Detection of Viroid and Viroid-like RNAs from Grapevine

Abstract

SUMMARYAnalysis by polyacrylamide gel electrophoresis of nucleic acid preparations, obtained from several varieties of grapevine by a procedure designed to isolate and purify viroids, revealed the presence of RNA species with some of the characteristic physical properties of viroids. Under non-denaturing conditions, a band with a mobility faster than that of citrus exocortis viroid (CEV) was detected, and under fully denaturing conditions two bands were observed, one co-migrating with the circular forms of CEV and a second migrating faster than the linear forms of this viroid. This RNA species did not hybridize with a cDNA probe to CEV. Some of the grapevine preparations were infective for Gynura aurantiaca, inducing symptoms similar to those caused by CEV, and the appearance of an RNA which had the same mobility as CEV in denaturing and non-denaturing electrophoretic systems and hybridized with cDNA to CEV. These results suggest that viroid-like and viroid RNAs can be recovered from grapevine, the former (with no detectable sequence homology to CEV) at a concentration sufficient to be observed as a physical entity in gels, and the latter (with close sequence homology to CEV) whose presence could only be revealed by bioassay. The possible involvement of these RNAs in some grapevine diseases of unknown aetiology is discussed.

Relationships among the viroids derived from grapevines

“…GYSVd and GVd-IB have been reported to induce yellow speckle disease symptoms and the distribution of GVd-I in grapevines also suggests an association with the disease (Flores et al, 1985).…”

“…W h e n a n i n o c u l u m p r e p a r a t i o n c o n t a i n i n g a p a r t i a l l y purified m i x t u r e o f only G V d -1 a n d G (Flores et al, 1985). Autoradiograph of a slot-blot containing pure CEVd-g, GVd-c and GVd-1 in three reaction sets of 1 x, 1/2 x, and 1/4 x after hybridization with cDNA probes synthesized to CEVd-g, GVd-I and GVd-c by the random priming procedure.…”

“…Other variants of HSVd-g have also been reported by Duran-Vila et al (1987) and Puchta et al (1988). Similarly, grapevine viroid-slow (GVd-s) (Flores et al, 1985) is a variant of citrus exocortis viroid (CEVd) designated as CEVdgrapevine (CEVd-g) by Garcia-Arenal et al (1987). The original designation of grapevine viroid-fast (GVd-f) (Flores et al, 1985) was recognized as GVd-1 after detection of a fourth grapevine viroid, GVd-2 (Semancik et al, 1987).…”

Hop Stunt Viroid Strains from Dapple Fruit Disease of Plum and Peach in Japan

“…After staining with ethidium bromide, the segment of the gel corresponding to the position where viroids (ranging from hop latent viroid to citrus exocortis viroid) were electrophoresed, was cut out from each lane and electrophoresed further on a 5 ~ gel containing 8 M-urea under denaturing conditions (Flores et al, 1985). A viroid-like RNA detected from both DF-plum and DF-peach AF, corresponding to the viroid-like circular RNAs in Fig.…”

“…(1984) with a slight modification. Frozen tissues were homogenized in 130 mMTris-HC1 pH 8.9, 17 mM-EDTA pH 7.0, 0-83 ~ SDS, 1 M-LiC1, 5 ~ polyvinylpyrrolidone and 1 ~ mercaptoethanol (Flores et al, 1985), instead of 1 M-K2HPO 4. After phenol extraction, 2-methoxyethanol extraction, cetyltrimethylammonium bromide precipitation, 2 M-LiCl fractionation and DNase I digestion, the RNA molecules were absorbed to CF-11 cellulose in a buffer containing 35~ ethanol, washed with the same buffer and then eluted with ethanol-free buffer.…”