Fungal proteinase expression in the interaction of the plant pathogen Magnaporthe poae with its host.

Infection by pathogenic fungi involves breaching the outer layer of the host by either mechanical or enzymatic means. Subtilisin-like proteinases are considered to be important in the infection process of entomopathogenic, nematophagous, and mycoparasitic fungi. Little is known regarding the expression of such proteinases by plant pathogenic fungi. Magnaporthe poae, a fungal pathogen of Kentucky bluegrass, expressed a subtilisin-like proteinase, proteinase Mp1, in the infected roots. Antibody was produced against the purified enzyme. From immunoblot analysis, expression of the proteinase in infected roots correlated with increasing severity of disease symptoms. Sequence analysis of a genomic clone indicated proteinase Mp1 was homologous to other fungal subtilisin-like proteinases. DNA gel blot analysis indicated proteinase Mp1 was encoded by a small gene family.

Incidence and Diversity of Double-Stranded RNAs Occurring in the Chestnut Blight Fungus, Cryphonectria parasitica, in China and Japan.

ABSTRACT Isolates of the chestnut blight fungus, Cryphonectria parasitica, were randomly sampled from 10 subpopulations in China and 8 subpopulations in Japan and screened for the presence of double-stranded (ds) RNA using an immunoblot procedure with a monoclonal antibody specific for dsRNA. The overall incidence of dsRNA in C. parasitica was 2 and 6% in China and Japan, respectively, much lower than the 28% found previously in North American populations. Genetic relatedness of dsRNAs within and among populations in China and Japan was examined using RNA-RNA hybridizations with labeled-dsRNA probes. The majority of Chinese and Japanese dsRNAs were members of a single hybridization group, related to Cryphonectria hypovirus 1 (CHV1) from Europe, and are referred to as CHV1-type dsRNAs. No evidence was obtained for genetic differentiation between CHV1-type dsRNAs sampled in China and Japan. Five Japanese isolates contained two genetically distinct dsRNAs. The larger segments (approximately 12 kilobases [kb]) were members of the CHV1 hybridization group, while the smaller segments (approximately 3 kb) did not hybridize with any known dsRNA from C. parasitica including the 2.7-kb dsRNA from isolate NB631 from New Jersey or dsRNA from isolate RC1 from Michigan. Two small dsRNA segments (approximately 1.8 and 2 kb) from one isolate sampled from Liaoning Province in northeastern China did not hybridize with any of the dsRNA probes tested including several described dsRNAs of similar size from C. parasitica in North America. Three dsRNAs from Anhui Province, China, hybridized to Cryphonectria hypovirus 2 (CHV2)-specific probes and are thus referred to as CHV2-type dsRNAs. Sequence analysis of 1,627 base pairs of these three CHV2-type dsRNAs from Anhui revealed that they were identical to each other in the region sequenced and very closely related to CHV2-NB58, isolated from New Jersey. We speculate that CHV2-NB58 may have been introduced into North America from this part of China. This is the first record of a North American C. parasitica dsRNA that is genetically related to a dsRNA from Asia.

First Report of Blueberry scorch virus on Highbush Blueberry in Connecticut and Massachusetts.

During June and July 2001, the Marucci Center received 33 foliage samples from healthy- and unhealthy-looking highbush blueberry (Vaccinium corymbosum L.) bushes from growers in Connecticut and Massachusetts, via local extension agents. Unhealthy bushes were reported to exhibit symptoms including leaf chlorosis and necrosis, blossom blight, tip dieback, or a general decline in vigor. Marginal leaf chlorosis, reddening, or necrosis characterized foliage samples from these bushes. Five-leaf samples from each bush were tested for Blueberry scorch virus (BlScV) (3) with Agri-Check detection kits (Hydros, Inc., Falmouth, MA). These kits use an indirect enzyme-linked immunosorbent assay (ELISA) protocol and antibodies developed at Rutgers University that are specific to the two eastern strains of BlScV (NJ1 and NJ2) (1). The ELISA extraction buffer was based on that used by Martin and Bristow (3) with 1% (wt/vol) nonfat dry milk powder added as a blocking agent. Fourteen samples from cvs. Blueray and Berkeley in both states and cvs. Elliott, Bluecrop, and Coville in Massachusetts tested positive for BlScV. These results were confirmed by a second test. Six of seven samples from symptomatic bushes and 8 of 26 samples from asymptomatic bushes harbored BlScV. Virus preparations extracted from five infected plants (two from Connecticut and three from Massachusetts) were examined using reverse transcription-polymerase chain reaction (RT-PCR) with oligonucleotide primers (5'-TGTGTCAAACAATATGGC-3' and 5'-GCATTTCGATGA-TTGCGG-3') designed to amplify a portion of the coat protein gene from any of the three known virus strains (1,2). Sequence analysis of fragments amplified from their coat protein genes revealed that two of the isolates from Massachusetts (GenBank Accession Nos. AY530957 and AY530958) and the two isolates from Connecticut (GenBank Accession Nos. AY530955 and AY530956) were similar but not identical to one another, and these four were most similar to strain NJ2. One isolate from Massachusetts (GenBank Accession No. AY530958) was most similar to strain NJ1. To our knowledge, this is the first report of BlScV on the east coast outside of New Jersey, where it was first reported in 1983 (4). These results indicate that the disease is now present in other blueberry-growing areas in the northeast and is likely to be spreading locally within those areas. Because blueberry scorch is symptomless in propagation material and may take several years to express symptoms in the field, the initial spread of the disease was probably due to the shipping of latently infected plants to BlScV-free areas before reliable testing was available. References: (1) T. D. Cavileer et al. J. Gen. Virol. 75:711, 1994. (2) B. T. Halpern and B. I. Hillman. Plant Dis. 80:219, 1996. (3) R. R. Martin and P. R. Bristow. Phytopathology 78:1636, 1988. (4) A. W. Stretch. (Abstr.) Phytopathology 73:375, 1983.

Synthesis of infectious transcripts of blueberry scorch carlavirus in vitro.

Blueberry scorch carlavirus (BBScV) is a filamentous virus with a polyadenylated, positive-sense RNA genome. A near full-length cDNA clone of BBScV was constructed by assembly of clones from a cDNA library. To generate a full-length cDNA clone, the 5' terminus was mutagenized by PCR to introduce nucleotides present in the wild-type virus and not in the near full-length clone, and then fused directly to the T7 bacteriophage RNA polymerase promoter at the 5' terminus. Capped and uncapped BBScV transcripts were synthesized in vitro from the full-length cDNA clone. Capped transcripts were infectious, producing systemic symptoms identical to those caused by the wild-type virus following inoculation onto Chenopodium quinoa leaves. Uncapped transcripts were substantially less infectious than capped transcripts. This represents the first report of infectious transcripts for a member of the carlavirus group.

In vitro translational analysis of genomic, defective, and satellite RNAs of Cryphonectria hypovirus 3-GH2.

Cryphonectria hypovirus 3-GH2 (CHV3-GH2) is a member of the fungal virus family Hypoviridae that differs from previously characterized members in having a single large open reading frame with the potential to encode a protein of 326 kDa from its 9.8-kb genome. The N-terminal portion of the ORF contains sequence motifs that are somewhat similar to papain-like proteinases identified in other hypoviruses. Translation of the ORF is predicted to release autocatalytically a 32.5-kDa protein. A defective RNA, predicted to encode a 91.6-kDa protein representing most of the N-terminal proteinase fused to the entire putative helicase domain, and two satellite RNAs, predicted to encode very small proteins, also are associated with CHV3-GH2 infected fungal cultures. We performed in vitro translation experiments to examine expression of these RNAs. Translation of three RT-PCR clones representing different lengths of the amino-terminal portion of the ORF of the genomic RNA resulted in autocatalytic release of the predicted 32.5-kDa protein. Site-directed mutagenesis was used to map the processing site between Gly(297) and Thr(298). In vitro translation of multiple independent cDNA clones of CHV3-GH2-defective RNA 2 resulted in protein products of approximately 92 kDa, predicted to be the full-length translation product, 32 kDa, predicted to represent the N-terminal proteinase, and 60 kDa, predicted to represent the C-terminal two-thirds of the full-length product. In vitro translation of cDNA clones representing satellite RNA 4 resulted in products of slightly less than 10 kDa, consistent with the predicted 9.4 kDa product.

Genome organization of a partitivirus from the filamentous ascomycete Atkinsonella hypoxylon.

We have identified viruses in several isolates of the filamentous ascomycete Atkinsonella hypoxylon. The virus from one isolate of the fungus, 2H, was selected for genomic characterization. Purified virus particles contained three dsRNAs with sizes estimated by gel electrophoresis to be 2.2, 2.1 and 1.8 kb. A library of cDNA clones representing the three dsRNA segments of isolate 2H was synthesized, mapped and sequenced. The three segments had no significant similarity to each other, as determined by Northern blot analysis, and had sizes of 2180, 2135 and 1790 nt as determined by nucleotide sequence analysis. Long open reading frames were deduced from the sequences of dsRNAs 1 (molecular mass 78 kDa) and 2 (74 kDa), but not from dsRNA 3. Both terminal regions of dsRNA 1 and dsRNA had similar nucleotide sequences, as determined from 5' RACE clones. Comparisons of the amino acid sequence deduced from dsRNA 1 revealed similarities with viral RNA-dependent RNA polymerases. Translation in vitro of full-length cDNA clones representing dsRNAs 1 and 2 each yielded single major products of > 70 kDa by analysis on polyacrylamide gels. Based on properties of its dsRNA segments, the virus of A. hypoxylon strain 2H fits into the Partitiviridae family, and represents the first member of this family from a fungal host completely characterized at the level of primary nucleotide sequence.

A small mitochondrial double-stranded (ds) RNA element associated with a hypovirulent strain of the chestnut blight fungus and ancestrally related to yeast cytoplasmic T and W dsRNAs.

A small double-stranded (ds) RNA element was isolated from a moderately hypovirulent strain of the chestnut blight fungus Cryphonectria parasitica (Murr.) Barr. from eastern New Jersey. Virulence was somewhat lower in the dsRNA-containing strain than in a virulent dsRNA-free control strain, but colony morphology and sporulation levels were comparable. A library of cDNA clones was constructed, and overlapping clones representing the entire genome were sequenced. The 2728-bp dsRNA was considerably smaller than previously characterized C. parasitica dsRNAs, which are 12-13 kb and ancestrally related to the Potyviridae family of plant viruses. Sequence analysis revealed one large open reading frame, but only if mitochondrial codon usage (UGA = Trp) was invoked. Nuclease assays of purified mitochondria confirmed that the dsRNA was localized within mitochondria. Assuming mitochondrial translation, the deduced amino acid sequence had landmarks typical of RNA-dependent RNA polymerases. Alignments of the conserved regions indicate that this dsRNA is more closely related to yeast T and W dsRNAs and single-stranded RNA bacteriophages such as Q beta than to other hypovirulence-associated dsRNAs.

A defective interfering RNA that contains a mosaic of a plant virus genome.

A symptom-modulating RNA associated with tomato bushy stunt virus (TBSV) was investigated with respect to physical and biological properties. Linear RNA of approximately 396 nucleotides was packaged in viral coat protein and was dependent on TBSV for replication. Coinoculation of the small RNA with TBSV resulted in the attenuation of TBSV-induced symptoms and depression of virus synthesis in whole plants. Nucleotide sequence analysis revealed that the symptom-modulating RNA was derived from 5', 3', and internal segments of the TBSV genome. The identification of this symptom-modulating RNA as a co-linear deletion mutant of the helper virus genome establishes it as the first definitive defective interfering RNA (DI RNA) to be identified in association with a plant virus.

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.

A viral dsRNA element of the chestnut blight fungus with a distinct genetic organization.

We have sequenced overlapping complementary DNA clones representing the viral double-stranded (ds) RNA from hypovirulent strain NB58 of the chestnut blight fungus Cryphonectria parasitica. Cryphonectria hypovirus 2-NB58 (CHV2-NB58) dsRNA contains 12,507 base pairs, excluding the poly(A) tail at the 3' end of the plus strand, and is organizationally similar to the largest dsRNA from the virus of strain EP713 (CHV1-713; identical to HAV; Shapira et al., (1991), EMBO J. 10, 731-739). CHV2-NB58 and CHV1-713 dsRNAs share approximately 60% nucleotide sequence identity. On the poly(A)-containing strand of CHV2-NB58, a 487-residue nontranslated region precedes two open reading frames, designated ORF A (438 codons) and ORF B (3291 codons). The connecting pentanucleotide sequence UAAUG (1802-1806) terminates ORF A and initiates ORF B. In contrast to the 69-kDa ORF A product of CHV1-713, the 50-kDa CHV2-NB58 ORF A product did not undergo autoproteolysis under the conditions tested, nor were motifs associated with cysteine proteases present in the CHV2-NB58 ORF A sequence. CHV2-NB58 ORF B products appear to be homologous with CHV1-713 ORF B products, and the motifs involved in autoproteolysis of the N-terminal 48 kDa of CHV1-713 ORF B were identified in the CHV2-NB58 ORF B product. Motifs associated with RNA polymerase and helicase activities were highly conserved between CHV2-NB58 and CHV1-713 and were found at similar genomic positions in the C-terminal half of ORF B.

Satellite and defective RNAs of Cryphonectria hypovirus 3-grand haven 2, a virus species in the family Hypoviridae with a single open reading frame.

Cryphonectria parasitica hypovirus 3-Grand Haven 2 (CHV3-GH2) is the most recently characterized member of the Hypoviridae family of viruses associated with hypovirulence of the chestnut blight fungus. Isolates of CHV3-GH2 contain either three or four double-stranded (ds) RNAs that are visible on ethidium bromide-stained agarose or polyacrylamide gels. Only the largest dsRNA appears to be required for virus infectivity, and was characterized previously (C. D. Smart et al., 1999, Virology 265, 66-73). In this study, we report the cloning, sequencing, and analysis of the other three dsRNAs. Sizes of the accessory dsRNAs are 3.6 kb (dsRNA 2), 1.9 kb (dsRNA 3), and 0.9 kb (dsRNA 4), compared to 9.8 kb for the genomic dsRNA segment (dsRNA 1). All three accessory dsRNA species are polyadenylated on the 3'-end of one strand, as is genomic dsRNA. DsRNA 2 represents a defective form of dsRNA 1, with the 5'-terminal 1.4 kb derived from the 5'-end of dsRNA 1 and the 3'-terminal 2.2 kb from the 3'-end of dsRNA 1. A single major open reading frame (ORF) is evident from deduced translations of dsRNA 2. The deduced translation product is a 91-kDa protein that represents a fusion consisting of the entire N-terminal protease and the entire putative helicase domain. DsRNAs 3 and 4 represent satellite RNAs that share very little sequence with dsRNA 1 and 2. DsRNA 4 is 937 nucleotides, excluding the poly(A)(+). The first AUG of the polyadenylated strand of dsRNA 4 occurs eight residues in from the 5'-terminus and would initiate the largest ORF on dsRNA 4, with the coding capacity for a 9.4-kDa protein. Within the deduced ORF and approximately 100 nucleotides from the 5'-end of dsRNA 4 is a 22-base sequence that is identical to sequences found in the nontranslated leaders of dsRNAs 1 and 2. DsRNA 3 accumulation in infected cultures varied, but it was less abundant than dsRNA 4. DsRNA 3 was found to represent a head-to-tail dimer of dsRNA 4 linked by a poly(A)/(U) stretch of 40-70 residues.

Autocatalytic processing of the 223-kDa protein of blueberry scorch carlavirus by a papain-like proteinase.

The first open reading frame of the blueberry scorch carlavirus (BBScV) genome encodes a putative replication-associated protein of 223 kDa (p223). A pulse-chase analysis of viral RNA translated in vitro in rabbit reticulocyte lysate revealed that p223 was proteolytically processed. Using a full-length ORF 1 cDNA clone in a coupled in vitro transcription/translation reaction, we confirmed that the ORF 1 gene product of BBScV processes autocatalytically. From sequence alignments with phylogenetically related viruses, including tymoviruses, we predicted that p223 contained a papain-like proteinase domain with a putative catalytic cysteine994 and histidine1075. A second possible proteinase domain, which contained cysteine895 and histidine984 residues with similar spacing but was otherwise less similar to the viral papain-like proteinases, was identified immediately upstream of the predicted catalytic site. The cleavage site of the proteinase was predicted to be between the putative helicase and the polymerase domains, possibly between or close to glycine1472 and alanine1473. Supporting these predictions, deletion of the 2091 nucleotides encoding the C-terminal region of p223, which contained the putative RNA polymerase domain and the putative cleavage site of the polyprotein, abolished autoproteolysis. Deletion of the 2061 nucleotides encoding the N-terminal region, which contained the putative methyltransferase domain, did not affect autoproteolysis. Alteration of cysteine994, histidine1075, or glycine1472 abolished autoproteolysis in vitro, supporting the involvement of these residues at the catalytic site and cleavage site. Alteration of the upstream cysteine895 and histidine984 residues did not affect processing in vitro. Capped BBScV full-length transcripts containing mutations in the codons for either cysteine994 or histidine1075 were not infectious in the systemic host plants Chenopodium quinoa and C. amaranticolor, whereas alteration of glycine1472 signficantly decreased but did not abolish infectivity. Transcripts containing mutations in the codons for either cysteine895 or histidine984 also were infectious, but resulted in delayed symptom expression in plants.

Structure of the gene encoding the M1 protein of sonchus yellow net virus.

The gene encoding the M1 protein of sonchus yellow net virus (SYNV), a plant rhabdovirus, has been sequenced and identified by Western blot analysis of SYNV proteins using antibodies directed against a fusion protein derived from a portion of the sequenced gene. The M1 gene is positioned between nucleotides 4039 and 5109 relative to the 3' end of the viral RNA and is the fourth gene from the 3' end of the genome. The 1071-nucleotide (nt) M1 gene lies between a putative nonstructural gene of unknown function and the gene encoding the glycoprotein and is bordered on either side by the same GG intergenic dinucleotide that separates other genes in the SYNV genome. The M1 mRNA (scRNA 6) consists of a 71-nt untranslated region at the 5' terminus followed by an 858-nt open reading frame (ORF) capable of encoding a protein with a calculated molecular weight of 31,779. The amino acid sequence deduced from this ORF is not highly homologous to those of other rhabdovirus matrix proteins, but has some localized regions of similarity. The UGA codon that terminates the M1 ORF is followed by a 3' untranslated region of 142 nt. The viral RNA (minus-sense) sequence corresponding to the extreme 3' end of the mRNA contains a 9-nt tract (3'-AUUGUUUUU-5') that is identical to the sequences at the termini of other SYNV genes.

Evidence for interspecies transmission of viruses in natural populations of filamentous fungi in the genus Cryphonectria.

Interspecies transmission is a significant evolutionary event that has allowed a variety of pathogens to invade new host species. We investigated interspecies transmission of viruses between the chestnut blight fungus, Cryphonectria parasitica, and a sympatric unidentified Cryphonectria species in Japan. Two isolates of Cryphonectria sp. were found to contain Cryphonectria hypovirus 1 (CHV-1), which has been typically found in C. parasitica. Three lines of evidence support the hypothesis of interspecies transmission of CHV-1. First, host species occur sympatrically and therefore have the opportunity to come into physical contact. Second, we transmitted CHV-1 between species experimentally in the laboratory. Third, phylogenetic analysis of 476 bp of the ORF B region of CHV-1 showed that sequences from Cryphonectria sp. were more closely related to those from C. parasitica than to each other. Local geographical subdivision of virus sequences from both host species argues against the alternative hypothesis of independent evolution of CHV-1 since speciation of their hosts. Based on these findings, we rule out the hypotheses that CHV-1 diverged from viruses in a common ancestor of the hosts, or that ancestral polymorphisms in CHV-1 persisted in the two host taxa. Estimating the direction and frequency of interspecies transmission in nature will require more extensive samples of CHV-1 from both host species.

A North American hypovirulent isolate of the chestnut blight fungus with European isolate-related dsRNA.

We have synthesized and mapped a cDNA library representing the one major dsRNA element associated with hypovirulence in strain NB58 of the chestnut blight fungus, Cryphonectira (=Endothia) parasitica, which was isolated from recovering chestnut trees in New Jersey, U.S.A. The linear dsRNA has a size of approximately 12.5 kbp and is polyadenylated at the 3' terminus of one strand. Molecular hybridization experiments indicate that there is sequence similarity between the NB58 dsRNA and dsRNAs from European isolates of C. parasitica, but not among dsRNAs of NB58 and those associated with other North American isolates. Hybridization experiments with mapped cDNA clones representing different regions of the 12.5 kbp dsRNA indicate that the termini and the 3'-proximal two-thirds (relative to the plus strand) are more conserved among NB58 and the European isolates than the rest of the 5'-proximal one-third. Nucleotide sequence analysis of the termini of NB58 dsRNA suggests common organizational features between it and the dsRNA from French-derived strain EP713.

The complete genome structure and synthesis of infectious RNA from clones of tomato bushy stunt virus.

The complete sequence of the genome of the cherry strain of tomato bushy stunt virus (TBSV), a member of the tombusvirus group, was determined. A full-length clone of the genome containing a bacteriophage T7 RNA polymerase promoter was assembled from partial cDNA clones. In vitro transcripts of the genome, either with or without a 5' cap structure, were highly infectious. In addition, a genomic clone modified to contain an EcoRI restriction site as a signature mutation was infectious. Five genes are encoded by the TBSV genome. The first ORF from the 5' terminus encodes a p33 protein as well as a p92 product translated by read-through of the amber terminator for p33. The capsid protein gene resides internally, and two ORFs for proteins of 19 and 22 kDa reside at the 3' terminus. These last three genes are expressed from two subgenomic RNAs. The genomic organization of TBSV agrees with previous models for tombusviruses. Computer alignments of TBSV proteins with those of two other tombusviruses suggest greater relatedness among the members of this group than previously reported.

The contribution of defective RNAs to the complexity of viral-encoded double-stranded RNA populations present in hypovirulent strains of the chestnut blight fungus Cryphonectria parasitica.

Hypovirulent strain EP713 of the chestnut blight fungus Cryphonectria (Endothia) parasitica harbors a family of viral encoded double-stranded (ds) RNAs thought to be responsible for the hypovirulence phenotype. These include L-dsRNA, described in the accompanying paper (Shapira et al., 1991); several prominent species in the estimated size range of 8 to 10 kb, referred to here as M-dsRNAs; and several smaller species designated S-dsRNAs which range in size from approximately 0.6 to 1.7 kb. The characterization of the M- and S-dsRNA species is the subject of this report. Results from polymerase chain reaction mapping and molecular hybridization analysis indicate that the M- and S-dsRNA species are internally deleted forms of L-dsRNA. Three different S-dsRNA species were cloned and sequenced. Each species contained a single deletion breakpoint and retained either 149, 155 or 156 bp of the terminus corresponding to the 5'-end of the coding strand and 440, 447 or 449 bp of the other terminus. Two of the S-dsRNA species contained, within the boundaries of the breakpoint, additional sequence information consisting of 42 bp or 95 bp that appeared to be unrelated to the L-dsRNA sequence. These results demonstrate that defective RNAs contribute significantly to the complexity of dsRNA populations found in hypovirulent strains of C. parasitica and provide a first approximation of the location of cis-acting signals involved in their replication.

The genome structure of turnip crinkle virus.

The nucleotide sequence of turnip crinkle virus (TCV) genomic RNA has been determined from cDNA clones representing most of the genome. Segments were confirmed using dideoxynucleotide sequencing directly from viral RNA, and the 3' terminal sequence was confirmed by chemical sequencing of end-labeled genomic RNA. Three open reading frames (ORFs) have been identified by examination of the deduced amino acid sequences and by comparison with the ORFs found in the genome of carnation mottle virus. ORF 1 initiates near the 5' terminus of the genome and is punctuated by an amber termination codon. Translation of ORF 1 would yield a 28-kDa protein and an 88-kDa read-through product. The read-through domain possesses amino acid sequence similarities with putative viral RNA polymerases. ORFs 2 and 3 encode products of 38 (coat protein) and 8 kDa, respectively, which are expressed from subgenomic mRNAs. The organization of the TCV genome suggests that TCV is closely related to carnation mottle virus and distinct from members classified in other small RNA virus groups, such as the tombus- and sobemoviruses.

Characterization of double-stranded RNA genetic elements associated with biological control of chestnut blight: organization of terminal domains and identification of gene products.

We have determined the organization within the terminal domains of the major large double-stranded RNA genetic elements associated with the hypovirulent strain EP713 of the chestnut blight pathogen Cryphonectria (Endothia) parasitica. Only the polyadenylated strand contained long open reading frames. Furthermore, only RNA of the same polarity as the polyadenylated strand was detectable in a single-stranded form, indicating that the polyadenylated strand is the coding or plus strand. The organization of the 5'-proximal portion of the plus strand consisted of a 495 nucleotide non-coding leader sequence followed by two overlapping open reading frames. The first, ORF1, extended 957 nucleotides while the second, ORF2, began 68 nucleotides upstream of the ORF1 termination codon and extended at least 1412 nucleotides. No open reading frames of significant size were detected within 0.8 kb of the poly(A) tail. In vitro translation of synthetic transcripts containing ORF1 yielded a polypeptide of Mr 29 kd. The ORF1 product was also detected in lysates of the hypovirulent strain but was absent in lysates of the isogenic virulent strain. It represents the first protein to be identified as a gene product encoded by a hypovirulence-associated double-stranded RNA genetic element.