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.

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.

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.

Variant dsRNAs associated with transmission-defective isolates of wound tumor virus represent terminally conserved remnants of genome segments.

Variant double-stranded RNAs are often associated with the genome of transmission-defective isolates of wound tumor virus. These RNAs are replicated and packaged into virus particles in systemically infected plants and are transcribed in vitro by the virion-associated transcriptase. Direct physical evidence that the variant RNAs are remnants of particular WTV genome segments was provided by molecular hybridization studies. Subsequently, ribonuclease T1 digestion products of 3'-end-labeled genome and remnant RNAs were analyzed by one- and two-dimensional electrophoretic techniques. One-dimensional partial and complete digestion patterns were indistinguishable, indicating that the guanosine positions relative to the 3' terminus of the corresponding strands of a particular genome segment and its remnant RNA are the same for at least 40 nucleotides from each end. Fingerprints of the 3' terminal ribonuclease T1-resistant fragments were identical, showing that the nucleotide composition of the 3' terminal ends of the corresponding strands of a particular genome segment and its remnant RNA are also identical. These results indicate that variant RNAs associated with transmission-defective WTV isolates are formed by deletion of an internal portion (as much as 85%) of genomic RNA segments yielding terminally conserved genomic remnants that are functional with respect to transcription, replication, and packaging.

Expression of wound tumor virus gene products in vivo and in vitro.

Infection of Agallia constricta vector cell monolayers with wound tumor virus results in the synthesis of 12 virus-specific polypeptides. Confirmation that these polypeptides are virus encoded rather than virus induced was obtained by cell-free translation of in vitro synthesized viral mRNA. In addition, transcription by purified wound tumor virus particles was coupled with translation of the resulting transcripts in a wheat embryo cell-free extract. Six previously described structural polypeptides, one presumptive structural polypeptide, and five previously unidentified nonstructural polypeptides were synthesized in infected vector cell monolayers, in cell-free extracts directed by in vitro synthesized viral mRNA, and in the homologous plant cell-free system, in which viral transcription was coupled with translation. Pulse-chase experiments revealed no evidence of precursor-product relationships for the wound tumor virus-specific polypeptides.

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.

Distinct roles for two G protein alpha subunits in fungal virulence, morphology, and reproduction revealed by targeted gene disruption.

Reduced accumulation of the GTP-binding protein G(i)alpha subunit CPG-1, due either to hypovirus infection or transgenic cosuppression, correlates with virulence attenuation of the chestnut blight fungus, Cryphonectria parasitica. The role of G protein-mediated signal transduction in fungal virulence was further examined by targeted disruption of the gene cpg-1, encoding CPG-1, and a second Galpha gene, cpg-2, encoding the subunit CPG-2. Disruption of cpg-1 resulted in a set of phenotypic changes similar to, but more severe than, those associated with hypovirus infection. Changes included a marked reduction in fungal growth rate and loss of virulence, asexual sporulation, female fertility, and transcriptional induction of the gene lac-1, encoding the enzyme laccase. In contrast, cpg-2 disruption resulted in only slight reductions in growth rate and asexual sporulation and no significant reduction in virulence, female fertility, or lac-1 mRNA inducibility. These results provide definitive confirmation of previous correlative evidence that suggested a requirement of CPG-1-linked signaling for a number of fungal processes, including virulence and reproduction, while demonstrating that a second Galpha, CPG-2, is dispensable for these processes. They also significantly strengthen support for the apparent linkage between hypovirus-mediated disruption of G protein signal transduction and attenuation of fungal virulence.

Methyl group analysis of virion-associated high-molecular-weight RNA synthesized in vitro by purified vaccinia virus.

The methylation pattern of virion-associated high-molecular-weight RNA synthesized in vitro by purified vaccinia virus has been determined. Analysis of purified high-molecular-weight RNA synthesized with S-[methyl-3H]-adenosylmethionine and alpha[32P]UTP as precursors gave the following results. (i) Eessentially all molecules contained blocked and methylated structures of the type m7G(5')ppp(5')Gm and m7G(5')ppp(5')Am. (ii) There was no detectable methylation at internal sites. (iii) Under several different conditions of synthesis, the ratio of molecules containing m7G(5')ppp(5')Gm to those containing m7G(5')ppp(5')Am was imilar for both the virion-associated high-molecular-weight RNA and the virion-released 8-12S mRNA.

Resistance to inhibitors of mammalian cell protein synthesis induced by preincubation in hypertonic growth medium.

Incubation of L-929 cells in growth medium containing excess NaCl induces cellular protein synthesis to become resistant to the inhibitory action of growth medium made hypertonic by the addition of Na-acetate, KCl, choline chloride, D-alanine, or sucrose. Hypertonic preincubation also induces resistance to the inhibitory action of the drugs dimethyl sulfoxide, ethanol, and L-1-tosylamido-2-phenylethyl chloromethyl ketone, but not to the action of NaF or the inhibitors of polypeptide chain elongation, emetine and cycloheximide. Induction is independent of the solute used to make the induction medium hypertonic and is not mediated by an elevated intracellular Na+ concentration. In a separate series of experiments, it was determined that the formation of functional methionyl-tRNAf.40 S ribosomal complexes is not impaired by exposure of uninduced cells to hypertonic growth medium. The combined results are evaluated with respect to the mechanism of action of hypertonic growth medium in the inhibition of polypeptide chain initiation and the adaptive mechanisms that animal cells have evolved to deal with alterations in their extracellular environments.