Characterization of Closterovirus‐like Particles Associated with Grapevine Leafroll Disease

Abstract

A purification procedure (Zeeet al. 1987) for closterovirus‐like particles associated with grapevine leafroll disease was improved after various sources of plant tissues, extraction buffers, pH values, and centrifugation techniques had been examined. An antiserum against the CA‐4 isolate was produced and used in ELISA. The virus‐like particles of the NY‐1 isolate measured in crude plant preparations were about 1,800 to 1,900 nm long. The molecular weight of coat protein of the NY‐1and NY‐2 isolates were ca. 43 × 103 daltons in SDS‐PAGE analysis; and bands having this molecular weight reacted in Western blotting tests with specific polyclonal and monoclonal antibodies. A large dsRNA molecule (ca. 10 × 106 Mr) along, with lower molecular weight species were isolated from leafroll diseased grapevines, but not from healthy grapevines. Seven leafroll isolates were tested for their serological relatedness in a protein A‐gold labelling immunosorbent electron microscopy, assay. Results indicated that serologically distinct serotypes existed, and mixed infection of grapevines with different scrotypes was common. A grouping of isolates into serotype I, II, III, IV is proposed.

Nucleotide sequence and genome organization of grapevine leafroll-associated virus-2 are similar to beet yellows virus, the closterovirus type member.

“…1 a). The yield of GLRaV-2 dsRNA was estimated to be 5-10 ng\15 g phloem tissue, which was much lower than that for GLRaV-3 (Hu et al, 1990 ; data not shown). Only the high molecular mass dsRNA that was purified from low melting point agarose gel was used for cDNA synthesis, cloning and establishment of the Lambda ZAPII cDNA library.…”

“…Pinot Noir that originated from a central New York vineyard served as the source for dsRNA isolation and cDNA cloning. DsRNA was extracted from phloem tissue of infected grapevines according to the method described by Hu et al (1990). Purification of the high molecular mass dsRNA (ca 15 kb) was carried out by electrophoretic separation of the total dsRNA on a 0n7 % low melting point agarose gel and extraction by phenol-chloroform following the method described by Sambrook et al (1989).…”

“…The MBP-CP fusion protein was induced by adding 0n3 mM IPTG and purified on a one-step affinity column according to the manufacturer's instructions (New England Biolabs). The MBP-CP fusion protein or the CP cleaved from the fusion protein were tested for reaction with specific antiserum to GLRaV-2 (kindly provided by Charles Greif of INRA, Colmar, France) on Western blot according to the method described by Hu et al (1990).…”

“…At least seven serologically distinct closteroviruses have been isolated from leafroll diseased vines and designated as grapevine leafroll-associated closteroviruses-1 to -7 (Gugerli et al, 1984 ;Rosciglione & Gugerli., 1986 ;Zee et al, 1987 ;Hu et al, 1990 ;Zimmermann et al, 1990 ;Gugerli & Ramel, 1993 ;Boscia et al, 1995 ;Choueiri et al, 1996). Grapevine leafrollassociated closterovirus-2 (GLRaV-2) was first reported by Author for correspondence : Dennis Gonsalves.…”

“…The coat protein (CP) of GLRaV-2 is ca. 22-26 kDa (Zimmermann et al, 1990 ;Gugerli & Ramel, 1993 ;Boscia et al, 1995), which is considerable smaller than other GLRaVs (35-43 kDa) (Zee et al, 1987 ;Hu et al, 1990 ;Zimmermann et al, 1990 ;Ling et al, 1997). Although G. P. Martelli (circular of ICTV Plant Virus Subcommittee Study Group on Closterolike Viruses, 1996) classified GLRaV-2 as a definitive member of the genus Closterovirus based on particle morphology and cytopathology, its molecular and biochemical properties are not well characterized.…”