Double-stranded RNA Elements Research Articles

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.

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.

Detection of double-stranded RNA molecules and virus-like particles in different Mucor species.

The presence of double-stranded RNA elements was examined in 123 strains representing 18 Mucor species. These genetic elements were found to be present in 6 strains: 1 M. aligarensis, 1 M. hiemalis, 2 M. corticolus, 1 M. mucedo and 1 M. ramannianus. Electrophoretic separation of the nucleic acids revealed 4 different RNA patterns, with 1 to 5 discrete dsRNA bands. The molecular weights corresponding to these bands were 1.42-4.15 x 10(6) D. Using electronmicroscopy, for the first time the presence of virus like particles in Mucor species has been revealed.

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.

Unencapsidated double-stranded RNA associated with membrane vesicles in isolates of Alternaria solani

Double-stranded RNA (dsRNA) elements of 8·3 and 5·5 kbp were detected in three of five geographically distinct isolates of Alternaria solani. Variation with respect to the number of dsRNA segments was detected among isolates of the fungus. No virus-like particles were purified from the mycelium, or observed in ultra-thin sections of hyphae. Utilizing differential centrifugation or density gradient centrifugation for subcellular fractionation, dsRNA was found associated specifically with fractions containing 80–100 nm spherical membrane vesicles. Similar vesicles were observed in cytoplasm of hyphae by electron microscopy. Treatment of dsRNA-containing fractions with ribonuclease A did not degrade the dsRNA, suggesting that it is located inside the vesicles. There was no correlation between the radial growth of the isolates in laboratory media and the presence or absence or dsRNA.

Detection of plasmid-like DNA and double-stranded RNA elements in some Canadian isolates of the oilseed rape pathogen Leptosphaeria maculans

Nine Canadian isolates of Leptosphaeria maculans were examined for the presence of plasmid-like elements. A single extrachromosomal DNA element, with an estimated size of 9 kb, was detected in undigested genomic DNA of a virulent isolate, Fairview 1. This element was susceptible to hydrolysis by DNAse I and exonuclease III, and it was shown to hybridize to DNA of all virulent isolates. In addition, another virulent strain, Saskatoon 8, was observed to contain four double-stranded RNA (dsRNA) segments, ranging from approximately 500 bp to 2.4 kb. The latter segments (dsRNA) were resistant to DNAse I and exonuclease III treatment, and to RNAse A in high-ionic-strength buffer, but were susceptible to RNAse A in the presence of low-ionic-strength buffer.Key words: Leptosphaeria maculans, Canola, double-stranded RNA, plasmids.

High-frequency occurrence of virus-like particles with double-stranded RNA genome in Fusarium poae

Fifty-five geographically different strains of Fusarium poae were assayed for the presence of extrachromosomal nucleic acid elements. All strains were found to harbour double-stranded RNA (dsRNA) elements and encapsidated virus-like particles (VLP). There were great individual differences in dsRNA patterns of the various strains, but numbers and sizes characteristic for a given isolate remained unchanged after repeated subculturing of the fungi. Morphological alterations or signs of degeneration were not observed in dsRNA-containing isolates. This is the first report on the ubiquitous occurrence of dsRNAs in a hyphomycete fungus species.

High-frequency occurrence of virus-like particles with double-stranded RNA genome in Fusarium poae.

Fifty-five geographically different strains of Fusarium poae were assayed for the presence of extrachromosomal nucleic acid elements. All strains were found to harbour double-stranded RNA (dsRNA) elements and encapsidated virus-like particles (VLP). There were great individual differences in dsRNA patterns of the various strains, but numbers and sizes characteristic for a given isolate remained unchanged after repeated subculturing of the fungi. Morphological alterations or signs of degeneration were not observed in dsRNA-containing isolates. This is the first report on the ubiquitous occurrence of dsRNAs in a hyphomycete fungus species.

Double-Stranded RNA Induces Sickle Erythrocyte Adherence to Endothelium: A Potential Role for Viral Infection in Vaso-Occlusive Pain Episodes in Sickle Cell Anemia

Vaso-occlusive pain episodes in sickle cell anemia are hypothesized to be precipitated by adherence of sickle erythrocytes to vascular endothelium in the microcirculation. Febrile episodes, thought to be viral in etiology, are frequently associated with vaso-occlusion; however, a direct link between viral infection and vascular occlusion has not yet been established. Many pathogenic viruses contain double-stranded RNA or replicate through double-stranded RNA intermediates. Double-stranded RNA has been shown to induce vascular cell adhesion molecule-1 (VCAM-1) protein expression on endothelial cells. Recently, a new adhesion pathway has been described between VCAM-1 expressed on cytokine stimulated endothelium and the alpha 4 beta 1 integrin complex expressed on sickle reticulocytes. Based on these observations, the hypothesis was developed that viral infection, through double-stranded RNA intermediates, increases endothelial VCAM-1 expression leading to sickle erythrocyte adhesion to endothelium via an alpha 4 beta 1-VCAM-1--dependent mechanism. In support of this hypothesis, endothelial cells exposed to the synthetic double-stranded RNA poly(I:C) or the RNA virus parainfluenza 1 (Sendai virus) express increased levels of VCAM-1 and support increased sickle erythrocyte adherence under continuous flow at 1.0 dyne/cm2 shear stress as compared with unstimulated endothelium. Blocking antibodies directed against either VCAM-1 on the endothelium or alpha 4 beta 1 on sickle erythrocytes inhibit nearly all of the increased sickle cell adherence caused by poly(I:C) or Sendai virus. These results support the hypothesis that viruses, through double-stranded RNA elements, can induce sickle erythrocyte adherence to endothelium through alpha 4 beta 1-VCAM-1--mediated adhesion and provide a potential link between viral infection and microvascular occlusion precipitating sickle cell pain episodes.

Spontaneous Appearance of Genetically Distinct Double-Stranded RNA Elements inRhizoctonia solani

A tuberborne sclerotium of the plant-pathogenic basidiomycete Rhizoctonia solani gave rise to a culture (Rhs 1AP) that exhibited a sector (Rhs 1A1) with reduced pigmentation and growth rate. Rhs 1AP is virulent on potato, which is the only known host plant for Rhs 1AP. In contrast, Rhs 1A1 is hypovirulent or nonpathogenic on potato. The virulent isolate Rhs 1AP contains two double-stranded RNAs (dsRNAs) of 23 and 6.54 kb, whereas the hypovirulent Rhs 1A1 possesses three dsRNAs in addition to the two found in Rhs 1AP [...]