Isolation of a Viroid-like RNA from Hop Different from Hop Stunt Viroid

Abstract

SUMMARYA viroid-like RNA was detected in nucleic acid preparations from two of the three commercial hop varieties grown in Spain. It had a size very close to that of avocado sunblotch viroid (ASBV), although dot-blot analysis revealed that it was very different in base sequence from ASBV, coconut cadang-cadang viroid, hop stunt viroid (HSV) and citrus exocortis viroid. In its physical and biological properties, the viroid-like RNA differed from the previously characterized HSV.Viroids are the only class of subviral agents endowed with autonomous replication whose molecular structure is well known. They comprise a single-stranded circular RNA which is highly self-complementary (Diener, 1979) and has a size range from 246 to 375 nucleotide residues (S~inger, 1984;Keese & Symons, 1985;Riesner & Gross, 1985). Viroids cause several diseases of higher plants (Riesner & Gross, 1985), although they can also replicate in other plants without detectable damage to the host (Owens et al., 1978). The last observation led some years ago to the proposal that an increasing number of viroid-like RNAs would be isolated from apparently healthy plants (Diener, 1979). This assumption has been confirmed recently in the case of grapevine (Sano et al., 1985(Sano et al., , 1986Flores et al., 1985). The detection of viroid-like RNAs has been greatly facilitated by the electrophoretic technique of Schumacher et aL (1983), which takes advantage of the very different mobilities of circular and linear RNA molecules of similar size in denaturing polyacrylamide gels. In the present communication we report that by applying this approach to RNA from hops, a new viroid-like RNA has been detected which is different from hop stunt viroid (HSV) described previously .Young leaves and flowers from individual hop plants (Humulus lupulus L.) were taken in the early summer of 1986 from different areas of Le6n (Spain). Samples were collected from the three hop varieties grown in Spain: H-3, H-7 and 'Fino de Alsacia'. Frozen tissue (20 g) was homogenized with a Willems Polytron (Kinematica, Kriens-Lucerne, Switzerland) in an extraction medium: 80 ml of water-saturated phenol, 16 ml 0.2 M-Tris-HC1 pH 8.9, 4 ml 0.1 M-EDTA pH 7, 4 ml 5 ~ SDS and 0.5 ml 2-mercaptoethanol (Semancik & Weathers, 1972). After centrifugation at 6000 g for 15 min the upper aqueous phases were re-extracted with 0.5 vol. of water-saturated phenol. The second aqueous phases were adjusted to a final volume of 40 ml with distilled water and the composition was adjusted to that of STE buffer (50 mM-Tris-HC1 pH 7.2, 100 mM-NaC1 and 1 mM-EDTA) and 35~ ethanol. Following the addition of 2 g of cellulose (CF-11, Whatman) (Franklin, 1966), the mixtures were gently shaken in centrifuge tubes for 10 to 15 min and centrifuged at 1000 g for 5 min. The cellulose pellets were washed three times with 60 ml of 35 ~ ethanol in STE and then with 20 ml of STE. The nucleic acids (and some other accompanying substances) eluted in the last wash were precipitated with 2.5 vol. of ethanol at -20 °C overnight,...

The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in theirin vivoRNA conformations

“…'Redonda morada') were extracted either with phenol-saturated buffer 69 or with a method avoiding organic solvents, 70 and then partitioned on non-ionic cellulose (CF11; Whatman) with STE (50 mM Tris-HCl, pH 7.2, 100 mM NaCl, 1 mM EDTA) containing 35% ethanol. The preparations were further clarified by removing polysaccharides with methoxyethanol.…”

“…71 RNAs were examined by non-denaturing PAGE in 5% gels with 1X TAE (Tris-acetate-EDTA), and by denaturing PAGE in 5% gels with 1X TBE (Trisborate-EDTA) and 8 M urea. 72 For preparative purposes, following double electrophoresis first in non-denaturing and then in denaturing conditions, 69 the second gel was stained with ethidium bromide and the bands of interest, identified with appropriate markers, were excised and the corresponding RNAs eluted.…”

Hop stunt viroid (HSVd) sequence variants from Prunus species: evidence for recombination between HSVd isolates.

“…Lastly, two isolates were provided by R. Flores (Instituto de Biologa Preparation of low molecular mass RNA. Low molecular mass RNA preparations were obtained by phenolic extraction of leaf samples followed by CF-11 cellulose chromatography, as described (Flores et al, 1985 ;Palla! s et al, 1987), or by a modification of the protocol of Flores et al (1991) which eliminates the CF-11 chromatography.…”

Arabidopsis thaliana has the enzymatic machinery for replicating representative viroid species of the family Pospiviroidae

“…Total RNA in the aqueous phase was recovered by ethanol precipitation and resuspended in 0.1 vol of 10 mM Tris⅐HCl (pH 8.0) containing 1 mM EDTA and 98% formamide. Alternatively, viroid-enriched RNAs were obtained by chromatography on nonionic cellulose (CF11, Whatman) (42). For dot-blot analysis, RNA preparations (5 l), boiled for 1.5 min and chilled on ice, were spotted on positively charged Nylon membranes (Roche Diagnostics) and fixed by UV irradiation.…”