Hypovirulence of chestnut blight fungus conferred by an infectious viral cDNA.

Strains of the chestnut blight fungus Cryphonectria parasitica that contain viral double-stranded RNAs often exhibit reduced virulence. Such hypovirulent strains act as biocontrol agents by virtue of their ability to convert virulent strains to hypovirulence after anastomosis. Transformation of virulent C. parasitica strains with a full-length complementary DNA copy of a hypovirulence-associated viral RNA conferred the complete hypovirulence phenotype. Cytoplasmic double-stranded RNA was resurrected from the chromosomally integrated complementary DNA copy and was able to convert compatible virulent strains to hypovirulence. These results establish viral double-stranded RNA as the casual agent of hypovirulence and demonstrate the feasibility of engineering hypovirulent fungal strains.

Attenuation of fungal virulence by synthetic infectious hypovirus transcripts.

Noninfectious, cytoplasmically transmissible viral double-stranded RNAs of the genus Hypovirus cause reduced virulence (hypovirulence) in the chestnut blight fungus Cryphonectria parasitica, providing the basis for virus-mediated biological control of a fungal disease. Synthetic transcripts corresponding to a full-length hypovirus RNA coding strand are infectious when introduced into fungal spheroplasts by electroporation. Hypovirus infections were readily established in Cryphonectria parasitica and in related fungal species not previously reported to harbor viruses. These results demonstrate the use of a synthetic mycovirus transcript to expand fungal host range, thereby broadening the potential application of virus-mediated hypovirulence to control fungal pathogenesis.

Targeted disruption of a fungal G-protein beta subunit gene results in increased vegetative growth but reduced virulence.

Targeted disruption of two G-protein alpha subunit genes in the chestnut blight fungus Cryphonectria parasitica revealed roles for the Gi alpha subunit CPG-1 in fungal reproduction, virulence, and vegetative growth. A second G alpha subunit, CPG-2, was found to be dispensable for these functions. We now report the cloning and targeted disruption of a C. parasitica G-protein beta subunit gene. The deduced amino acid sequence encoded by this gene, designated cpgb-1, was found to share 66.2, 65.9, and 66.7% amino acid identity with G beta homologues from human, Drosophila, and Dictyostelium origins, respectively, but only 39.7% identity with the Saccharomyces cerevisiae G beta homologue STE4 product. Low stringency Southern hybridization failed to detect any related G beta subunit genes in C. parasitica. Targeted disruption of cpgb-1 resulted in several of the changes previously reported to accompany disruption of the C. parasitica Gi alpha subunit gene cpg-1. These included very significant reductions in pigmentation, asexual sporulation, and virulence. In contrast to results obtained for Gi alpha gene disruption, the reduction in virulence resulting from the disruption of a G beta gene was accompanied by increased, rather than decreased, vegetative growth on synthetic medium. The relevance of these results to mechanisms of fungal virulence is considered.

Mutagenesis of putative acylation sites alters function, localization, and accumulation of a Gi alpha subunit of the chestnut blight fungus Cryphonectria parasitica.

Targeted disruption of cpg-1, a gene encoding the G protein Gi alpha subunit, CPG-1, in the chestnut blight fungus, Cryphonectria parasitica, results in reduced mycelial growth, reduced orange pigmentation, loss of virulence, loss of asexual sporulation, and female infertility. We report the development of a complementation system for cpg-1 null mutants and its use to evaluate the in vivo consequences of mutating conserved putative CPG-1 myristoylation (G2) and palmitoylation (C3) sites. Independent mutations of the two putative acylation sites differentially altered complex fungal biological processes, including virulence, and modified CPG-1 membrane association. Results of combined Northern (RNA) and Western (immunoblot) analysis also indicated a role for lipid modification in post-transcriptional regulation of CPG-1 accumulation.

Molecular analysis of the laccase gene from the chestnut blight fungus and selective suppression of its expression in an isogenic hypovirulent strain.

The gene encoding laccase in the chestnut blight fungus, Cryphonectria parasitica, has been cloned and characterized. The predicted C. parasitica laccase amino acid sequence (591 aa) was 57% identical to the Neurospora crassa laccase sequence and contained four potential copper-binding regions that are conserved in a number of copper-binding proteins. Treatment of a virulent C. parasitica strain with 3 microM cycloheximide resulted in a marked increase in laccase mRNA accumulation, whereas identical treatment of an isogenic strain that contained a hypovirulence-associated virus failed to significantly increase laccase mRNA levels. In contrast, the accumulation of mRNAs encoding beta-tubulin, actin, or glyceraldehyde-3-phosphate dehydrogenase was not appreciably altered by either the presence of a hypovirulence-associated virus or treatment with cycloheximide. These results provide evidence that the expression of a specific fungal gene encoding a known protein product is selectively modulated by a hypovirulence-associated virus.

Identification of a Cryphonectria parasitica laccase gene promoter element involved in cycloheximide-inducible, hypovirus-repressible transcriptional activation.

The gene lac-1, encoding the enzyme laccase, is the best characterized of a number of genes in the chestnut blight fungus, Cryphonectria parasitica, that are repressed by hypoviruses, a group of virulence-attenuating mycoviruses. lac-1 has also been shown to be transcriptionally activated by low concentrations of the translational inhibitor cycloheximide (CHX) and by the immunosuppressant cyclosporin A. We now report the identification of a CHX responsive element within the lac-1 promoter region. Gel-mobility shift analysis revealed a 111-bp fragment located 1.8kb upstream of the lac-1 transcriptional start point that exhibited protein binding activity. Insertion of this element within a basal lac-1 promoter sequence conferred CHX responsive transcriptional activation. Moreover, this activation was prevented by hypovirus infection. A 22-bp sequence with an imperfect dyad symmetry located within the 111-bp element was found to be essential for sequence-specific protein binding and, thus, represents a putative target for interactions between the lac-1 promoter and proteins that are involved in mediating CHX inducible activation of lac-1 transcription.

Molecular analysis and overexpression of the gene encoding endothiapepsin, an aspartic protease from Cryphonectria parasitica.

The gene, epn-1, encoding endothiapepsin (Epn), an aspartic protease (AspP) synthesized and secreted by the ascomycete fungus responsible for chestnut blight, Cryphonectria (Endothia) parasitica, was identified and characterized. Inspection of the nucleotide and deduced amino acid (aa) sequences revealed perfect agreement with the experimentally derived 330-aa sequence of mature Epn [Barkholt, Eur. J. Biochem. 167 (1987) 327-338] and an additional 89 aa of putative preprosequence. Of the nine fungal AspP characterized to date, Epn was found to be most closely related to aspergillopepsin and penicillopepsin (52% and 55% identity, respectively), proteases produced by the ascomycetes Aspergillus awamori and Penicillium janthinellum, and least related to proteases produced by the yeasts Candida albicans and Saccharomyces cerevisiae (27% and 26% identity, respectively). Epn production was found to be the same in isogenic virus-free and virus-containing strains, indicating that this AspP is not down-regulated by the presence of a hypovirulence-associated viral double-stranded RNA, as has been reported for several other secreted C. parasitica gene products. Strains containing multiple copies of epn-1 were obtained by transformation with a plasmid vector containing the cloned epn-1. One of these strains was shown to produce seven to ten times more Epn than the parental wild-type strain.

cDNA probes of individual genes of human rotavirus distinguish viral subgroups and serotypes.

The use of cDNA probes for detection of rotaviruses has been investigated using plasmids containing inserts specific for each of the eleven genes of human rotavirus strain Wa. In a dot-blot detection system in which radioactive DNA probes were hybridized to viral RNA extracted from cultivatable rotavirus strains, cDNAs of genes 7, 8, 10 and 11, were found to be the most reliable probes for detecting a range of rotavirus strains. Unexpectedly, rotaviruses could be distinguished with respect to subgroup and subtype specificities when cDNAs of genes 6 and 9, which encode the immunologically relevant proteins VP6 (group-specific antigen) and VP7 (type-specific antigen), were used as probe, even though the nucleic acid sequences of these genes are known to have a high degree of sequence homology.

Hypovirus Transmission to Ascospore Progeny by Field-Released Transgenic Hypovirulent Strains of Cryphonectria parasitica.

ABSTRACT Strains of the chestnut blight fungus, Cryphonectria parasitica, have been genetically engineered to contain an integrated full-length cDNA copy of the prototypic virulence-attenuating hypovirus CHV1-EP713. Unlike natural hypovirulent C. parasitica strains, these transgenic hypovirulent strains are able to transmit virus to ascospore progeny under laboratory conditions. This ability provides the potential to circumvent barriers to cytoplasmic virus transmission imposed by the fungal vegetative incompatibility system. During July 1994, transgenic hypovirulent strains were introduced into a Connecticut forest site (Biotechnology Permit 94-010-01). Subsequent analysis of the release site confirmed hypovirus transmission from transgenic hypovirulent strains to ascospore progeny under field conditions. Additionally, it was possible to recover transgenic hypovirulent strains from the test site as long as 2 years after the limited, single-season release. Evidence also was obtained for cytoplasmic transmission of transgenic cDNA-derived hypovirus RNA, including transmission to mycelia of a virulent C. parasitica canker after treatment with conidia of a transgenic strain. Finally, a transgenic hypovirulent strain was recovered from a superficial canker formed on an untreated chestnut tree. Genetic characteristics of the recovered strain suggested that the canker was initiated by an ascospore progeny derived from a cross involving an input transgenic hypovirulent strain. The durability of a molecular marker for field-released cDNA-derived hypovirus RNA is discussed.

Characterization of South African Cryphonectria cubensis Isolates Infected with a C. parasitica Hypovirus.

ABSTRACT Cryphonectria cubensis is the causal agent of a serious canker disease of Eucalyptus spp. in tropical and subtropical parts of the world. In this study, a South African C. cubensis isolate was transfected by electroporation with a synthetic RNA transcript corresponding to the full-length coding strand of the C. parasitica hypovirus (CHV1-EP713). Hypovirus infection resulted in pronounced morphological changes that included a striking increase in bright yellow-orange pigment production, a reduction in mycelial growth rate, and reduced sporulation. Greenhouse studies revealed that the virus-containing strain was significantly less virulent than the original virulent C. cubensis isolate. Although the hypovirus was not transmitted through conidia produced by infected C. cubensis, the virus was readily transmitted via hyphal anastomosis to C. cubensis isolates representing a broad range of vegetative compatibility groups. These results suggest that vegetative incompatibility may not pose a strong barrier against virus transmission in South African isolates of C. cubensis and that hypovirus-mediated biological control could provide opportunities to reduce the impact of Cryphonectria canker in South Africa.

Biological control of chestnut blight: an example of virus-mediated attenuation of fungal pathogenesis.

Environmental concerns have focused attention on natural forms of disease control as potentially safe and effective alternatives to chemical pesticides. This has led to increased efforts to develop control strategies that rely on natural predators and parasites or that involve genetically engineered microbial pest control agents. This review deals with a natural form of biological control in which the virulence of a fungal pathogen is attenuated by an endogenous viral RNA genetic element: the phenomenon of transmissible hypovirulence in the chestnut blight fungus, Cryphonectria parasitica. Recent progress in the molecular characterization of a hypovirulence-associated viral RNA has provided an emerging view of the genetic organization and basic expression strategy of this class of genetic elements. Several lines of evidence now suggest that specific hypovirulence-associated virus-encoded gene products selectively modulate the expression of subsets of fungal genes and the activity of specific regulatory pathways. The construction of an infectious cDNA clone of a hypovirulence-associated viral RNA represents a major advancement that provides exciting new opportunities for examining the molecular basis of transmissible hypovirulence and for engineering hypovirulent strains for improved biocontrol. These developments have significantly improved the prospects of using this system to identify molecular determinants of virulence and elucidate signal transduction pathways involved in pathogenic responses. In addition, novel approaches are now available for extending the application of transmissible hypovirulence for management of chestnut blight and possibly other fungal diseases.

Cyclophilin-dependent stimulation of transcription by cyclosporin A.

Exposure to cyclosporin A (CspA) increased laccase (lac-1) transcript accumulation in the chestnut blight fungus Cryphonectria parasitica. This response was suppressed by compounds that interfere with calcium-dependent signal transduction and by the presence of a virulence-attenuating mycovirus. CspA stimulated the accumulation of mRNA from a nonhomologous reporter fused to the lac-1 promoter, indicating that the increased transcript levels resulted from an increase in promoter activity. Based on the current model for the regulation of lac-1 transcription, these results suggest that CspA interferes with a negative regulatory pathway that normally constrains lac-1 promoter activity. Significantly, CspA did not stimulate lac-1 transcription in mutant strains deficient in CspA binding activity, directly demonstrating a requirement for the interaction of CspA and cyclophilin in the modulation of lac-1 transcription. Our results establish that CspA treatment can stimulate gene transcription and that cyclophilin is the cellular receptor that mediates this activity.

Induction of a Cryphonectria parasitica cellobiohydrolase I gene is suppressed by hypovirus infection and regulated by a GTP-binding-protein-linked signaling pathway involved in fungal pathogenesis.

Extracellular cellulase activity is readily induced when the chestnut blight fungus Cryphonectria parasitica is grown on cellulose substrate as the sole carbon source. However, an isogenic C. parasitica strain rendered hypovirulent due to hypovirus infection failed to secrete detectable cellulase activity when grown under parallel conditions. Efforts to identify C. parasitica cellulase-encoding genes resulted in the cloning of a cellobiohydrolase (exoglucanase, EC 3.2.1.91) gene designated chb-1. Northern blot analysis revealed an increase in cbh-1 transcript accumulation in a virus-free virulent C. parasitica strain concomitant with the induction of extracellular cellulase activity. In contrast, induction of cbh-1 transcript accumulation was suppressed in an isogenic hypovirus-infected strain. Significantly, virus-free C. parasitica strains rendered hypovirulent by transgenic cosuppression of a GTP-binding protein alpha subunit were also found to be deficient in the induction of cbh-1 transcript accumulation.

Altered transcriptional response to nutrient availability in hypovirus-infected chestnut blight fungus.

The gene lac-1, encoding the enzyme laccase, is one of several genes of the chestnut blight fungus, Cryphonectria parasitica, that are suppressed by virulence-attenuating mycoviruses of the hypovirus group. Two antagonistic regulatory pathways have been shown to govern the activity of the lac-1 promoter: a positive pathway that stimulates transcription and a negative pathway that represses transcription. We now report that these two regulatory pathways respond independently to specific changes in the nutritional environment. These newly defined conditions were used to confirm that a hypovirus suppresses the activity of the positive regulatory pathway, and to implicate calmodulin and calcineurin as components of the signal transduction cascades regulating lac-1 transcription. Significantly, lac-1 transcript accumulation was shown to be affected by amino acid availability. Further analysis revealed that transcriptional repression mediated by the negative regulatory pathway is relieved under conditions of amino acid deprivation. Thus, by blocking the positive pathway, hypovirus infection prevents increased lac-1 transcript accumulation in response to amino acid deficiency. These observations are consistent with the hypothesis that hypoviruses alter the transcriptional response of the host fungus to changes in nutrient availability.

Gene expression by a hypovirulence-associated virus of the chestnut blight fungus involves two papain-like protease activities. Essential residues and cleavage site requirements for p48 autoproteolysis.

Proteolytic processing plays a fundamental role in gene expression of a recently characterized viral-like double-stranded RNA associated with biological control of the chestnut blight fungus. Polypeptide p29, a papain-like protease, was shown to autocatalytically release itself from the NH2 terminus of the polyprotein specified by the first of two encoded open reading frames, ORF A (Choi, G. H., Shapira, R., and Nuss, D. L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1167-1171; Choi, G. H., Pawlyk, D. M., and Nuss, D. L. (1991) Virology 183, 747-752). The characterization of a second autocatalytic protease, p48, which is encoded by ORF B, is the subject of this report. Deletion analysis revealed that the catalytic domain resides within the carboxyl-terminal region, while site-specific mutational analysis identified Cys-341 and His-388 as residues essential for autoproteolysis. Autoproteolytic processing by p48 was also demonstrated when expressed in Escherichia coli and microsequence analysis of the recovered COOH-terminal cleavage product indicated that cleavage occurred between Gly-418 and Ala-419. The requirements for a functional cleavage site, including confirmation of the cleavage dipeptide, were defined by amino acid substitution analysis. Similarities between p29 and p48 suggest that the respective coding domains could have arisen as a result of a gene duplication event.

Regulation of expression of the wound tumor virus genome in persistently infected vector cells is related to change in translational activity of viral transcripts.

The interaction between a plant virus and its insect vector was studied at the molecular level by examining wound tumor virus (WTV) gene expression in cultured cells derived from its leafhopper vector. Infection of vector cells by WTV is noncytopathic and results in an acute phase (through day 5), followed by persistence beginning with the first cell passage. Viral-specific polypeptide synthesis and viral genome RNA accumulation increased to a maximum level during the first 5 days following inoculation and then decreased as infected cells were passaged (to 5 to 20% of the level observed during the acute phase by passages 10 to 15). In contrast, viral-specific mRNAs were present at approximately the same level in the acute phase and in the early stage (passage 10) of the persistent phase of infection. Although viral transcripts isolated at different times after inoculation exhibited identical electrophoretic migration patterns, they had different functional activities in cell-free translation systems. Transcripts isolated from persistently infected cells were inefficiently translated in vitro, reflecting the situation in infected cells. These results indicate that the decline in the level of viral polypeptide synthesis associated with the persistent phase of WTV infection is related to a change in the translational activity of viral transcripts.

A viral gene confers hypovirulence-associated traits to the chestnut blight fungus.

A viral double-stranded (ds)RNA associated with reduced virulence (hypovirulence) and the accompanying biological control of the chestnut blight fungus, Cryphonectria parasitica, was shown recently to contain two contiguous coding domains designated ORF A and ORF B. We report here that transformation of an isogenic virulent, dsRNA-free C. parasitica strain with a cDNA copy of ORF A conferred traits similar to those exhibited by the dsRNA-containing hypovirulent strain: characteristics included reduced pigmentation, reduced laccase accumulation and suppressed conidiation. However virulence was not reduced, indicating an apparent uncoupling of associated traits from hypovirulence. These results establish a direct cause and effect relationship between a viral dsRNA genetic element present in a hypovirulent C. parasitica strain and specific phenotypic traits. They demonstrate further that these traits are not the result of a general reaction of the fungus to the presence of the replicating viral RNA, but are caused by a specific viral coding domain.