagation in the chestnut blight fungus Cryphonectria parasitica.

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.

Characterization of hypovirus-derived small RNAs generated in the chestnut blight fungus by an inducible DCL-2-dependent pathway.

The disruption of one of two dicer genes, dcl-2, of the chestnut blight fungus Cryphonectria parasitica was recently shown to increase susceptibility to mycovirus infection (G. C. Segers, X. Zhang, F. Deng, Q. Sun, and D. L. Nuss, Proc. Natl. Acad. Sci. USA 104:12902-12906, 2007). We now report the accumulation of virus-derived small RNAs (vsRNAs) in hypovirus CHV1-EP713-infected wild-type and dicer gene dcl-1 mutant C. parasitica strains but not in hypovirus-infected dcl-2 mutant and dcl-1 dcl-2 double-mutant strains. The CHV1-EP713 vsRNAs were produced from both the positive and negative viral RNA strands at a ratio of 3:2 in a nonrandom distribution along the viral genome. We also show that C. parasitica responds to hypovirus and mycoreovirus infections with a significant increase (12- to 20-fold) in dcl-2 expression while the expression of dcl-1 is increased only modestly (2-fold). The expression of dcl-2 is further increased ( approximately 35-fold) following infection with a hypovirus CHV1-EP713 mutant that lacks the p29 suppressor of RNA silencing. The combined results demonstrate the biogenesis of mycovirus-derived small RNAs in a fungal host through the action of a specific dicer gene, dcl-2. They also reveal that dcl-2 expression is significantly induced in response to mycovirus infection by a mechanism that appears to be repressed by the hypovirus-encoded p29 suppressor of RNA silencing.

Enhanced hypovirus transmission by engineered super donor strains of the chestnut blight fungus, Cryphonectria parasitica, into a natural population of strains exhibiting diverse vegetative compatibility genotypes.

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.

Large-scale expressed sequence tag analysis for the chestnut blight fungus Cryphonectria parasitica.

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.

Hypovirulence: mycoviruses at the fungal-plant interface.

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.

Extending chestnut blight hypovirus host range within diaporthales by biolistic delivery of viral cDNA.

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.

Characterizing the roles of Cryphonectria parasitica RNA-dependent RNA polymerase-like genes in antiviral defense, viral recombination and transposon transcript accumulation.

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.