Download Particles of Rice Gall Dwarf Virus in Thin Sections of Diseased Rice

J. gen. Virol.
(1985), 66, 2581-2587. Printed in Great Britain
2581
Key words: RGDV[insect/particles
Particles of Rice Gall Dwarf Virus in Thin Sections of Diseased Rice
Plants and Insect Vector Cells
ByTOSHIHIRO
O M U R A , 1. H I R O Y U K I H I B I N O , 2 H I T O S H I I N O U E 3 AND
TSUNEOTSUCHIZAKI
1
~National Agriculture Research Center, Tsukuba Science City, Ibaraki 305, Japan,
2The International Rice Research Institute, P.O. Box 933, Manila, Philippines and
3gyushu National Agricultural Experiment Station, Chikugo, Fukuoka 833, Japan
(Accepted 5 September 1985)
SUMMARY
Rice gall dwarf virus (RGDV) was studied in infected rice plants and in its principal
insect vector, the green leafhopper Nephotenix nigropictus, by electron microscopy. In
plants, virions were restricted to the cytoplasm and vacuoles of phloem-related cells
and of gall cells generated from the phloem. In the insects, virions occurred in the
cytoplasm of many different kinds of cells including those of the salivary gland, the gut,
the fat body, muscles and nerves. Virions in both plant and insect cells had dense cores
40 to 50 nm in diameter which were surrounded by a less dense region; the total
diameter was 60 to 70 nm. Viroplasms, crystals and tubules were observed in cells both
of plants and insects. Although virions often occupied most of infected plant cells, they
occurred only in restricted areas of insect cells. The restriction of virions to phloemrelated cells in infected plants probably explains why yields of purified RGDV are
usually about 1/30 of those of rice dwarf virus, which invades many types of cell. About
3 % of the virus particles seen in thin sections of infected plants appeared to be empty.
Empty particles were purified from infected rice plants and were found to lack nucleic
acid and much of the 56000 tool. wt. protein present in intact particles.
INTRODUCTION
Rice gall dwarf virus (RGDV) is a recently described phytoreovirus (Omura et al., 1980, 1982)
which is transmitted in a persistent manner by leafhoppers (Inoue & Omura, 1982; Morinaka et
al., 1982). RGDV possesses polyhedral particles about 65 nm in diameter, a genome of 12
segments of dsRNA (Hibi et al., 1984), seven capsid proteins (Omura et al., 1985) and an RNAdependent RNA polymerase (Yokoyama et al., 1984).
In the present paper, we report the morphology and distribution of RGDV particles in
infected plants and in viruliferous Nephotettix nigropictus, and compare RGDV with other plant
reoviruses. The distribution in infected plants of RGDV was compared with that of another
phytoreovirus, rice dwarf virus (RDV) (Shikata, 1966), in an attempt to explain the lower yield
(1/30 to 1/50) of RGDV compared with that of RDV (T. Omura, unpublished data).
In previous work (Omura et al., 1985), core particles were prepared by removing outer capsid
proteins using CsC1, and it was concluded that the 150 000 mol. wt. (150K) and 45K proteins are
located on the surface of the outer capsid, that the 183K and 120K proteins are on the surface of
the core, that the 165K and 56K proteins are inside the core, and that the 143K protein is either
inside the outer capsid or inside the core. During the course of our observations of thin sections
of RGDV-infected cells, the inner part of the virus particles appeared to be electron-dense and
may therefore correspond to the core particle. Also, a few particles lacked the electron-dense
inner part and appeared to be empty. Such particles were obtained from infected plants when
large amounts of material were used for purification. We have studied proteins and nucleic acids
of intact and empty particles and cores to determine the location of nucleic acids and proteins
among the virus particle types with reference to the forms observed in ultrathin sections.
0000-6692 © 1985 SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:30:03
2582
T. OMURA AND OTHERS
METHODS
h!li,cted plants and insects. A virus-free colony of N. nigropictus was maintained on rice plants in an air-
conditioned room (23 to 27 "C). Viruliferous insects were obtained by releasing second or third instar nymphs on
diseased phmts for 2 days and then maintaining them for 12 days on healthy seedlings. Seedlings of rice (var.
Taichung Native 1) at the second to third leaf stage were inoculated with RGDV using the viruliferous insects, and
the inoculated seedlings were grown in a controlled-environment greenhouse kept at 24 to 28 °C.
Electron microscopy. Leaf samples were collected from plants 30 days after inoculation, or from healthy plants.
Samples were fixed with 2-5% glutaraldehyde in 0-1 M-phosphate buffer pH 7-0 (PB) for 2 h at 0 °C, washed with
cold PB, post-fixed m 2% osmium tetroxide in PB for 3 h at 6 °C, dehydrated in an acetone series, and embedded in
Epon 812. Thin sections were cut transversely from samples using a diamond knife mounted on a Sorvall MT2-B
ultramicrotome, stained with uranyl acetate and lead citrate and examined in a Hitachi H-500 electron
microscope.
Sections about I ~tm thick were stained with 0.1% toluidine blue in 1% sodium borate for 10 min and examined
in a light microscope.
Viruliferous insects were cut open in a drop of 0.01 M-PB containing 0.850/0 sodium chloride and 2.5~
glutaraldehyde and their heads, salivary glands and abdomens were excised and kept in fixative at 6 °C for 1 h.
After washing with 0.85~ sodium chloride in PB at 6 °C, tissues were post-fixed with 1~ osmium tetroxide and
processed as described for plant tissue.
Purified preparations were negatively stained with 2% neutralized phosphotungstic acid (PTA). The
appearance of the particles was confirmed using 2% uranyl acetate.
Preparation (?/'empty and core particles. Virus was purified from infected plants as described by Omura et al.
(1982). When large amounts of tissue were processed, a faint band was observed about 3/20 of the length of the
sucrose density gradient above the band containing intact particles. This band contained empty particles (Fig. 9c).
Core particles (Fig. 9h) were prepared as described by Omura et al. (1985).
E.vtraction and eh,ctrophoresis O! mlcleic acMs aml proteins. R NA was released by adding I ~ SDS and 0.1~
EDTA to purified virus preparations, and the solution was applied directly to the top of aerylamide gels (Reddy &
Black, 1973). Electrophoresis was as described by Omura et af. (1983).
Dissociation of particles in SDS and electrophoresis of the viral polypeplides were as described by Omura et al.
(1985).
RESULTS
Thin sections ~?[plant tissues
Gall tissues occurred on the abaxial side of the vascular bundles (Fig. 1), and resulted f r o m
hyperplasia of the phloem. Gall tissue was c o m p o s e d of virus-infected cells, p a r e n c h y m a cells,
sieve tubes, xylem elements and disintegrated cells with very little protoplasm. Cells c o n t a i n e d
either m a n y virus particles or none (Fig. 2). P a r e n c h y m a cells surrounding xylem elements were
free from virus particles. T h e virions were found mostly in the cytoplasm, rarely in the vacuole,
and were not observed in nuclei, chloroplasts or m i t o c h o n d r i a . Virions were either distributed in
crystalline arrays (Fig. 3) or dispersed. Large aggregates of virions were frequently found in gall
cells (Fig. 2); some aggregates were tightly packed to form crystalline arrays and others were
m o r e loose.
Virions were occasionally found in tubules about 100 n m in d i a m e t e r with walls about 5 n m in
thickness (Fig. 4), and s o m e t i m e s several particles were enclosed in a long tubule. In a given cell,
some particles were surrounded by tubules, and others were not. T h e shapes and d i m e n s i o n s o f
the particles in either situation were the same.
A b o u t 3 ~ of the particles were devoid of the dense core and were scattered a m o n g n o r m a l
particles in infected plant cells (Fig. 3). Such particles were only very rarely found in insect cells.
Viroplasm-like matrices were found in infected cells (Fig. 5). T h e m a t r i c e s generally had
sharply defined edges.
Virions had dense cores 40 to 50 n m in d i a m e t e r surrounded by a less dense region giving total
d i a m e t e r of 60 to 70 n m (Fig. 3, 7).
Thin sections o f insect tissues
Virions were found in nerve, gut (Fig. 6), salivary gland (Fig. 8), fat body and muscle cells.
Salivary gland and fat body cells were the most frequently affected. T h e particles were the s a m e
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:30:03
RGD V in thin sections
2583
Fig. 1 to 5. RGDV in infected plants.
Fig. 1. Light mierograph of a cross-section of a gall showing hyperplasia of phloem cells toward the
undersurface of the leaf.
Fig. 2. Part of a vascular bundle from which a gall had formed, showing a cell full of virus particles (V),
parenchyma cells (P), sieve tubes (S) and a xylem cell (X). Bar marker represents 4 btm.
Fig. 3. Crystalline array of virus particles in a gall cell. Bar marker represents 300 nm
Fig. 4. Virions in tubules in a root cell. Bar marker represents 300 nm.
Fig. 5. Viroplasm and scattered virus particles in a root cell. Bar marker represents 150 nm.
size and shape as those found in plants and were found in the c y t o p l a s m but not in nuclei or
m i t o c h o n d r i a . Virions n e v e r occupied m o s t of the cell volume, as they did in plant cells.
A l t h o u g h large aggregates of particles were f r e q u e n t (Fig. 8), unlike in plant cells, these
occupied only a small v o l u m e of the insect cells (Fig. 6). S o m e cells c o n t a i n e d only a few
scattered virions, in contrast to plant cells w h i c h usually c o n t a i n e d either m a s s i v e aggregates of
virus particles or none at all. Scattered particles were associated with bundles of m i c r o f i l a m e n t s
(Fig. 8) as observed in Laodelphax striatellus infected with m a i z e rough d w a r f virus (Vidano,
1970). Often particles were seen surrounding m u l t i m e m b r a n o u s structures, or enclosed in
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:30:03
2584
T. OMURA AND OTHERS
Fig. 6 to 8, RGDV in viruliferous insects.
Fig. 6. Virions in phagocytic vesicle-like matrices bound with membrane in a gut cell. Bar marker
represents 1 ~tm.
Fig. 7. A high magnification of virions in crystalline array. Note non-uniform distribution of electron
density in the cores. Bar marker represents 100 nm.
Fig. 8. Densely packed crystals of virus particles in a salivary gland ceil. Bar marker represents 1 ~tm.
vesicles (not shown). Viroplasm-like forms similar to that observed in insects infected with
wound tumour virus (WTV) (Shikata & Maramorosch, 1967) were frequently observed.
None of the above-mentioned structures was found in healthy plants or in insects not exposed
to the virus.
Nucleic acid and protein components of purified particles
Intact virus particles, empty particles and cores were purified and examined by negative
staining and for their component nucleic acids and proteins. Empty particles resembled those
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:30:03
2585
RGD V in thin sections
(e)
183K-"~
165K'-"b
150K----~
143K----~
120K"'~
56K----~
45K----~
Fig. 9. (a, b, c) R G D V particles negatively stained with PTA : (a) intact particles, (b) core particles and
(c) empty particles. Bar markers represent 100 nm. (d) Electrophoresis of R G D V nucleic acids in a 10~o
polyacrylamide gel: left lane, intact particles; centre lane, core particles; right lane, empty particles. (e)
Electrophoresis of RG DV proteins in a 10~o polyacrylamide gel: left lane, intact particles; right lane,
empty particles. Molecular weights of the proteins are shown to the left.
seen in thin sections and contained little or no nucleic acid (Fig. 9e, d). Electrophoresis of
nucleic acids released from equal volumes of suspensions of intact particles and cores of equal
A 280, showed that nucleic acids from cores gave more pronounced bands than nucleic acids from
intact particles (Fig. 9d).
Comparison of protein from intact and empty particles by electrophoresis showed that empty
particles contained much less 56K protein than intact particles (Fig. 9e). No other differences
were apparent between the protein components of the particle types.
DISCUSSION
The common features produced by R G D V in plant and insect cells were restriction of virions
to the cytoplasm and absence from cell organelles, formation of crystalline arrays of virions,
formation of viroplasm-like matrices and enclosure of virions in tubules. The notable differences
were that virions were restricted to phloem-related cells in the plant, whereas they were observed
in many kinds of insect cells, and that virions frequently occupied most of the cell in the plant,
but were found only in restricted areas in insect cells.
The present study shows that the distribution of R G D V in infected plants is quite different
from that of RDV, but similar to WTV. Particles of R G D V and WTV (Nagaraj & Black, 1961 ;
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:30:03
2586
T. O M U R A
AND OTHERS
Shikata & Maramorosch, 1966) are restricted to vascular bundle-related cells, whereas RDV can
be found in a wide range of cells (Fukushi et al., 1962; Iida et al., 1972; Shikata, 1966).
Furthermore, in plants infected with RGDV not all the cells, even in phloem tissue, contain
virus. This difference is probably the reason for the yield of purified R G D V being about 30 to 50
times lower than that of RDV, the scarcity of R G D V particles in dip preparations (T. Omura,
unpublished data), and the relatively low dilution endpoint for serological detection of R G D V
(Omura et al., 1984).
There is a correlation between the formation of hyperplasia and the distribution of plant
reovirus particles in infected hosts. Plant reoviruses (Boccardo & Milne, 1984) produce
neoplastic tissues derived from the phloem and virus particles are restricted to vascular bundlerelated cells; the exception is RDV which does not cause hyperplasia and invades mesophyll
cells as well as vascular tissues. The present study therefore shows that R G D V resembles the
majority of plant reoviruses in this property.
Also, except in sugar-cane infected with Fiji disease virus and in rice infected with rice blackstreaked dwarf virus, both insect and plant cells infected with plant reoviruses contain tubules
(Boccardo & Milne, 1984). RGDV therefore also resembles most plant reoviruses in that it too
produces tubules in both plant and insect cells. However, the biological meaning of the tubules is
not clear.
Intact particles observed in ultrathin sections are composed of an electron-dense inner part
and a less dense outer layer; the apparently empty particles are composed of only the outer
layers. Core particles isolated in vitro contained all the RNA segments, while empty particles
had little RNA (Fig. 9d). The electron-dense inner part in ultrathin sections is apparently
composed of nucleic acids (Fig. 7).
The particles devoid of the dense core observed in thin sections were considered to be the
empty particles found in leaf dip preparations from infected plants. Purified empty particle
preparations contained no nucleic acid. It is unlikely that empty particles are the products of
degeneration, because the proportion of virions that were intact in the infected plants was the
same at 30 days and 70 days after inoculation (T. Omura, unpublished data). The results further
suggest that the capsid proteins can be built into a stable shell in the absence of nucleic acids.
The relative amount of the 56K protein, thought to be located inside the core (Omura et al.,
1985), was much reduced in empty particles. The 56K protein, therefore, seems not to affect the
construction of protein shells and may be associated with nucleic acid in intact particles and
involved in nucleic acid-related activity in the intact virus particles.
The authors are grateful to D r R. G. M i l n e for fruitful discussions.
REFERENCES
BOCCARDO, G. & MILNE, R. G. (1984). Plant reovirus group, Commonwealth Mycological Institute~Association of
Applied Biologists Descriptions ~71Plant Viruses, no. 294.
FUKUSHI, T., SHIKATA, E. & KIMURA, I. (1962). Some m o r p h o l o g i c a l c h a r a c t e r s of rice d w a r f virus. Virology 18, 192205.
ninl, T., OMURA, T. & SANTO,V. (i 984). Dou ble-stranded R N A o f rice gall d w a r f virus. Journal oJGeneral Virology65,
1585-1590.
IIDA, T., SHINKAI, A. & KIMURA, I. (I972). Rice d w a r f virus. Commonweahh Mycological Institute~Association of
Applied Biologists Description ~71Plant Vh'uses, no. 102.
INOUE, H. & OMURA, T. (1982). T r a n s m i s s i o n of rice gall d w a r f virus by the green rice leafhopper. Plant Disease 66,
57 59.
MOR1NAKA, T., INOUE, H., OMURA, T., SAITO, Y., PUTTA, M., CHETTANACHIT, D., PAREJAREARN, A. & DISTHAPORN, S.
(1982). T r a n s m i s s i o n of rice gall d w a r f virus by cicadellid leafhoppers Recilia dorsalis and Nephotettix
nigropietus in T h a i l a n d . Plant Disease 66, 703 704.
NAGARAJ, A. N. & BLACK, L. M. (1961). L o c a l i z a t i o n of wound t u m o r virus a n t i g e n in p l a n t t u m o r s by the use of
fluorescent antibodies. Virology 15, 289 294.
OMURA, T., INOUE, H., MORINAKA, T., SAITO, Y., CHETTANACHIT, D., PUTTA, M., PAREJAREARN, A. & DISTHAPORN, S.
(1980). Rice gall dwarf, a new virus disease. Plant Disease 64, 795-797.
OMURA, T., MOR~NAKA, T., INOUE, H. & SAITO, Y. (1982). Purification and some p r o p e r t i e s of rice gall d w a r f virus, a
new Phytoreovirus. Phytopathology 72, 1246 1249.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:30:03
R G D V in thin sections
2587
OMURA, T., MINOBE,Y., KIMURA,L, HIBINO,H., TSUCH1ZAKI,T. & SAITO,Y. (1983). Improved purification procedure
and RNA segments of rice ragged stunt virus. Annals t~lthe Phytopathologieal Society of Japan 49, 670-675.
OMURA, T., HIBINO,H., USUGI,T., INOUE,H., MORINAKA,T., TSURUMACHI,S., ONG, C. A., PUTTA~M., TSUCHIZAKI~T. &
SAITO, V. (1984). Detection of rice viruses in plants and individual insect vectors by latex flocculation test.
Plant Disease 68, 374-378.
OMURA,T., MINOBE,Y., MATSUOKA,M., NOZU, Y., TSUCHIZAKI,T. & SAITO,Y. (1985). Location of structural proteins in
particles of rice gall dwarf virus. Journal q[ General Virology 66, 811-815.
REDDY, O. V. R. & BLACK,I.. M. (1973). Electrophoretic separation of all components of the double-stranded R N A of
wound tumor virus. Virology 54, 557-562.
SHIKATA, E. (1966). Electron microscopic studies on plant viruses. Journal of the Faculo" ol'Agriculture, Hokkaido
University 55, l 101.
SHIKATA,E. & MARAMOROSCH,K. (1966). An electron microscope study of plant neoplasia induced by wound tumor
virus. Journal o["the National Cancer Institute 36, 97-116.
SHIKATA,E. & MARAMOROSCH,K. (1967). Electron microscopy of wound tumor virus assembly sites in insect vectors
and plants. Virology 32, 363 377.
VIDAYO,C. (1970). Phases of maize rough dwarf virus multiplication in the vector Laodelphax striatellus (Fallen).
Virology 41, 218-232.
YOKOYAMA,M., NOZU, Y., HASHIMOTO,J. & OMURA, Y. (1984). In vitro transcription by R N A polymerase associated
with rice gall dwarf virus. Journal o! General Virology 65, 533 538.
(Received 16 M a y 1985)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:30:03