dsRNA Elements Research Articles

P.307 Search for sporadic acute viral hepatitis cases with type-4 hepatitis E virus in western India

A full-length cDNA clone was developed from a 5.3 kb double-stranded (ds) RNA element present in strain CKP of the plant pathogenic fungus Chalara elegans. The complete nucleotide sequence was 5310 bp in length and sequence analysis revealed that it contained three large putative open reading frames (ORFs). ORF1 was initiated at nucleotide position 329 and encoded a putative coat protein, which shared some homology (35–45% amino acid identity) to other dsRNAs in the family Totiviridae. Both ORF2 and ORF3 were initiated at nucleotide positions 2619 and 4071, respectively, and encoded a putative RNA-dependent RNA polymerase (RdRp). Sequence comparison using deduced amino acid sequences of both ORF2 and ORF3 revealed that all RdRp conserved motifs shared highest homology (41% identity) to that of SsRNA1 of Totiviridae. This dsRNA in C. elegans was designated Chalara elegans RNA Virus 1 (CeRV1). During the development of the full-length cDNA clone of CeRV1, several partial cDNA clones from an additional dsRNA fragment in strain CKP were obtained, which when aligned with each other, produced one linear fragment which was 2336 bp long. Northern blot and sequence analysis of this second clone showed it differed in sequence composition from CeRV1. This dsRNA in C. elegans was designated Chalara elegans RNA Virus 2 (CeRV2). Sequence analysis of CeRV2 showed it contained all conserved motifs and shared some homology (45% amino acid identity) to RdRp regions of Totiviridae. The nucleotide and amino acid sequences of the conserved motifs of the RdRp regions between CeRV1 and CeRV2 showed an identity of 56% and 50%, respectively. These findings suggest that co-infection of two distinct totivirus-like dsRNAs (CeRV1 and CeRV2) in C. elegans, a first report in this fungus. Transmission electron microscopy of strain CKP of C. elegans revealed the presence of putative virus-like particles in the cytoplasm, which were similar both in shape and size to viruses in the Totiviridae.

Double-stranded RNA mycoviruses inFusarium culmorumandFusarium graminearumisolates

The presence of double-stranded RNA (dsRNA) elements in Fusarium culmorum and F. graminearum isolates of different origin, and the possible effect of these elements on pathogenicity and toxin production of the isolates were examined. Altogether 40 F. culmorum and 38 F. graminearum isolates were involved in this study, together with F. cerealis and F. pseudograminearum strains. Double-stranded RNA elements indicative of mycovirus infection were detected for the first time in 5 F. culmorum isolates. The isolates originated from the United States, The Netherlands, Australia and Israel. The dsRNA nature of the fragments was proved by RNase, DNase and S1 nuclease treatments. The sizes of the dsRNA elements varied between 0.6–3.95 kbp. A dsRNA element of 3.0 kbp in size was also detected in a F. graminearum isolate came from South Africa. None of the Central-European isolates examined were found to carry such elements. We also examined the mycotoxin producing abilities and pathogenicity of the dsRNA infected isolates. Four of the mycovirus infected F. culmorum isolates produced deoxynivalenol and zearalenone (chemotype I), while one of the isolates came from the USA produced nivalenol and fusarenone X (chemotype II). The dsRNA-containing F. graminearum isolate produced deoxynivalenol and zearalenone. In general, dsRNA-containing isolates were found to be as pathogenic to two wheat cultivars as dsRNA free isolates. RAPD analysis and sequence analysis of a putative reductase gene fragment indicated that dsRNA-containing isolates are scattered among dsRNA free isolates. Further work is in progress in our laboratory to characterize these dsRNA elements by sequence analysis.

Low TRBP Levels Support an Innate Human Immunodeficiency Virus Type 1 Resistance in Astrocytes by Enhancing the PKR Antiviral Response

Acute human immunodeficiency virus type 1 (HIV-1) replication in astrocytes produces minimal new virus particles due, in part, to inefficient translation of viral structural proteins despite high levels of cytoplasmic viral mRNA. We found that a highly reactive double-stranded (ds) RNA-binding protein kinase (PKR) response in astrocytes underlies this inefficient translation of HIV-1 mRNA. The dsRNA elements made during acute replication of HIV-1 in astrocytes triggers PKR activation and the specific inhibition of HIV-1 protein translation. The heightened PKR response results from relatively low levels of the cellular antagonist of PKR, the TAR RNA binding protein (TRBP). Efficient HIV-1 production was restored in astrocytes by inhibiting the innate PKR response to HIV-1 dsRNA with dominant negative PKR mutants, or PKR knockdown by siRNA gene silencing. Increasing the expression of TRBP in astrocytes restored acute virus production to levels comparable to those observed in permissive cells. Therefore, the robust innate PKR antiviral response in astrocytes results from relatively low levels of TRBP expression and contributes to their restricted infection. Our findings highlight TRBP as a novel cellular target for therapeutic interventions to block productive HIV-1 replication in cells that are fully permissive for HIV-1 infection.

Co-infection by two distinct totivirus-like double-stranded RNA elements in Chalara elegans ( Thielaviopsis basicola)

A full-length cDNA clone was developed from a 5.3 kb double-stranded (ds) RNA element present in strain CKP of the plant pathogenic fungus Chalara elegans. The complete nucleotide sequence was 5310 bp in length and sequence analysis revealed that it contained three large putative open reading frames (ORFs). ORF1 was initiated at nucleotide position 329 and encoded a putative coat protein, which shared some homology (35–45% amino acid identity) to other dsRNAs in the family Totiviridae. Both ORF2 and ORF3 were initiated at nucleotide positions 2619 and 4071, respectively, and encoded a putative RNA-dependent RNA polymerase (RdRp). Sequence comparison using deduced amino acid sequences of both ORF2 and ORF3 revealed that all RdRp conserved motifs shared highest homology (41% identity) to that of SsRNA1 of Totiviridae. This dsRNA in C. elegans was designated Chalara elegans RNA Virus 1 (CeRV1). During the development of the full-length cDNA clone of CeRV1, several partial cDNA clones from an additional dsRNA fragment in strain CKP were obtained, which when aligned with each other, produced one linear fragment which was 2336 bp long. Northern blot and sequence analysis of this second clone showed it differed in sequence composition from CeRV1. This dsRNA in C. elegans was designated Chalara elegans RNA Virus 2 (CeRV2). Sequence analysis of CeRV2 showed it contained all conserved motifs and shared some homology (45% amino acid identity) to RdRp regions of Totiviridae. The nucleotide and amino acid sequences of the conserved motifs of the RdRp regions between CeRV1 and CeRV2 showed an identity of 56% and 50%, respectively. These findings suggest that co-infection of two distinct totivirus-like dsRNAs (CeRV1 and CeRV2) in C. elegans, a first report in this fungus. Transmission electron microscopy of strain CKP of C. elegans revealed the presence of putative virus-like particles in the cytoplasm, which were similar both in shape and size to viruses in the Totiviridae.

A Satellite RNA of Ophiostoma novo-ulmi Mitovirus 3a in Hypovirulent Isolates of Sclerotinia homoeocarpa

ABSTRACT Two genetically distinct double-stranded RNA (dsRNA) elements were identified in hypovirulent isolates of Sclerotinia homoeocarpa, the causal agent of dollar spot of turfgrass. The large dsRNA (L-dsRNA) was consistently present in all hypovirulent isolates, whereas the small dsRNA (S-dsRNA) was found only in some hypovirulent isolates. Virulence comparisons revealed that there was no significant difference between isolates containing one or both dsRNAs. Therefore, the L-dsRNA appears to be the genetic determinant of hypovirulence, while the S-dsRNA is not essential for hypovirulence in S. homoeocarpa. The L-dsRNA in hypovirulent isolate Sh12B of S. homoeocarpa was previously characterized as a fungal mitochondrial virus and designated Ophiostoma novo-ulmi mitovirus 3a-Sh12B (OnuMV3a-Sh12B) because it was conspecific with O. novo-ulmi mitovirus 3a-Ld from O. novo-ulmi, the causal agent of Dutch elm disease. In the present study, the nucleotide sequences of the S-dsRNAs (738 to 767 nucleotides) in hypovirulent isolates Sh12B, Sh279B, and Sh286B were determined. Nucleotide sequence analysis indicated that the S-dsRNA was not derived from the OnuMV3a dsRNA and it could not encode an RNA-dependent RNA polymerase. These results are consistent with biological data that the S-dsRNA was always associated with the L-dsRNA and was never found independently. Therefore, the S-dsRNA can be regarded as a satellite RNA of OnuMV3a in S. homoeocarpa. Northern blotting analysis indicated that nucleic acid extracts from isolate Sh12B of S. homoeocarpa contained more single (+) stranded RNA than dsRNA for this satellite RNA. The 5'- and 3'-terminal sequences of the positive strand of the S-dsRNA each could be folded into a stem-loop structure and the terminal 21 nucleotides were complementary to each other, potentially forming a panhandle structure.

Diversity and vertical transmission of double-stranded RNA elements in root rot pathogens of trees, Helicobasidium mompa and Rosellinia necatrix

The diversity and vertical transmission of double-stranded (ds) RNA in Helicobasidium mompa and Rosellinia necatrix was examined by electrophoresis and Northern hybridization. These two fungi share the similar niche as root rot pathogens of trees in forests and orchards, and had diverse dsRNAs. The detection frequency of dsRNA in both fungi was different; in H. mompa, 68.4% (132 out of 193 MCGs; mycelial compatibility groups) had dsRNA, whereas 20.9% (53 out of 254 MCGs) in R. necatrix. dsRNA banding patterns and Northern blot analyses revealed the presence of various dsRNA elements in both fungi. Hyphal tip isolation was mostly unsuccessful to remove dsRNA with some exceptions. Sexual reproduction functioned to remove dsRNA in both fungi since dsRNA was not detected from single sexual spore cultures. Possible explanations for the difference in the detection frequency of dsRNA are discussed in terms of the differences in their sexual reproduction and other factors.

Molecular epidemiological analysis of Cryptosporidium isolates from humans and animals by using a heteroduplex mobility assay and nucleic acid sequencing based on a small double-stranded RNA element.

Two extrachromosomal double-stranded RNA (dsRNA) elements occur in Cryptosporidium parvum. A heteroduplex mobility assay (HMA) was developed for the rapid characterization of sequence diversity in a 173-bp fragment of the small dsRNA element of Cryptosporidium with either a natural sequence from Cryptosporidium meleagridis or a synthetic sequence as reference DNA. The 173-bp fragment was generated from 265 samples of whole feces (242 from humans and 18 from livestock with C. parvum genotype 1 or 2, 4 from humans with Cryptosporidium felis, and 1 from a human with C. meleagridis). The HMA method identified 21 patterns in C. parvum (8 in genotype 1, 12 in genotype 2, and a type common to both genotypes), 4 patterns in C. felis, and 1 pattern in C. meleagridis. All patterns were confirmed as distinct by DNA sequencing. For genotype 1, a single HMA type was found in 89% of samples: 64 of 65 cases from three waterborne outbreaks, all 16 cases from eight intrafamilial outbreaks, and 17 of 28 sporadic cases. Among the remaining 11 sporadic cases due to genotype 1, seven other HMA types were detected. For genotype 2, a single HMA type was found in 72% of samples: 36 of 43 cases from three waterborne outbreaks, 11 of 15 cases from seven intrafamilial outbreaks, 44 of 75 sporadic cases, and all 18 samples from livestock. Within the intrafamilial outbreaks, two other HMA types were identified: the same HMA type was detected in samples from cases within the same outbreak. Among the sporadic cases due to genotype 2, 10 additional HMA types were detected.

Antibodies raised against Bcvir15, an extrachromosomal double-stranded RNA-encoded protein from Babesia canis, inhibit the in vitro growth of the parasite.

As part of a search for homologous members of the Plasmodium falciparum Pf60 multigene family in the intraerythrocytic protozoan parasite Babesia canis, we report here the characterization of a cDNA of 1,115 bp, which was designated Bcvir for its potential viral origin. The Bcvir cDNA contained two overlapping open reading frames (ORFs) (ORF1 from nucleotide [nt] 61 to 486 and ORF2 from nt 417 to 919), where Bcvir15, the deduced ORF1 peptide (M(1) to I(141)), is the main expressed product. The Bcvir cDNA was derived from an extrachromosomal dsRNA element of 1.2 kbp that was always found associated with a double-stranded RNA (dsRNA) of 2.8 kbp by hybridization, and no copy of this cDNA sequence was found in B. canis genomic DNA. Biochemical characterization of Bcvir15, by using polyclonal rabbit sera directed against recombinant proteins, indicated that it is a soluble protein which remained associated with the cytoplasm of the B. canis merozoite. Interestingly, purified immunoglobulins from the anti-glutathione S-transferase-Bcvir15 (at a concentration of 160 micro g/ml) induced 50% inhibition of the in vitro growth of B. canis, and the inhibitory effect was associated with morphological damage of the parasite. Our data suggest that the extrachromosomal dsRNA-encoded Bcvir15 protein might interfere with the intracellular growth of the parasite rather than with the process of invasion of the host cell by the merozoite. Epitope mapping of Bcvir15 identified three epitopes that might be essential for the function of the protein.

A rapid method for identifying diversity within PCR amplicons using a heteroduplex mobility assay and synthetic polynucleotides: application to characterisation of dsRNA elements associated with Cryptosporidium

A 173-bp fragment of the small extra-chromosomal double-stranded RNA (dsRNA) element of Cryptosporidium parvum was generated by reverse transcriptase PCR from nucleic acid extracted from whole faeces of 18 epidemiologically unrelated cases of cryptosporidiosis. Eleven different sequences were detected and two selected as reference DNA in a heteroduplex mobility assay (HMA). Although sequence diversity was detected, this was difficult to characterise because of the similarity in electrophoretic mobility of the homo- and heteroduplex bands. A PCR method was devised to generate synthetic polynucleotides of greater sequence diversity for use in the HMA. The presence of the synthetic 173-bp fragments was enriched by using, as template for the PCR, material excised from the area of the heteroduplex bands in stained electrophoresis gels. Nine novel sequences were generated and evaluated as reference sequences in the HMA. One of these with 20 bp different from the original sequence was selected for use in the HMA for improved resolution of heteroduplex and homoduplex bands and number of patterns easily resolved (nine different patterns corresponding to different DNA sequences). This method may be useful for analysis of DNA where there is limited natural variation or little sequence variation is described.

Double-stranded RNA elements associated with the MVX disease of Agaricus bisporus

Double-stranded RNA (dsRNA) has been isolated from Agaricus bisporus fruit bodies exhibiting a wide range of disease symptoms. The symptoms which occurred singularly or in combination included; bare cropping areas on commercial beds (primordia disruption), crop delay, premature veil opening, off- or brown-coloured mushrooms, sporophore malformations and loss of crop yield. All symptoms were associated with loss of yield and/or product quality. Collectively, these symptoms are described as mushroom virus X (MVX) disease. The dsRNA titre was much lower than that previously encountered with the La France viral disease of mushrooms and a modified cellulose CF11 protocol was used for their detection. A broad survey of cultivated mushrooms from the British industry identified dsRNA elements ranging between 640 bp and 20.2 kbp; the majority have not previously been described in A. bisporus. 26 dsRNA elements were identified with a maximum of 17, apparently non-encapsidated dsRNA elements, in any one sample. Three dsRNAs (16.2, 9.4 and 2.4 kbp) were routinely found in mushrooms asymptomatic for MVX. Previously, La France disease was effectively contained and controlled by minimising the on-farm production and spread of basidiospores. Our on-farm observations suggest that MVX could be spread by infected spores and/or mycelial fragments.

Double-stranded RNA elements in Rhizoctonia solani AG 3

All isolates of Rhizoctonia solani (teleomorph Thanatephorus cucumeris ) anastomosis group (AG) 3 from potato tubers in commercial fields in Scotland were found to contain more than one double-stranded RNA (dsRNA) element ranging in size from 0.3 to 15 kb. Strains of other AGs (2–1, 3, 4, 5) from culture collections also contained dsRNA elements. Isolates of AG 3 from different black scurf lesions on single tubers showed similar dsRNA profiles and, when opposed on agar plates, underwent compatible hyphal fusion where the colonies met. Isolates from different tubers in the same commercial field often showed somatic incompatibility (mean 44%) leading to cytoplasmic death when colonies fused. Isolates from different fields always showed somatic incompatibility after hyphal fusion, suggesting that the complex population structure of AG 3 could significantly limit the transmission of dsRNA between strains. The incidence of different dsRNA elements in AG 3 isolates could not be related to pathogenicity in seedling tests on tomato, carrot, lettuce, radish, cress or onion. Double-stranded RNA elements could not be eliminated from AG 3 isolates by repeated hyphal-tip subculture or extended (8 week) incubation on agar containing cycloheximide. Some isolates lost the smaller dsRNA elements (1.0 to 1.6 kb) during these procedures, and one isolate (of AG 4) lost a 6.5 kb dsRNA element, but the larger elements (up to 9.5 or 12 kb) were always retained. When assessed by low stringency hybridisation, the dsRNA elements of R. solani AG 3 and of strains from AGs 2–1, 4 and 5 showed no homology with a plasmid containing cDNA of the complete hypovirus dsRNA genome of Cryphonectria parasitica .

Double-strnaded RNA elements and virus-like particles in aspergilli

Mycoviruses with double-stranded RNA (dsRNA) genomes are frequently encountered in Aspergillus isolates. A detailed study of such dsRNA elements in black Aspergillus isolates collected worldwide was carried out, and the data were analysed. The results indicate that about 10% of black Aspergilli are infected. However, the geographic distribution of infected isolates exhibits large variations; 3-13% of the isolates collected from different continents were found to carry dsRNA elements. Hybridization experiments indicated that electrophoretic banding patterns are not reliable tools for estimating the diversity of these mycovirus genomes. Among strains representing other Aspergillus sections, dsRNA segments indicative of mycovirus infection were observed for the first time in 4 species (A. leporis, A. petrakii, A. fumigatus and A. primulinus). The latter species is able to reproduce sexually. This is the second report on the detection of naturally-occurring dsRNAs in sexually reproducing Aspergillus species. The presence of virus-like particles in these and other Aspergilli was also examined by electron microscopy. Most infected Aspergillus isolates examined were found to carry virus-like particles in the size range 36-40 nm.

Genetically related isolates of the entomopathogenic fungus Metarhizium anisopliae harbour homologous dsRNA viruses

Several isolates of the entomopathogenic Metarhizium anisopliae that harboured dsRNA viruses of similar electrophoretic band sizes (1.8 and 2.0 kbp) were assessed for homologies of the dsRNA by Northern analysis. The isolates were also characterised genetically by RAPD and VCG. Similarly sized dsRNA, as visualised by electrophoresis, were not always homologous, suggesting that the comparison of dsRNA based solely on electrophoretic banding patterns is an unreliable method of dsRNA characterisation. Several isolates, but not all, harbouring multiple dsRNA patterns, including a 1.8 and 2.0 kbp doublet, also showed homologies to strains harbouring only the dsRNA banding doublet. This suggests that mixed infections of different dsRNA elements are found in M. anisopliae. Genetically similar fungi, based on RAPD banding patterns and vegetative compatibility, were more likely to harbour genetically related dsRNA. The findings suggested that dsRNA elements in M. anisopliae are horizontally transferred to genetically related isolates or are maintained through clonal lineages.

Detection of double-stranded RNA in Macrophomina phaseolina

One hundred ten isolates of Macrophomina phaseolina, the causal agent of charcoal rot of many different plant species, were obtained from Mexico, Somalia, and several locations in the United States, and analyzed for the presence of double-stranded RNA (dsRNA). Twenty-one isolates from the United States and Mexico contained dsRNA elements. The sizes of the dsRNAs ranged from ca 0.4 kbp to 10 kbp and the number of dsRNAs present among the isolates ranged from one to ten. Attempts to detect virus particles by sucrose gradient centrifugation were unsuccessful. Three isolates having dsRNA and three dsRNA-free isolates were evaluated for virulence on sorghum at two different field locations and for mycelial growth in PDA and V8 agar. For both locations, the isolates that had dsRNA were significantly less virulent (P < 0.05) than the dsRNA-free isolates. Differences between locations were not significant (P < 0.05). The contrasts between means of isolates with and without dsRNA suggest a strong association between the presence of dsRNA and a reduction in both mycelial growth and the capacity to cause disease. These results suggest an association between the presence of dsRNA and hypovirulence in the fungus.

Double-stranded RNA and virus-like particles in the edible basidiomycete Flammulina velutipes (Enokitake)

One of the commercial strains of Flammulina velutipes was analyzed for the presence of double-stranded RNA (dsRNA) elements to examine the underlying mechanism of strain degeneration. As a result, two dsRNA elements sized 1.9 and 1.8 kb were detected in mycelium derived from spontaneously brown-colored fruit body. They were not detected in the normal strains or in fruiting-impaired degenerative isolates. The dsRNAs were not in the nuclear or mitochondrial fractions, but were located in the cytoplasmic fraction. The presence of virus-like particles of ca. 50 nm diameter associated with the dsRNAs was confirmed by electron microscopic observation.

Double-stranded RNA and virus-like particles in the edible basidiomycete Flammulina velutipes (Enokitake).

One of the commercial strains of Flammulina velutipes was analyzed for the presence of double-stranded RNA (dsRNA) elements to examine the underlying mechanism of strain degeneration. As a result, two dsRNA elements sized 1.9 and 1.8 kb were detected in mycelium derived from spontaneously brown-colored fruit body. They were not detected in the normal strains or in fruiting-impaired degenerative isolates. The dsRNAs were not in the nuclear or mitochondrial fractions, but were located in the cytoplasmic fraction. The presence of virus-like particles of ca. 50 nm diameter associated with the dsRNAs was confirmed by electron microscopic observation.

Genetic interrelationships and genome organization of double-stranded RNA elements of Fusarium poae.

The similar sized double-stranded RNA (dsRNA) elements present in vegetatively compatible strains of Fusarium poae were always genetically related, while vegetatively incompatible strains of the fungus contained either homologous or non-homologous dsRNAs of the same size. Electron microscopic observations revealed the co-existence of encapsidated and naked dsRNA elements in the same host. A mycovirus, named FUPO-1 was purified from strain A-11 and was found to contain two kinds of dsRNA segments, dsRNA 1 and dsRNA 2. The dsRNA genome of these segments was converted to cDNA clones by reverse transcription and the clones were subjected to sequence analysis. The single long open reading frame deduced from the sequence of dsRNA 1 showed similarities to the putative coat protein genes known from other mycoviruses, while conserved motifs of an RNA-dependent RNA polymerase were identified in the predicted amino acid sequence of dsRNA 2. The genome organization and certain sequence motifs of FUPO-1 show similarities to that of the Atkinsonella hypoxylon 2H virus and the FusoV mycovirus, members of the Partitiviridae family.

Double-stranded RNA and virus-like particles in the grass endophyte Epichloë festucae

Two double-stranded ribonucleic acid (dsRNA) elements were detected in an isolate of the clavicipitaceous grass endophyte Epichloe festucae obtained from an asymptomatic Festuca rubra plant. No dsRNA was detected in three other isolates of E. festucae. The size of the two dsRNA elements isolated from mycelium was of 5.2 and 3.2 kbp. Hybridization experiments with a cDNA probe complementary to the 5.2 kbp dsRNA showed that there was no sequence similarity between the dsRNA elements. Isometric virus-like particles of about 50 nm in diameter were observed by electron microscopy in sucrose-gradient purified virus preparations from the isolate containing dsRNA. The 5.2 kbp dsRNA element was present in the partially purified virus preparations, suggesting that this element is encapsidated. There was no difference with respect to growth rate or colony morphology between the dsRNA containing and dsRNA-free isolates. The presence of dsRNA and virus-like particles suggests the presence of a mycovirus in the Vit5 strain of E. festucae.

A double-stranded RNA element from a hypovirulent strain of Rhizoctonia solani occurs in DNA form and is genetically related to the pentafunctional AROM protein of the shikimate pathway.

M2 is a double-stranded RNA (dsRNA) element occurring in the hypovirulent isolate Rhs 1A1 of the plant pathogenic basidiomycete Rhizoctonia solani. Rhs 1A1 originated as a sector of the virulent field isolate Rhs 1AP, which contains no detectable amount of the M2 dsRNA. The complete sequence (3,570 bp) of the M2 dsRNA has been determined. A 6.9-kbp segment of total DNA from either Rhs 1A1 or Rhs 1AP hybridizes with an M2-specific cDNA probe. The sequences of M2 dsRNA and of PCR products generated from Rhs 1A1 total DNA were found to be identical. Thus this report describes a fungal host containing full-length DNA copies of a dsRNA element. A major portion of the M2 dsRNA is located in the cytoplasm, whereas a smaller amount is found in mitochondria. Based on either the universal or the mitochondrial genetic code of filamentous fungi, one strand of M2 encodes a putative protein of 754 amino acids. The resulting polypeptide has all four motifs of a dsRNA viral RNA-dependent RNA polymerase (RDRP) and is phylogenetically related to the RDRP of a mitochondrial dsRNA associated with hypovirulence in strain NB631 of Cryphonectria parasitica, incitant of chestnut blight. This polypeptide also has significant sequence similarity with two domains of a pentafunctional polypeptide, which catalyzes the five central steps of the shikimate pathway in yeast and filamentous fungi.

Movement of a small mitochondrial double-stranded RNA element of Cryphonectria parasitica: ascospore inheritance and implications for mitochondrial recombination.

Movement via somatic fusion and inheritance of a small mitochondrial double-stranded (ds) RNA element was examined in Cryphonectria parasitica. The 2.7-kb dsRNA from the C. parasitica strain NB631 encodes a putative RNA-dependent RNA polymerase when the mitochondrial code (UGA = Trp) is invoked. All progeny from asexual spores (conidia) of strain NB631 examined for dsRNA contained the 2.7-kb element. Unlike other C. parasitica dsRNAs, which are cytoplasmic, the dsRNA in strain NB631 was transmitted through the sexual cycle (ascospores) if the strain containing the element acted as the female in crosses. Movement of the 2.7-kb dsRNA was also observed through hyphal anastomosis. Transfer by anastomosis was accompanied by mitochondrial movement and recombination of the mitochondrial genome as determined by RFLP analysis. In control pairings between isolates lacking dsRNA, mitochondrial movement and recombination were also observed. Transfer by anastomosis allowed the generation of infected and uninfected isogenic lines, and permitted us to evaluate the effects of the dsRNA element on virulence of the host. Bark virulence assays on American chestnut suggest that NB631 dsRNA decreases the virulence of C. parasitica, but not to the level associated with members of the Hypoviridae.