RESUMO
BACKGROUND: Mycoviruses were recently discovered in the white pine blister rust (WPBR) fungus Cronartium ribicola (J.C. Fisch.). Detection and characterization of their double stranded RNA (dsRNA) would facilitate understanding of pathogen virulence and disease pathogenesis in WPBR systems. METHODS: Full-length cDNAs were cloned from the dsRNAs purified from viral-infected C. ribicola, and their cDNA sequences were determined by DNA sequencing. Evolutionary relationships of the dsRNAs with related mycoviruses were determined by phylogenetic analysis. Dynamic distributions of the viral RNAs within samples of their fungal host C. ribicola were investigated by measurement of viral genome prevalence and viral gene expression. RESULTS: In this study we identified and characterized five novel dsRNAs from C. ribicola, designated as Cronartium ribicola totivirus 1-5 (CrTV1 to CrTV5). These dsRNA sequences encode capsid protein and RNA-dependent RNA polymerase with significant homologies to dsRNA viruses of the family Totiviridae. Phylogenetic analysis showed that the CrTVs were grouped into two distinct clades. CrTV2 through CrTV5 clustered within the genus Totivirus. CrTV1 along with a few un-assigned dsRNAs constituted a distinct phyletic clade that is genetically distant from presently known genera in the Totiviridae family, indicating that CrTV1 represents a novel genus in the Totiviridae family. The CrTVs were prevalent in fungal samples obtained from infected western white pine, whitebark pine, and limber pines. Viral RNAs were generally expressed at higher levels during in planta mycelium growth than in aeciospores and urediniospores. CrTV4 was significantly associated with C. ribicola virulent pathotype and specific C. ribicola host tree species, suggesting dsRNAs as potential tools for dissection of pathogenic mechanisms of C. ribicola and diagnosis of C. ribicola pathotypes. CONCLUSION: Phylogenetic and expression analyses of viruses in the WPBR pathogen, C. ribicola, have enchanced our understanding of virus diversity in the family Totiviridae, and provided a potential strategy to utilize pathotype-associated mycoviruses to control fungal forest diseases.
Assuntos
Basidiomycota/virologia , Micélio/patogenicidade , Pinus/microbiologia , Doenças das Plantas/microbiologia , RNA de Cadeia Dupla/fisiologia , Totiviridae/fisiologia , Basidiomycota/genética , Basidiomycota/crescimento & desenvolvimento , Basidiomycota/patogenicidade , Genoma Viral/genética , Micélio/genética , Micélio/crescimento & desenvolvimento , Micélio/virologia , Filogenia , Pinus/classificação , RNA de Cadeia Dupla/classificação , RNA de Cadeia Dupla/genética , RNA Viral/genética , Totiviridae/classificação , Totiviridae/genética , Transcrição Gênica , Proteínas Virais/genética , VirulênciaRESUMO
Three double-stranded RNA (dsRNA) segments from Aspergillus ochraceus isolate FA 0611, designated as AoR1, AoR2, and AoR3, were cloned and sequenced. AoR1 was identical with AoV dsRNA 1 previously reported from A. ochraceus ATCC 28706, which putatively encoded an RNA-dependent RNA polymerase of an unidentified mycovirus. AoR2 was found to encode a putative viral capsid protein (CP) with 63% similarity to that of Penicillium stoloniferum virus S, which was detected from P. stoloniferum ATCC 14586. The function of AoR3 was unknown. The three segments were found to contain a conserved sequence at their 5' termini, while an identical sequence was only found at the 3' termini of AoR1 and AoR2. It is suggested that AoR1, AoR2, and AoR3 originate from an independent partitivirus infecting A. ochraceus. The novel virus is suggested to be Aspergillus ochraceus virus 1, AoV1.
Assuntos
Aspergillus ochraceus/virologia , Genoma Viral , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/isolamento & purificação , Sequência de Bases , Proteínas do Capsídeo/genética , China , Dados de Sequência Molecular , Filogenia , RNA de Cadeia Dupla/classificação , Alinhamento de SequênciaRESUMO
Dicers are associated with double-stranded RNA-binding proteins (dsRBPs) in animals. In the plant, Arabidopsis, there are four dicer-like (DCL) proteins and five potential dsRBPs. These DCLs act redundantly and hierarchically. However, we show there is little or no redundancy or hierarchy amongst the DRBs in their DCL interactions. DCL1 operates exclusively with DRB1 to produce micro (mi)RNAs, DCL4 operates exclusively with DRB4 to produce trans-acting (ta) siRNAs and 21nt siRNAs from viral RNA. DCL2 and DCL3 produce viral siRNAs without requiring assistance from any dsRBP. DRB2, DRB3 and DRB5 appear unnecessary for mi-, tasi-, viral si-, or heterochromatinising siRNA production but act redundantly in a developmental pathway.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/genética , Elementos de DNA Transponíveis , MicroRNAs/biossíntese , MicroRNAs/química , Mutação , Conformação de Ácido Nucleico , Fenótipo , Filogenia , RNA de Cadeia Dupla/classificação , RNA de Cadeia Dupla/genética , RNA de Plantas/biossíntese , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/química , RNA Viral/biossíntese , RNA Viral/química , Proteínas de Ligação a RNA/classificação , Proteínas de Ligação a RNA/genética , Ribonuclease III/classificação , Ribonuclease III/genéticaRESUMO
We surveyed native populations of the chestnut blight fungus, Cryphonectria parasitica, in Japan and China, and C. nitschkei, a sympatric species on chestnut trees in Japan, to learn more about the diversity of hypoviruses and other double-stranded (ds) RNA viruses. In a sample of 472 isolates of C. parasitica and 45 isolates of C. nitschkei from six prefectures in Japan, we found 27 containing one or more dsRNAs. Twelve isolates of C. parasitica and two isolates of C. nitschkei were infected with Cryphonectria hypovirus 1 (CHV-1); four of these 12 C. parasitica isolates also contained other dsRNAs that did not hybridize to CHV-1. In China, only one of 85 C. parasitica isolates was CHV-1-infected; no dsRNAs were detected in the other isolates from China. No other known hypoviruses were found in this study. However, we found two previously undescribed dsRNAs in Japan approximately 9kb in size that did not hybridize to each other or to any known dsRNAs from C. parasitica. We also found three additional groups of dsRNAs, one of which represents the genome of a new member of the virus family Chrysoviridae and was found only in C. nitschkei; the other two dsRNAs were found previously in isolates of C. parasitica from Japan or China. The most significant result of this survey is the discovery of novel dsRNAs that can be characterized in future research.
Assuntos
Ascomicetos/virologia , Vírus de RNA/classificação , Sequência de Aminoácidos , Biodiversidade , China , Fagaceae/microbiologia , Japão , Dados de Sequência Molecular , Peso Molecular , Doenças das Plantas , Vírus de RNA/genética , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/classificação , RNA de Cadeia Dupla/genética , RNA Viral/química , RNA Viral/classificação , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Alinhamento de Sequência , Especificidade da Espécie , Proteínas Virais/genéticaRESUMO
Cherry chlorotic rusty spot (CCRS) and Amasya cherry disease (ACD) display similar symptoms and are associated with a series of dsRNAs. However, a direct comparison has been lacking. Here, a side-by-side analysis confirmed that both diseases were symptomatologically very similar, as were the number (10-12) and size of their associated dsRNAs. Sequence determination of four of these dsRNAs revealed that they were essentially identical for CCRS and ACD. The largest (3399 bp), which potentially encoded a protein of 1087 aa with the eight motifs conserved in RNA-dependent RNA polymerases of dsRNA mycoviruses, had the highest similarity to those coded by dsRNA 1 of viruses belonging to the genus Chrysovirus and was termed CCRS or ACD chrys-dsRNA 1. The three closely migrating dsRNAs had the properties of the other components of a chrysovirus and in CCRS and ACD versions, respectively, were chrys-dsRNA 2 (3125 and 3128 bp), chrys-dsRNA 3 (2833 bp) and chrys-dsRNA 4 (2499 and 2498 bp), potentially encoding the major capsid protein (993 and 994 aa) and two proteins (884 and 677 aa, respectively) of unknown function. The four 5'- and 3'-UTRs shared internal similarities and had conserved GAAAAUUAUGG and AUAUGC termini, respectively. The 5'-UTRs contained the 'Box 1' motif followed by a stretch rich in CAA, CAAA and CAAAA repeats, characteristic of chrysovirus dsRNAs. Because species of the genus Chrysovirus have only been described as infecting fungi, this suggests a fungal aetiology for CCRS and ACD, a proposal supported by the properties of two other CCRS- and ACD-associated dsRNAs (see accompanying paper by Coutts et al., 2004, in this issue).
Assuntos
Genoma Viral , Penicillium chrysogenum/classificação , Doenças das Plantas/virologia , Vírus de Plantas/classificação , Vírus de RNA/classificação , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas do Capsídeo/genética , Dados de Sequência Molecular , Penicillium chrysogenum/genética , Filogenia , Vírus de Plantas/genética , Vírus de RNA/genética , RNA de Cadeia Dupla/classificação , RNA de Cadeia Dupla/genética , RNA Polimerase Dependente de RNA/genética , Alinhamento de SequênciaRESUMO
A recent work identifies a distinct class of siRNAs derived from transgenes and endogenous retroelements in plants (Hamilton et al., 2002). This class has slower electrophoretic mobility than previously characterized siRNAs and may play an important role in transgene-induced systemic silencing and in methylation of endogenous retroelement DNA.
Assuntos
Genes de Plantas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Metilação de DNA , Genes Reporter , Modelos Genéticos , RNA de Cadeia Dupla/classificação , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/classificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Retroelementos , TransgenesRESUMO
Genetic relatedness was examined among putative viral double-stranded RNA (dsRNA) genomes of Eimeria acervilina, E. maxima, and E. necatrix using Northern hybridization. No cross-hybridization was found among these dsRNAs, revealing little sequence homology, if any. In addition, dsRNAs from eight E. acervulina strains collected from different localities in the United States were also examined for genetic relatedness. The putative viral dsRNAs from these eight strains, including the Guelph strain, hybridized with one another to varying degrees, indicating that they are related but divergent.
Assuntos
Galinhas/parasitologia , Coccidiose/parasitologia , Eimeria/virologia , Doenças das Aves Domésticas/parasitologia , Vírus de RNA/genética , RNA de Cadeia Dupla/análise , RNA Viral/análise , Animais , Northern Blotting , Genoma Viral , Hibridização de Ácido Nucleico , Vírus de RNA/isolamento & purificação , RNA de Cadeia Dupla/classificação , RNA Viral/classificaçãoRESUMO
Sequences were recently obtained from four double-stranded (ds) RNAs from different plant species. These dsRNAs are not associated with particles and as they appeared not to be horizontally transmitted, they were thought to be a kind of RNA plasmid. Here we report that the RNA-dependent RNA polymerase (RdRp) and helicase domains encoded by these dsRNAs are related to those of viruses of the alpha-like virus supergroup. Recent work on the RdRp sequences of alpha-like viruses raised doubts about their relatedness, but our analyses confirm that almost all the viruses previously assigned to the supergroup are related. Alpha-like viruses have single-stranded (ss) RNA genomes and produce particles, and they are much more diverse than the dsRNAs. This difference in diversity suggests the ssRNA alpha-like virus form is older, and we speculate that the transformation to a dsRNA form began when an ancestral ssRNA virus lost its virion protein gene. The phylogeny of the dsRNAs indicates this transformation was not recent and features of the dsRNA genome structure and translation strategy suggest it is now irreversible. Our analyses also show some dsRNAs from distantly related plants are closely related, indicating they have not strictly co-speciated with their hosts. In view of the affinities of the dsRNAs, we believe they should be classified as viruses and we suggest they be recognized as members of a new virus genus (Endornavirus) and family (Endoviridae).
Assuntos
Evolução Molecular , Vírus de Plantas/genética , Plantas/virologia , Vírus de RNA/genética , RNA de Cadeia Dupla/genética , Replicon/genética , Vírus Defeituosos/genética , Filogenia , Vírus de Plantas/classificação , Plantas/genética , RNA Helicases/química , RNA Helicases/genética , Vírus de RNA/classificação , RNA de Cadeia Dupla/classificação , RNA Viral/genética , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genéticaRESUMO
Human rotaviruses from the states of Rio de Janeiro, São Paulo and Pará of Brazil were analysed by RNA electrophoresis. At least some bands characteristic of rotavirus double-stranded RNA were detected in 138 (86.8%) of 159 faecal samples in which the presence of rotavirus had been demonstrated by enzyme immunoassay. Of the RNA-positive samples, 18 (13.0%) were classified as subgroup 1, 94 (68.1%) as subgroup 2, and 26 (18.8%) could not be classified due to absence of visible bands 10 and 11. Subgroup 2 was more frequent in the three states. All strains of subgroup 1 detected in Rio de Janeiro were associated with a single short-lived school outbreak. All strains of subgroup 1 resembled each other in electrophoretic pattern, irrespective of geographical origin, although minor differences could be detected by co-electrophoresis. Subgroup 2, on the other hand, showed a great degree of electrophoretic heterogeneity and could be divided into several sub-categories.
