Grapevine phloem-limited
specific cell organelle
viruses
induce
vesiculation
of a
F.Faoro, S.Sant
CNR, Centre Miglioramento Sanitario Colture Agrarie, Via Celoria 2,20133 Milano, Italy
Grapevine fleck virus (GFKV) and grapevine leafroll closteroviruses 1 and 3 (GLRV 1, GLRV 3; are
three world-wide spread grapevine-phloem limited viruses. The former is a taxonomically still
unclassified isometric virus, 30 nm in dia, while the others are two species of the genus
Closterovirus (family: Closteroviridze) with filamentous particles of 12x1800-2200 nm. All of them
are ssRNA+ viruses that induce vesiculation of cell organelles, possibly as a consequence of the
binding of the replication complex (Helicase-RNA dependent RNA polymerase) to the cell
membranes [ 11. In the case of GFKV it was reported that vesicles can be originated Corn both
mitochondria and plastids [2], while for GLRV 1 and 3 it seemed that the vesiculating membranes
were mainly those of mitochondria [3]. However it could not be excluded that vesicles derived also
from the endoplasmic reticulum (ER) and the tonoplast [3]. As it has been recently demonstrated that
in the case of some Tonzbusviridae the binding of the replication complex to a membrane is highly
specific [4] we have examined the ultrastructure of numerous grapevine plants of different cultivars
infected either with GFKV or GLRV 1 or GLRV 3, in order to establish for each virus species
whether the vesiculation process involves only a specific cell organelle rather than different cell
membranes.
Leaf midribs were excised from infected plants grown either in controlled experimental
fields or in vitro. They were cross-cut in 0.5-lmm pieces, fixed in a mixture of 2% glutaraldehyde
(Glu) +2.5% paraformaldehyde, postfixed in 1% osmium tetroxide (OS), dehydrated in ethanol and
embedded in Epon-Araldite or London Resin White. Part of the samples were not osmicated to allow
gold labelling experiments with sera against virus coat proteins or with RNAse. Alternatively,
samples were simultaneously fixed in a mixture of 2%Os + 2%Glu + 0.15% picric acid+l% sucrose
to improve vesicle resolution.
Ultrastructural investigations demonstrated that all the above viruses induce vesiculation
of mitochondrial membranes only. This was particularly evident in parenchyma and companion cells
showing early and intermediate stages of infection when all cell organelles were still recognisable
and large aggregates of virus particles were not present yet. GFKV-induced vesicles were doublemembrane bound (Fig.l), thus easily distinguishable from those induced by GLRV 1 and 3 that were
surrounded by a single membrane (Figs.2,3). Furthermore vesiculating mitochondria induced by
GLRV 1 (Fig.2) were slightly different from those found in GLRV 3 infected cells (Fig.3). The
finely stranded content of vesicles was labelled by RNAse-gold confirming that it was mainly
composed of RNA strands, possibly the viral replicative form. The above data, besides demonstrating
that also these grapevine viruses replicate only on a specific cell membrane, point out that the
different types of vesiculating mitochondria can be used for diagnosis .
References
1.
2.
3.
4.
Buck K.W., Advances in Virus Research,47 (1996) 159.
Castellano M.A. et al., Phytopathologia mediterranea, 24 (1985) 165
Faoro F., Filamentous viruses of woodyplants,p.29 Monette P. ed., Research Signpost, Trivandrum, India (1997)
Burgyan J. et al, J.GeneraZ Virology, 77 (1996) 1967.
FIGS. 1-3 - Cross sections of parenchyma phloem cells of grapevine cv. “Barbera” infected with
GFKV, GLRV 1 and GLRV 3 respectively, and showing mitochondria in early (a), intermediate (b)
and late (c) stages of vesiculation; CW= cell wall, V= virus particles; all bars = 100 nm.
FIG. 1 - Vesicles are double-membrane bound being generated by invagination of both membranes
of the mitochondrial envelope (arrows)
FIG. 2 - Vesicles form a characteristic crown around the mitochondria.
FIG. 3 - Vesicles are formed between the two mitochondrial membranes (arrows)