J. gen. Virol. (1985), 66, 333 338. Printedin Great Britain
333
Key words: viroid/grapevine/hopstunt viroid
A Viroid-like RNA Isolated from Grapevine Has High Sequence Homology
with Hop Stunt Viroid
By T. S A N O , *
I. U Y E D A ,
E. S H I K A T A , T. M E S H I , 1 T. O H N O z AND
Y. O K A D A 1
Department of Botany, Faculty of Agriculture, Hokkaido University, Sapporo 060, 1Department
of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Tokyo 113 and
"-Department of Agricultural Chemistry, Faculty of Agriculture, Tokyo University of Agriculture
and Technology, Fuchu 183, Japan
(Accepted 8 October 1984)
SUMMARY
Viroid-like R N A was isolated from some samples of grapevine leaves (Vitis vinifera
L.). W h e n inoculated to cucumber, this R N A induced symptoms that were identical to
those induced by hop stunt viroid (HSV). It had a molecular size similar to that of HSV,
and hybridized with a 32p-labelled D N A probe for the cucumber isolate o f H S V (HSVc, pCP-55). This hybridization suggests high ( > 5 5 ~ ) sequence homology with HSV.
The HSV-related sequence was detected not only in the infected cucumber plants but
also directly in R N A extracted from grapevine plants. The result indicates that viroidlike R N A , closely related to HSV, is present in grapevines in Japan.
INTRODUCTION
Viroids are infectious low molecular weight R N A molecules that can cause serious diseases of
higher plants (Diener, 1979). So far, more than t0 viroids, including isolates, have been
sequenced and have been found to comprise 250 to 370 nucleotides (Siinger, 1982; Visvader &
Symons, 1983; Kiefer et al., 1983; Ohno et al., 1983; SanD et al., 1984). A m o n g the viroids
reported from various countries in the world, hop stunt viroid (HSV), which has 297 nucleotides,
was endemic only to Japan (Yamamoto et al., 1970), although most commercial hops in Japan
were introduced from Europe or America. Sasaki & Shikata (1977a, b) showed that cucumber
pale fruit viroid (CPFV), reported only in Europe, was biologically identical to HSV. Recently,
we determined the sequence extending to 303 nucleotides and the secondary structure of C P F V
R N A , and found it to have 9 5 ~ sequence homology with HSV (Ohno et al., 1983; SanD et al.,
1984). We therefore concluded that C P F V was a cucumber isolate of HSV (HSV-c).
In this paper, we report the finding of a viroid-like R N A in grapevines, which also has a high
sequence homology with HSV. This is the first report of a viroid-like R N A in grapevines.
METHODS
Sources of grapevines. Leaves of grapevine plants (Vitis vinifera L.), samples A, B, C, D (Table 1), were collected
in Yamanashi Prefecture and samples E, F, G, H were from lwate Prefecture. Table 1 shows the virus symptoms
in the source plants. Samples A and B are different plants of the same variety. Sample C was from a meristemcultured plant derived from the plant which gave sample D and was free from fan leaf, leaf roll and fleck diseases
(Iri et al., 1982). Samples F and H were from plants not showing virus symptoms but which were not meristemcultured plants (Table 1).
Extraction of low molecular weight RNA. Low mol. wt. RNA was extracted from the grapevine leaves and is
hereafter called grapevine RNA-A, grapevine RNA-B, etc. as described previously (Uyeda et al., 1984). Extracts
from each grapevine sample or sap from cucumber infected with HSV and HSV-c were mechanically inoculated to
cucumber cotyledons (Cucumis satit~usL. cv. Suyo). Low mol. wt. RNA was extracted from these cucumber plants
to give cucumber RNA-C, RNA-D, RNA-HSV and RNA-HSV-c, respectively.
Polyacrvlamidegelelectrophoresis ( PAGE). This was carried out in 15o~,gel in 0.04 M-Tris,0-02M-acetate, 0.001 MEDTA (Loening, 1967). After electrophoresis, gels were stained with ethidium bromide (0-5 ~tg/ml)and observed
using a u.v.-transiUuminator.
0000-6239 (~) ]985 SGM
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334
T. S A N O A N D
OTHERS
Table 1. Infection of cucumber plants by low tool. wt. RNA extracted from grapevines
Sample
A
B
C
D
E
F
G
H
Grapevine cv.
Cabernet Sauvignon
Cabernet Sauvignon
Zenkoji
Zenkoji
Chardonne
Alpha
Weiserblugnder
Weiserblugnder
Symptoms in vine
Virus-free*
Leaf roll, fleck
Virus-free*
Leaf roll, fleck
Leaf roll
Symptomless:~
Leaf roll
Symptomless
Infectivity for cucumber
0/6~
6/6
0/18
13/13
0/3
3/4
4/4
4/4
Virus-free: viruses were eliminated by meristem culture (sample C) or heat therapy (sample A), and plants
were shown to be free from leaf roll, fleck and fan leaf diseases.
t Number of cucumber plants infected/no, inoculated, 6 weeks after inoculation_ Infected plants showed
stunting, leaf curling and vein clearing.
Symptomless: plants showed no symptoms, but virus had not been deliberately eliminated.
*
Northern blot hybridization. 3-~P-labelledDNA probe for HSV-c (pCP-55; Sano et al., 1984), with a specific
activity of 2 x 107 c.p.m./lag DNA, was prepared by 5'-end labelling (Maniatis et al., 1982). The low tool. wt.
RNA was glyoxalated and electrophoresed in 2~ agarose gel (Carmichael & McMaster, 1980), transferred to
nitrocellulose paper (Schleicher & Schfill) and hybridized with the 3zp-labelled DNA probe for HSV-c (5 × 105
c.p.m./ml) (Thomas, 1980). The nitrocellulose papers were then washed, dried and exposed to X-ray films at
70 °C. Fragments of pBR322 DNA formed by HpaII digestion were used as size markers.
-
RESULTS
Symptoms on cucumber plants inoculated with low mol. wt. RNA from grapevines
Samples of low mol. wt. R N A extracted from grapevines were inoculated to cucumber plants.
Those inoculated with grapevine R N A - B , -D, -F, -G and -H became stunted, and showed leaf
curling and vein clearing by 21 to 30 days after inoculation (Table 1). Each extraction induced
similar symptoms (Fig. 1) which were identical to those induced by HSV. The cucumber plants
inoculated with grapevine R N A - A , -C and -E showed no symptoms.
Detection of the specific RNA by PAGE
Grapevine R N A - C , -D, cucumber R N A - D , -HSV and -HSV-c were electrophoresed on a
15 ~ gel (Fig. 2). Cucumber R N A - D (lane d) contained a specific band (fraction 5) which could
not be detected in healthy cucumber R N A (lane c) and which migrated at the same rate as HSV
and HSV-c (lanes a, b, arrow v). However, grapevine R N A - D contained at least three extra
bands (lane f, fractions 1, 2 and 3) which could not be detected in grapevine R N A - C (lane e) but
none of them migrated like the specific band in cucumber R N A - D (arrow v). G r a p e v i n e R N A D did not appear to contain a band corresponding to the specific band of cucumber R N A - D or
the viroid bands of HSV and HSV-c (lane a, b, arrow v).
Infectivity assays of gel fractions
After electrophoresis, gels of cucumber R N A - D and grapevine R N A - D (Fig. 2d, f ) were
sliced into seven fractions as indicated in Fig. 2. R N A was eluted by homogenizing the gel slices
in 0-5 M-ammonium acetate, 0-01 M-magnesium acetate, 0-1 °/o SDS (pH not adjusted). Eluted
R N A was precipitated by ethanol, dried, dissolved in 0.1 M-Tris-HC1 p H 7.5, 0-01 M-EDTA,
0 . 0 5 ~ bentonite and inoculated to cucumber plants.
As shown in Table 2, most infectivity was associated with fraction 5 of either extract and thus
corresponded with the specific band in cucumber R N A - D (Fig. 2, arrow v) but not with any
band detected in grapevine R N A - D by the ethidium bromide staining method. There was no
infectivity associated with the three extra bands in grapevine R N A - D .
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Viroid-like RNA from grapevine
335
Fig. t. Cucumber plants (cv. Suyo) 6 weeks after inoculation with grapevine RNA-D (left), or no
inoculation (right).
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 2. Electrophoresis in 15 ~/opolyacrylamide gel of cucumber RN A-HSV (a), cucumber R N A-HSV-c
(b), healthy cucumber R N A (c), cucumber R N A - D (d), grapevine RNA-C (e) and grapevine R N A - D
(f). All samples were electrophoresed in the same gel. Arrow v indicates the position of HSV and
HSV-c RNA. Gels were sectioned into seven slices as indicated.
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336
T. S A N O A N D O T H E R S
(a)
(b)
(c)
(d)
(:3
(e)
O--~
--622
--527
--404
--309
--242
--217
Fig. 3. Northern blot hybridization analysis of cucumber RNA-HSV (a), healthy cucumber RNA (b),
cucumber RNA-HSV-c (c), grapevine RNA-C (d), grapevine RNA-D (e) and cucumber RNA-D (f).
About 20 ~tg RNA was electrophoresed in each lane. O indicates the origin of electrophoreses. The
nucleotide numbers in co-electrophoresed glyoxalated DNA fragments of HpalI-digested pBR322 are
shown on the right.
Table 2. Infectivity of gel fractions from sample D on cucumber plants
Fraction no.
A
t
Sample
Grapevine RNA-D*
Cucumber RNA-D x 1t
Cucumber RNA-D x 101"
1
0:~
1
0
2
0
0
0
3
0
2
0
4
0
3
1
5
2
4
4
6
0
0
0
0
0
0
* Low mol. wt. RNA extracted from grapevine sample D.
t Low mol. wt. RNA extracted from cucumber inoculated with grapevine sample D undiluted ( x 1) or diluted
10-fold (x 10).
Number of cucumber plants infected 8 weeks after inoculating four plants with each sample.
Cross-hybridization among the infectious RNA in grapevine extracts and cDNA of HSV-c
Samples of 20 ~tg of grapevine R N A - C , grapevine R N A - D , cucumber R N A - D , -HSV and
-HSV-c were denatured and electrophoresed in 2 ~ agarose in 0.1 M-sodium phosphate buffer
pH 7.0. After electrophoresis, the R N A was transferred to nitrocellulose paper and hybridized
with the 3zp-labelled D N A probe for HSV-c. The probe hybridized with cucumber R N A - H S V
and -HSV-c, and with both cucumber and grapevine R N A - D , but not with grapevine R N A - C or
healthy cucumber R N A (Fig. 3). These results suggested that the infectious R N A species in
grapevine plants were related to HSV.
In further tests, R N A eluted from gel fractions 1 to 6 of cucumber R N A - D (Fig. 2d), which
were the same preparations used for infectivity assays in Table 2, were electrophoresed
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337
Viroid-like R N A from grapevine
2
3
4
5
6
7,
0--~
-622
-527
-404
--309
--242
-217
Fig. 4. Northern blot hybridization analysisof RNA eluted from each of six fractions of the 15% polyacrylamide gel in (d) of Fig. 2. Lanes 1 to 6, RNA eluted from gel fractions 1 to 6; lane 7, cucumber
RNA-D. O indicates the origin of electrophoresis. Markers are as in Fig. 3.
separately in a 2 ~ neutral agarose gel, blotted and hybridized as above. As shown in Fig. 4, the
probe hybridized only with R N A from fraction 5.
The molecular size of the RNA that hybridized with c D N A to HSV-c was about 300
nucleotides. Fig. 3 shows that some species of RNA about 600 nucleotides and larger were
detected in addition to the major component of 300 nucleotides.
DISCUSSION
We have extracted low molecular weight RNA from eight different grapevines and five of
these preparations, when inoculated to cucumber, induced symptoms identical to those induced
by HSV.
PAGE revealed that a specific band in cucumber RNA-D migrated at the same rate as HSV.
Furthermore, infectivity was associated with this band or, in grapevine extracts, with
presumptive RNA having the mobility of this band but not detected, probably because of its low
concentration in the grapevine plants used.
Northern blot hybridization analysis demonstrated sequence homology between the
infectious RNA from grapevine and HSV-c, and that this infectious R N A can be detected using
a cDNA probe for HSV-c. In previous work (Sano et al., 1984), we found that, under the same
hybridization conditions as used in this work, a D N A probe for H S ¥ could detect HSV-c but not
potato spindle tuber viroid, which have 95 ~ and 55 ~ sequence homology to HSV respectively.
Accordingly, we consider the sequence homology between the infectious RNA of grapevine and
HSV is high, because it is more than 55 ~. We therefore conclude that this R N A is a viroid
because (i) it is infectious, (ii) its molecular size is about 300 nucleotides and (iii) it hybridizes
with a D N A probe for HSV-c.
The RNA species of about 600 nucleotides and larger detected by Northern blot hybridization
in grapevine and cucumber RNA-D in Fig. 3 may correspond to the longer than unit length
RNA species reported for HSV and other viroids (Ishikawa et al., 1984).
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338
T. SANO AND OTHERS
W e c a n n o t be sure that the infectious R N A causes a p a r t i c u l a r disease, because only eight
g r a p e v i n e plants were e x a m i n e d in these experiments. M o r e o v e r , a l t h o u g h four had l e a f roll
a n d / o r fleck diseases, no infectious R N A was isolated in sample E infected w i t h leaf roll disease
and s a m p l e H, s h o w i n g no visible s y m p t o m , c o n t a i n e d infectious R N A . F u r t h e r w o r k is n e e d e d
to survey the o c c u r r e n c e of the infectious R N A in g r a p e v i n e s and to assess its disease potential.
This and e x p e r i m e n t s i n v o l v i n g inoculation of infectious R N A into i n d i c a t o r plants for
g r a p e v i n e viruses h a v e been initiated but m a y take 2 to 3 years to complete.
Because the infectious R N A was not found in the virus-free g r a p e v i n e sample C, w h i c h was
d e r i v e d f r o m sample D, it m a y be possible to e l i m i n a t e the infectious R N A f r o m g r a p e v i n e by
m e r i s t e m culture.
As well as the infectious R N A , three other low m o l e c u l a r w e i g h t R N A species were d e t e c t e d
in the g r a p e v i n e affected with leaf roll and fleck diseases. T h e s e R N A s did not infect c u c u m b e r
plants and their nature and roles are u n k n o w n .
As far as we are aware, this is the first report o f a viroid-like R N A in g r a p e v i n e .
We thank Dr K. Ueno and Mr M. Iri, Mann's Wine Co. Ltd., for supplying virus-free and diseased grapevines.
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