Infection of Protoplasts from Chenopodium quinoa with Cowpea ...

J. gen. Virol. (1984), 65, 1851-1855. Printed in Great Britain
Key words: Chenopodium quinoa/protoplasts/cowpea mosaic virus~cymbidium ringspot virus
Infection of Protoplasts from Chenopodium quinoa with Cowpea Mosaic
and Cymbidium Ringspot Viruses
By AMARILIS DE VARENNES, MARCELLO RUSSO'~
ANDREW J. MAULE*
John Innes Institute, Colney Lane, Norwich NR4 7UH, U.K.
(Accepted I0 July 1984)
SUMMARY
AND
Mesophyll protoplasts were prepared from Chenopodium quinoa plants and infected
with cowpea mosaic virus (CPMV) or cymbidium ringspot virus (CyRSV) using
inocula containing either polyethylene glycol or poly-L-ornithine. The level of infection
was estimated by fluorescent antibody staining and by spot hybridization assay. About
25~ of the protoplasts became infected with CPMV and about 3590 with CyRSV.
Yields of progeny RNA were about 700 ng of CPMV RNA and 3 ~g of CyRSV RNA
per 106 protoplasts. Cytopathic structures found in C. quinoa protoplasts inoculated
with CPMV or CyRSV were similar to the structures found in CPMV-infected cowpea
protoplasts or in CyRSV-infected C. quinoa plants respectively.
Protoplasts from several plant species can be infected with plant viruses (for review, see
Harrison & Mayo, 1983; Takebe, 1983). Although there is one report of infection of
Chenopodium hybridum protoplasts with brome mosaic virus (Okuno & Furusawa, 1979), to our
knowledge no attempt has yet been made to exploit Chenopodium quinoa, a well known host for
many plant viruses, for protoplast work. We studied the possibility of obtaining protoplasts from
this species, maintaining them in culture and infecting them with the comovirus cowpea mosaic
(CPMV) and the tombusvirus cymbidium ringspot (CyRSV).
C. quinoa plants were grown in a peat-vermiculite mixture in a glasshouse at 25 °C. During
winter, natural light was supplemented by 16 h illumination with 400 W sodium lamps giving
8 klx or 100 ~tE/mZ/s. Plants suitable for protoplast isolation were approximately 2 weeks old,
with five or six leaves. Only middle leaves, 2-5 to 3 cm long, were used. The leaves were surface-
sterilized by briefly rinsing in 70% ethanol and immersing in a 59o solution of commercial
bleach with 0"03~o 'Hederol' detergent for l0 min, followed by three washes in sterile distilled
water. The lower epidermis was removed by peeling. Tissue was plasmolysed in 0.6 M-mannitol
for 30 min and then digested with 2~ Cellulase Onozuka R-10 (Kinki Yakult Manuf. Co.), 0.1 o~
Macerozyme R-10 (Kinki Yakult Manuf. Co.) in 0-6 i-mannitol, pH 5.5, at 30 ~C in a shaking
water-bath at 60 excursions/min. Although after 3 h the leaves appeared digested and the
resulting isolated protoplasts became infected upon inoculation, these protoplasts tended to
stick irreversibly to the incubation vials during culture. This was prevented by digesting leaves
for 5 to 6 h. The protoplasts were separated from tissue debris by filtration through a 66 ~tm
nylon mesh and washed three times with 0.6 M-mannitol. Protoplast yields were from 4 × 106 to
6 × 106 protoplasts per gram of tissue.
Freshly prepared protoplasts (Fig. t a) were inoculated with CPMV or CyRSV using
polyethylene glycol (PEG) as described by Maule (1983) and 20 ~g of virus per 10 ~ protoplasts.
Protoplasts were washed three times with 0-6 M-mannitol containing 10 mM-CaCI, and cultured
at 3 × 105 to 5 × l05 cells/ml of incubation medium (Rottier et al., 1979) at 25 °C with
continuous lighting at 2-5 klx. Protoplast viability, assessed using phenosafranine (Widholm,
1972), had dropped to 80 to 85% after 48 h and to 50 to 55% after 66 h. After longer periods of
t Present address: Centro di Studio sui Virus e le Virosi delle Colture Mediterranee, c/o Dipartimento di
Patologia Vegetale, Universitfi degli Studi, Bari, Italy.
0022-1317/84/0000-6189 $02.00 © SGM
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1852 Short communication
Fig. 1, (a) C. quinoa protoplasts resuspended in 0.6 M-mannitol. (b, c) Fluorescence micrographs of C.
quinoa protoplasts infected with CyRSV (b) or CPMV (c) and stained with fluorescent antibody. (d, e)
Electron micrographs of C. quinoa protoplasts infected with (d) CPMV, showing part of an inclusion
body (IB), and (e) CyRSV, showing two multivesicular bodies (MVB) and a vesiculated mitochondrion
(inset). Bar markers represent 20 ~tm for (a) and (b), 15 ~tm for (c) and 200 nm for (d), (e) and inset.
incubation the protoplasts were extensively clumped. Virus infection was assessed by indirect
fluorescent antibody staining (Maule et al., 1980) and by spot hybridization (Thomas, 1980).
C. quinoa protoplasts could also be infected with CPMV and CyRSV when poly-t-ornithine
(PLO) was included in the inocula. Under the conditions tested (1 rtg/ml PLO, 10 ~tg/ml virus
and 10 raM-potassium citrate, pH 5-2, in 0.6 M-mannitol), the proportion of protoplasts infected
using inocula containing PLO was similar to that using inocuta containing PEG. Generally, the
PEG method was used in preference because of its simplicity.
The proportion of protoplasts stained with fluorescent antibody increased from 1 to 5°/o 12 h
post-inoculation to about 25~ 36 to 60 h post-inoculation for CPMV, and from 15~ at 12 h to
about 35~o 48 to 60 h post-inoculation for CyRSV, The appearance of the fluorescence in the

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Fig. 2. Spot hybridization assay of CPMV and CyRSV RNA in extracts of infected protoplasts.
Variation in the concentration of viral RNA is shown at various times (h) after inoculation with CPMV
(T) or CyRSV (11), as an autoradiograph (a) or as a graphical representation (b) using the calculated
concentration of viral RNA. Values are the mean of duplicate spots. Each spot is equivalent to 2.5 ×
105 protoplasts.
stained protoplasts differed strikingly between infections by the two viruses. Fluorescing
material in protoplasts infected with CyRSV was diffusely spread in the cytoplasm forming a
network between the chloroplasts (Fig. 1 b). In protoplasts infected with CPMV, fluorescent
masses were localized in discrete areas of the cytoplasm (Fig. 1 c) and only occasionally, late in
infection, was the fluorescence distributed throughout the cytoplasm as described for CPMV-
infected cowpea protoplasts by Hibi et al. (1975).
RNA was extracted from infected protoplasts disrupted in the presence of 5~ Sarkosyl
(Sigma) in 40 mM-Tris-HC1, 80 mM-NaC1, 4 mM-EDTA, pH 8.5, by treatment with a buffer-
saturated mixture of phenol and chloroform (8 : 1, v/v) and recovered by ethanol precipitation.
When RNA was spotted onto nitrocellulose filters (Thomas, 1980) and probed with 32p-labelled
cDNA (Maule et al., 1983) prepared to each viral RNA by random priming (Taylor et al., 1976),
it was observed that the accumulation of viral RNA paralleled the accumulation of virus antigen
(as assessed by fluorescent antibody staining). For quantification of viral RNA, the radioactive
areas were cut from the nitrocellulose filters, assayed by scintillation counting, and compared
with blots of known quantities of purified RNA. As shown in Fig. 2, CPMV-infected
protoplasts contained about 700 ng of progeny viral RNA per 106 protoplasts and CyRSV-in-
fected protoptasts, more than 3 ~tg of progeny RNA per 106 protoplasts. The molecular weight
distribution of virus RNA detected in this assay was assessed using Northern blots (Thomas,
1980). RNA extracted from protoplasts was denatured with 70~ (w/v) formamide in the pres-
ence of t M-glyoxal (Covey et aL, 1983) and electrophoresed in 1X agarose gels. After
electrophoresis the gels were blotted onto nitrocellulose filters and probed with 32p-labelled
cDNA. Both species of CPMV RNA (B-RNA and" M-RNA) were detected in RNA from
CPMV-infected protoplasts (Fig. 3 a); a major species, corresponding to the genomic RNA, was
detected in RNA from protoplasts infected with CyRSV (Fig. 3b), together with smaller species
whose significance is at present under investigation (D. Gallitelli, personal communication).
Samples of healthy and infected protoplasts were collected by centrifugation, resuspended in
4~ glutaraldehyde in 0.05 M-phosphate buffer pH 7.0, containing 0.6M-mannitol, and
processed for electron microscopy essentially as described by Martelli & Russo (1981). Thin
sections of CPMV-infected protoplasts (Fig. I d) showed the presence of the cytopathic structure
typical of infections by this virus both in cowpea mesophyll tissue (De Zoeten et al., 1974) and
protoplasts (Hibi et al., 1975). In thin sections of CyRSV-infected protoplasts (Fig. l e)
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(a) 0 12 24 36 48 60 72 H (b) H 0 12 24 36 48
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RNA
Fig. 3. Northern blot of CPMV RNA (a) or CyRSV RNA (b) in infected protoplasts, taken at various
times (h) after inoculation. H, Healthy protoplasts. Positions of the genomic RNAs of CPMV (B, M)
and CyRSV (arrow) are marked.
peroxisomes appeared to have been transformed into multivesicular bodies (MVB), a
characteristic cytopathological feature of infections by this virus (Russo et aI., 1983). Also in
these protoplasts, a small proportion of the mitochondria contained fibril-containing vesicles
between the two mitochondrial membranes (Fig. 1 e, inset), a feature peculiar to CyRSV
infection of C. quinoa, also found in leaf tissue, but not in several other plants infected with the
same virus (A. Di Franco & M. Russo, unpublished results).
In conclusion, we have shown that protoplasts from C. quinoa can be used for virus infection
studies, including those of CyRSV for which C. quinoa is a local lesion host. C. quinoa protoplasts
are easily produced and are stable. They look promising as an experimental system to be tried
with other viruses belonging to different groups.
We thank Dr J. Davies for helpful discussions. A. de Varennes holds a studentship from the National Institute
of Scientific Research, Portugal.
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(Received 14 May 1984)