K.W. Buck

Semi-conservative replication of double-stranded RNA by a virion-associated RNA polymerase

Abstract 5-Bromo-UTP was found to replace UTP efficiently as a substrate for the virion-associated double-stranded RNA replicase of Penicillium stoloniferum virus PsV-S. The double-stranded RNA product of the replication reaction with 5-bromo-UTP as a substrate gave in equilibrium caesium sulphate density gradient centrifugation a single band with a buoyant density of 1.647 g/ml, consistent with that of a hybrid double-stranded RNA consisting of one brominated and one unbrominated strand. After the reaction none of the original unbrominated double-stranded RNA (buoyant density 1.606 g/ml) could be detected. It is concluded that replication of double-stranded RNA in virions of PsV-S takes place by a semi-conservative mechanism.

Negatively supercoiled DNA from plants infected with a single-stranded DNA virus

A method for isolating covalently closed circular double-stranded DNA from plants infected with the geminivirus, tomato golden mosaic virus, is described. Ethidium bromide titration showed this DNA to be negatively supercoiled with a superhelical density of -0.062. The presence of S1 nuclease-sensitive secondary structure in the supercoiled DNA was demonstrated by its conversion to the open circular and linear DNA forms on treatment with this enzyme.

RNA polymerase activity in double-stranded ribonucleic acid virus particles from Aspergillus foetidus

Abstract RNA polymerase activities have been detected in purified particles of Aspergillus foetidus viruses S and F. Incorporation of [3H]-UTP into acid insoluble RNA was dependent on ATP, GTP, CTP and magnesium ions. No pretreatment of the particles was required and the rate of reaction was proportional to the amount of virus added. In the conditions used RNA synthesis by A. foetidus virus S was complete in 4 h. The reaction could be stimulated by Triton X-100, but was unaffected by heat shock, dithiothreitol, potassium chloride or ammonium chloride; it was inhibited by ethidium bromide but not by actinomycin D. The major reaction product was single-stranded RNA, as indicated by its sensitivity to degradation by ribonuclease A. This is the first report of synthesis of single-stranded RNA by a double-stranded RNA mycovirus.

Characterisation of multimeric DNA forms associated with tomato golden mosaic virus infection

SummaryHomodimeric and trimeric double-stranded DNA forms of both components of the genome of the geminivirus, tomato golden mosaic virus have been isolated from infectedNicotiana tabacum plants and characterised.

Cassava latent virus specific DNAs in mosaic diseased cassava of Nigerian origin

SamenvattingHet genoom van het ‘cassava latent’ virus (CLV), een geminivirus, bestaat uit twee cirkelvormige, enkelstrengige DNA-moleculen nl. DNA 1 (2,78 kb) en DNA 2 (2,72 kb). DNA, verkregen uitNicotiana benthamiana-planten, die mechanisch waren geïnoculeerd met sap van natuurlijk geïnfecteerde cassaveplanten, bevat naast het enkelstrengige genoom-DNA en de corresponderende open, lineaire en via covalent-bindingen gesloten cirkelvormige, extra-getwiste, dubbelstrengige vormen, een enkelstrengig DNA, dat kleiner is dan het genoom-DNA (c. 1,3 kb). Van dit DNA is aangetoond, dat het fungeert als een defect DNA. Het kleinere DNA interfereert met de functies van het genoom-DNA waardoor de plant minder hevige ziektebeelden gaat vertonen. Het was echter nog niet bekend of dezelfde DNA-vormen in het veld voorkomen in natuurlijk geïnfecteerde cassaveplanten met mozaïeksymptomen. In het hier beschreven onderzoek is aangetoond, dat de DNA-vormen in natuurlijk geïnfecteerde cassaveplanten overeenkomen met die in kunstmatig met het CLV geïnfecteerdeN. benthamiana-planten. De hoeveelheid DNA, kleiner dan het genoom, wordt echter sterk verhoogd door passage van het virus van cassave naarN. benthamiana. De mogelijkheid, dat de hoeveelheid van het DNA, kleiner dan het genoom, in mozaïek-vertonende cassaveplanten gecorreleerd is met de mate van symptoomexpressie wordt besproken.

The behaviour of tomato golden mosaic virus DNA in cultured cells isolated from systemically infected tobacco leaves

When callus tissue was cultured from leaf pieces taken from a Nicotiana tabacum cv. Xanthi nc. plant systemically infected with tomato golden mosaic virus (TGMV), TGMV-specific DNA persisted for up to 6 months in culture. Analysis of TGMV-specific intracellular DNA forms indicated a decrease in double-stranded relative to single-stranded forms and an increase in sub-genomic relative to genomic single-stranded DNA species in the callus tissue compared to those in the original leaf explant. The implications of the results with regard to TGMV replication are discussed.

Transcriptase activity assoclated with a type 2 double-stranded RNA mycovirus

Abstract It is shown that the virion-associated RNA polymerase of Phialophora virus A, a type 2 double-stranded RNA mycovirus with a genome consisting of three RNA species, is a transcriptase. Synthesis of single stranded RNA in vitro continues for a least 24 hours and after this time two full length transcripts are produced, on average, per dsRNA molecule, i.e.re-initiation of transcription occurs in this in vitro system. Analysis of the products by polyacrylamide gel electrophoresis indicated that the efficiences of transcription of all three double-stranded RNA species were similar. The transcriptase activity of Phialophora virus A differs from the replicase activity of Penicillium stoloniferum virus S, the only other type 2 double-stranded RNA mycovirus whose RNA polymerase has been characterised.

Systemic infection of petunia by mechanical inoculation with tomato golden mosaic virus

SamenvattingAangetoond werd datPetunia hybrida systemisch kan worden geïnfecteerd met het ‘tomato golden mosaic virus’ (TGMV), een virus dat behoort tot de groep van de geminivirussen. Mechanische inoculatie van petuniaplanten met TGMV gaf in de systemisch geïnfecteerde bladeren symptomen, die eerder in een aantal andere Solanaceae waren waargenomen. Daar in eerdere proeven petunia niet met TGMV kon worden geïnfecteerd en DNA-replicatie en symptoomontwikkeling wel optrad in, voor de beide genomen van het virus, transgene planten, werd gesuggereerd dat het hier een geval betrof van uitbreiding van de waardplantenreeks.De hier gepresenteerde resultaten kunnen echter tot andere conclusies leiden. Het is namelijk mogelijk, dat bepaalde F1-hybriden van petunia resistenter zijn tegen het virus. Verschillen in de symptoomontwikkeling zijn echter ook niet uit te sluiten en zouden veroorzaakt kunnen worden door premunitie als gevolg van de aanwezigheid van het manteleiwit in opnieuw geïnfecteerde cellen.

Apparently identical viruses from Gaeumannomyces graminis var. Tritici and Phialophora sp. (lobed hyphopodia)

Two double-stranded RNA viruses were obtained from a field isolate of Gaeumannomyces graminis var. tritici (GGT). One of these was shown to be related to previously described GGT viruses, the other was indistinguishable from a virus obtained from an isolate of Phialophora sp. (lobed hyphopodia). The implications of this finding for the epidemiology of virus infection in GGT are discussed.

Widespread inhibitor production in culture by isolates of Gaeumannomyces graminis var. tritici

Isolates of the take-all fungus, Gaeumannomyces graminis var. tritici (GGT), produce a diffusible fungal growth inhibitor (Q factor) in the pH range 3.5–5.0. On potato dextrose agar (PDA) medium this property is limited to about 14% of field isolates. However on glucose/asparagine (LB) and sucrose/urea (MB-50) agar media at pH 4.0, Q factor was produced by 15 out of 16 GGT isolates which did not produce it on PDA medium. Out of a total of 30 GGT isolates tested, 29 produced Q factor on LB and MB-50 media. Single conidial isolates of GGT which were infected with double-stranded RNA viruses from three different groups produced similar amounts of Q factor to derived isochromosomal ascospore isolates which were completely free from virus particles and double-stranded RNA. In the conditions in which the inhibitor was produced the GGT isolates showed negligible growth and there is evidence that Q factor inhibits the isolates which produce it. Three isolates of a Phialophora sp. with lobed hyphopodia and one isolate of G. graminis var. graminis grew well at pH 4.0 on LB and MB-50. None of these produced Q factor, but all were sensitive to the inhibitory action of Q factor produced by GGT isolates. In LB liquid medium at pH 4.0 all of seven GGT isolates tested produced Q factor which in culture filtrates showed no loss of activity after 30 days at 4 °C.