63
J. gen. ViroL (1978) 4 I , 63-75
Printed in Great Britain
Factors Influencing the Infection of Barley Mesophyll Protoplasts
with Brome Mosaic Virus R N A
By T E T S U R O O K U N O AND I W A O F U R U S A W A
Laboratory of Plant Pathology, Faculty of Agriculture, Kyoto University,
Kyoto 6o6, Japan
(Accepted 4 May I978)
SUMMARY
One of the most critical factors for the infection of barley protoplasts with brome
mosaic virus (BMV) RNA was the osmotic strength of the medium during protoplast isolation and inoculation. Infection was most efficient when protoplasts
were isolated and inoculated in 0"5 M-mannitol and then washed with o. 7 Mmannitol, less infection occurred when the protoplasts were isolated in o'5 or
o'7 M-mannitol and inoculated and washed in o'7 M-mannitol. Other conditions
optimal for infection were inoculum containing I #g/ml BMV RNA or less, t #g/ml
poly-L-ornithine, 2o mM-potassium citrate buffer at pH 4"7 and o'5 mM-CaC12, and
inoculation at o °C. Up to 9o % of protoplasts were infected in these conditions.
Osmotic shock produced by increase of mannitol concentration immediately before
inoculation induced invagination suggestive of pinocytosis, and this seemed to be
associated with efficient infection of barley protoplasts with BMV. The discrepancy
between the effect of this osmotic shock on the infection of barley protoplasts
with BMV and its RNA, and the mechanism of infection are discussed.
INTRODUCTION
Leaf mesophyll protoplasts have been infected with various kinds of viruses and the
conditions optimal for infection have been studied intensively (see Takebe, I975, I977;
Alblas & Bol, I977; Okuno & Furusawa, 1978a, b). In contrast there have been fewer
studies on the infection of protoplasts with virus RNA (Aoki & Takebe, I969; Motoyoshi
et al. I973; Motoyoshi & Hull, i974; Motoyoshi et al. I974a; Sarkar et al. I974; Beier
& Bruening, I976). The use of virus RNA as an inoculum in protoplast systems would be
particularly useful for the study of multicomponent viruses whose nucleoproteins cannot
easily be separated from each other, provided that the protoplasts can be infected efficiently
using low concentrations of virus RNA.
Okuno & Furusawa 0978b) reported that barley leaf protoplasts became efficiently
infected with brome mosaic virus (BMV) when osmotic shock was produced in the cells
by increasing the osmotic pressure of the medium and that the effect of osmotic shock on
the infection of barley protoplasts with BMV was opposite to that on the infection of
bacterial protoplasts (Engelhardt & Zinder, 1964), mammalian cells (Koch, I973) or
tobacco leaf protoplasts (Aoki & Takebe, i969) with virus RNA. In this paper we examine
some factors affecting the infection of barley protoplasts with BMV RNA, and we compare
the effects of osmotic shock on infection by BMV and BMV RNA. The infection mechanism was also studied by means of electron microscopy.
5
VIR 41
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64
T. O K U N O
AND
I. F U R U S A W A
METHODS
Preparation o f B M V nucleoprotein and RNA. A standard strain of BMV (ATCC 66) was
used. Virus growth, purification and storage were as described (Okuno & Furusawa, 1978 a).
BMV RNA was isolated from purified virus by the phenol-sodium dodecyl sulphatebentonite method described by Hiruki (I969) with a slight change. Ethanol-precipitated
RNA was suspended in distilled water and used with cr without dialysis against distilled
water. RNA concentration was calculated using the absorbance value 22 cm2/mg at
260 nm.
Inoculation ofprotoplasts. Protoplasts of leaf mesophyll cells were isolated from the first
leaves of barley (Hordeum vulgate L. cv. Moore) as reported by Okuno & Furusawa 0977).
For inoculation with BMV RNA, protoplasts isolated in o'5 M-mannitol were washed
three times with 0"5 M-mannitol by centrifugation (pre-washing) and were suspended in
cold o-5 M-mannitol. The suspension was immediately mixed with an equal volume of cold
40 raM-potassium citrate buffer, pH 4"7, containing I #g/ml BMV RNA, 2 #g/ml poly-Lornithine, I mu-CaC12 and 0"5 M-mannitol, which had been previously incubated together
at o °C for Io min. After t5 rain at o °C, inoculated protoplasts were collected and washed
twice with o'7 u-mannitol containing Io mM-CaC12 by centrifugation (post-washing) and
then incubated as described by Okuno & Furusawa (I977). Inoculation of protoplasts
with BMV was as described by Okuno & Furusawa (r978b).
Fluorescent antibody staining. Infection was assessed by fluorescent antibody staining as
described (Okuno et al. I977), with a slight change. Specimens were stained with BMVspecific fluorescent antibody at 5 °C overnight instead of at 37 °C for 60 min. A Nikon
fluorescence microscope was used to examine the stained specimens. Barley protoplasts
contain an unknown substance which occurs as yellow fluorescent specks (T. Hibi, personal
communication; Okuno et al. I975; see Takebe, I977). However, this does not cause any
difficulty in determining the incidence of infection by staining with fluorescent antibody
for viruses which, like BMV, are dispersed in the cytoplasm; indeed the auto-fluorescence
was hardly visible when the improved conditions of isolating and incubating barley protoplasts were used (Okuno & Furusawa, I977)- A fluorescence micrograph of barley protoplasts is shown in Fig. I.
Infectivity assay. Protoplasts (about 5 x io 5 cells) were collected by centrifuging at 2oog
for 5 rain and frozen at - 2 o °C until use. Each sample was homogenized with I ml o.oI Msodium acetate buffer, pH 5"0 containing I mM-MgC12 and after suitable dilution was
assayed on Io half leaves of Chenopodium hybridum. The number of lesions formed per half
leaf was multiplied by the dilution factor.
Electron microscopy. Protoplasts were fixed overnight at o °C with 2"5 % glutaraldehyde
in o"o5 M-phosphate buffer, pH 6-8, containing 0"5 or 0"7 M-mannitol. After washing with
cold phosphate buffer several times by centrifugation, protoplasts were post-fixed overnight
at o °C with 1% OsO4. After washing with distilled water, protoplasts were wrapped in
lens paper and dehydrated through an ethanol series. Samples were then placed in propylene oxide and embedded in Spurr low-viscosity medium (Polysciences Inc.). Ultrathin
sections were stained with uranyl acetate for 3o rain and then with lead citrate for 30 min.
The samples were examined using a transmission electron microscope, JEOL Model JML 7.
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Infection o f protoplasta with B M V R N A
65
Fig. I. A fluorescencemicrograph of barley protoplasts incubated for 24 h after inoculation with
BMV RNA and stained with fluorescent antibody to BMV. Arrow indicates non-stained
protoplast.
RESULTS
Influence of osmolarity on infection of barley protoplasts with B M V and B M V RNA
The influence of mannitol concentration throughout isolation and inoculation of protoplasts is shown in Fig. 2. Infection with BMV R N A was greatest when the mannitol concentration was 0"5 M which is the minimum suitable for subsequent incubation of barley
protoplasts (Okuno & Furusawa, I977). With increasing mannitol concentration, the
efficiency of infection with BMV R N A decreased sharply, whereas infection with BMV was
hardly affected provided the concentration was kept constant. Influence of osmotic shock
by increase of mannitol concentration immediately before inoculation is shown in Fig. 3The efficiency of infection with BMV R N A decreased with increasing mannitol concentration from o. 5 to 0.8 M, in contrast to that of infection with BMV. The results in Table I
show that it was not the osmotic shock produced by either increase or decrease of mannitol
concentration immediately before inoculation but the mannitol concentration during
isolation a n d / o r inoculation of the protoplasts that greatly influenced infection with BMV
RNA. It was found, however, that infection with BMV RNA, as with BMV, was enhanced
by increasing the mannitol concentration at post-inoculation washing (Table 2). The
efficiency of infection increased with increasing interval between mixing protoplasts with
BMV R N A and osmotic shock treatment and was greatest when the treatment was at
post-inoculation washing (data not shown).
It was reported recently that ribonuclease (RNase) activity increased in isolated tobacco
leaf protoplasts during incubation in hypertonic mannitol solution and that this increase
was completely inhibited by adding cycloheximide (Premecz et al. I977); also, RNase
5-2
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66
T. OKUNO AND I. FURUSAWA
|
I
I
I
|
60
I
I
t
80
60
"~ 40
O
©
"3 40
m
©
20
20
o
I
I
I
I
0.5
0-6
0-7
0.8
Mannitol concentration (M)
Fig. 2
I
I
I
I
0.5
0-6
0-7
0-8
Mannitol concentration (M)
Fig. 3
Fig. 2. Influence of mannitol concentration througbout the isolation and inoculation of barley
protoplasts on their infection with BMV RNA (O--O) and BMV ( 0 - - 0 ) . Protoplasts were
inoculated either with 0"25 #g/ml BMV RNA together with I #g/ml poly-L-ornithine in I0 mMcitrate buffer, pH 5'0, at 0 °C, or with 2o #g/ml BMV together with 0-2 #g/ml poly-L-ornithinein
o'I raM-citrate buffer, pH 5"0, at 20 °C. Infection was determined by fluorescent antibody staining
after incubation for 27 h.
Fig. 3- Influence of increase of mannitol concentration immediately before inoculation on the
infection of barley protoplasts with BMV RNA (O--O) and BMV (O--Q). For the other
conditions of inoculation, refer to the legend of Fig. 2. Infection was determined by fluorescent
antibody staining after incubation for 44 h.
activity in isolated oat protoplasts was greatly reduced by pre-treatment of the source
leaves with cycloheximide or L-lysine (Sawhney et al. ~977). To investigate the effect of
these substances on infection of barley protoplasts with BMV RNA, protoplasts were
isolated in the presence of cycloheximide or L-lysine and then inoculated with BMV RNA.
Table 3 shows that the presence of these substances during isolation of the protoplasts
considerably enhanced infection. Also, infection with BMV R N A of protoplasts pre-treated
with 2"5/zg/ml cycloheximide during isolation was hardly affected by increasing the
mannitol concentration immediately before inoculation (data not shown). Barley protoplasts treated during isolation with cycloheximide at concentrations above I # g / m l often
deteriorated during their subsequent incubation while L-lysine (to raM) appeared to decrease
senescence of protoplasts (Sawhney et al. I977). However, under the optimum conditions
described in this paper, 7o to 9o % of protoplasts became infected with BMV R N A without
cycloheximide or L-lysine treatment.
Other factors affecting the infection of barley protoplasts with B M V R N A
Buffer salts, p H and eoncentr ation
Table 4 shows the effect of buffer salts, pH and concentration on the infection of barley
protoplasts with BMV RNA. Infection was more efficient in citrate buffer than in phosphate
buffer, unlike that in some other protoplast-virus systems (Kubo et al. I976; Alblas & Bol,
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Infection o f protoplasts with B M V R N A
67
T a b l e L Effect of osmotic shock immediately before inoculation on the infection of barley
protoplasts with B M V RNA*
Mannitol molarity during
c
~"
~
Isolation Inoculation
o'5
o'5
o.6
0'6
0'7
0"7
0.8
0'8
0"5
o'7
o'6
0.8
0"7
0"5
0'8
0.6
% fluorescing
protoplastst
58
36
19
II
7
12
7
7
* For the other conditions of inoculation, refer to the legend of Fig. 2.
-~ Determined by fluorescent antibody staining after incubation for 27 h.
T a b l e 2. Effect of osmotic shock at post-inoculation washing on the infection of barley
protoplasts with B M V and B M V RNA*
,
Mannitol molarity during
~
,
Inoculum
Isolation and
Post-inoculation - - ~ ' - inoculation
washing
BMW BMV RNA
05
o5
O'5
O'5
0"5
o-6
O'7
0'8
38"~
42
53
55
29
3t
46
45
* Protoplasts were inoculated either with 0'8 #g/ml BMV RNA together with I #g/ml poly-L-ornithine
in zo raM-citrate buffer, pH 5'0, at o °C or with BMV (see legend to Fig. 2).
"~ Figures are % fluorescing protoplasts determined by fluorescent antibody staining after incubation for
44 h.
T a b l e 3. The effect of exposure to cycloheximide and L-lysine during protoplast isolation on
infection with B M V RNA*
Cycloheximide
t-lysine
concentration concentration
(/zg/ml)
(raM)
Inoculation medium
IO raM-citrate buffer, pH 5"o
containing I #g/ml poly-Lornithine and o'5 M-mannitol
IOO mM-phosphate buffer,
pH 6"0, containing 25/zg/ml
protamine sulphate and
0"4 M-mannitol
-I
)
"1
t
o
O'I
o
o
o.I
o
o
O
to
o
o
lo
Fluorescing
protoplasts~
(%)
36
65
50
5
15
1I
* Protoplasts isolated in 0"5 M-mannitol were inoculated with I #g/ml BMV RNA at o °C.
t Determined by fluorescent antibody staining after incubation for 44 h.
I977). T h e o p t i m u m p H in citrate buffer was p H 4"7 a n d p H 6-0 i n p h o s p h a t e buffer
( T a b l e 4, Expt. I, 2 a n d 3); similar results are r e p o r t e d i n the t o b a c c o p r o t o p l a s t - t o b a c c o
r a t t l e virus s y s t e m ( K u b o et al. I976). T h e o p t i m u m c o n c e n t r a t i o n o f citrate buffer was
2o mM a n d at o t h e r c o n c e n t r a t i o n s , especially at 5o mM or a b o v e , i n f e c t i o n efficiency
decreased drastically. I n f e c t i o n i n Ioo raM-citrate buffer was n o t i m p r o v e d b y r e d u c i n g the
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68
T. O K U N O A N D I. F U R U S A W A
T a b l e 4. Effect of buffer ion, p H and concentration on the infection of barley protoplasts
with B M V RNA*
Expt.
t
Buffer
Citrate
2
Citrate
3
Phosphate
4
Citrate
Citrate
Phosphate
Buffer
concentration
(mM)
Buffer
pH
BMV RNA
concentration
(#g/ml)
Fluorescing
protoplastst
(%)
IO
4"7
2"7
I7
IO
IO
Io
I0
5"0
5-2
5'7
6"2
2' 7
2' 7
2"7
2"7
15
I2
II
9
Io
Io
Io
Io
IO
25
25
25
25
2o
5
Io
2o
50
4"7
5'0
5"2
5"7
6"2
5"4
6-0
7"O
8"O
5"o
5"o
5"0
5"0
5'0
o'9
0"9
0"9
o'9
0' 9
0"4
0"4
O'4
O'4
o'4
o'8
0-8
0'8
0.8
52
34
34
34
36
I9
24
7
9
45
3o (1)
36 (5)
42 (8)
8 (to)
IO0
5,0
0-8
2
Ioo:~
IO
25
50
Ioo
5"0
6-0
6-o
6.o
6.0
0.8
0"8
o.8
o-8
0'8
3
2o
]6
i6
I7
* Protoplasts isolated in 0"5 M-mannitol were inoculated with BMV RNA together with I #g/ml poly-Lornithine in 0"5 M-mannitol at o °C.
f" Determined by fluorescent antibody staining after incubation for 44 h. Results in parentheses are for
samples inoculated with 4 #g/ml BMV RNA.
$ Final mannitol concentration of inoculation mixture was 0"4 M.
final m a n n i t o l c o n c e n t r a t i o n to 0"4 M, a n d when 4 / ~ g / m l B M V R N A was used as i n o c u l u m
instead o f 0.8 # g / m l , infection efficiency g r a d u a l l y increased with increase o f buffer conc e n t r a t i o n f r o m 5 to 50 mM (Table 4, Expt. 4)- Infection was less affected b y c o n c e n t r a t i o n
o f p h o s p h a t e buffer t h a n b y t h a t o f citrate buffer.
S a r k a r et aL (t974) r e p o r t e d t h a t t o b a c c o p r o t o p l a s t s were efficiently infected with
T M V R N A at high p H in high ionic strength media. Barley p r o t o p l a s t s were i n o c u l a t e d
with B M V R N A u n d e r the c o n d i t i o n s described b y S a r k a r et al. (I974) b u t o n l y a small
p r o p o r t i o n o f p r o t o p l a s t s (2 to 4 % ) were infected regardless o f the presence or absence o f
poly-L-ornithine (0'2 to I /~g/ml).
Concentrations of poly-L-ornithine and B M V RNA
I n general, p o l y c a t i o n s are essential for infection of p r o t o p l a s t s with viruses w h o s e
particles are c h a r g e d negatively u n d e r the i n o c u l a t i o n c o n d i t i o n s (see T a k e b e , I975;
A l b l a s & Bol, I977; O k u n o & F u r u s a w a , I 9 7 8 a ). I n contrast, infection o f p r o t o p l a s t s with
virus R N A can occur even in the absence o f p o l y c a t i o n s which, however, greatly e n h a n c e
infection ( A o k i & T a k e b e , I969; S a r k a r et al. i974; Beier & Bruening, I976). Poly-L-
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Infection o f protoplasts with B M V R N A
69
Table 5- Effect of different concentrations of poly-L-ornithine and B M V RNA on infection of
barley protoplasts*
Expt.
I
BMV R N A
Poly-L-ornithine Fluorescing
concentration
concentration protoplastst
(#g/ml)
(#g/ml)
(~)
o'I
o'I
o-I
O'I
o-5
o'5
0. 5
0-5
o
o.I
0"5
4
4
I5
1
49
o
o.i
0. 5
I
4
3
20
48
0.003
0"03
0-3
3
9
i
I
t
I
I
4
I7
34
22
17
* In 2o mM-citrate buffer, pH 5'0 (Expt. I), or Io mM-citrate buffer, pH 5"0 (Expt. 2).
t Determined by fluorescent antibody staining after incubation for 26 h (Expt. I) or 44 h (Expt. 2).
Table 6. Effect of divalent cations on infection of barley protoplasts with B M V RNA*
Concentration
(mM)
MgCI~
CaCI2
o
O'I
I
IO
43t
48
47
28
43
53
52
30
* Protoplasts were inoculated with t #g/ml BMV RNA together with r #g/ml poly-L-ornithine in lO raMcitrate buffer, pH 5"o.
t Figures are % fluorescing protoplasts determined by fluorescent antibody staining after incubation for
44 h.
ornithine greatly stimulated the infection of barley protoplasts with BMV RNA although
a few protoplasts became infected in its absence (Table 5, Expt. I). RNA concentration
was rather important. Usually o.t to I #g/ml BMV RNA was used as inoculum and on
either side of this range the efficiency of infection decreased (Table 4 and Table 5, Expt. 2).
When protoplasts were inoculated in phosphate buffer instead of citrate buffer, infection
efficiency increased with increasing BMV RNA concentration from 0"3 to 9/zg/ml regardless of whether poly-L-ornithine (I #g/ml) or protamine sulphate (25 #g/ml) were used
(data not shown).
Divalent cations
Divalent cations such as Ca 2+ and Mg z+ favour the infection of tobacco protoplasts
with TMV RNA (Aoki & Takebe, 1969; Sarkar et aL 1974). The infection of barley protoplasts with BMV RNA was slightly increased by adding CaCl~ or MgCl~ (o.I to I mM)
to the inoculation medium (Table 6), and a considerable proportion of protoplasts were
infected in the presence of IO mM-CaC12 or MgC12, conditions which completely inhibit
infection with BMV (Furusawa & Okuno, I978; Okuno & Furusawa, I978a ).
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7o
T. O K U N O
A N D I. F U R U S A W A
Table 7- Effect of temperature and pre-inoculation incubation on infection of barley protoplasts
with B M V RNA*
Pre-inoculation
incubation
(min)
Expt.
Temperature
(°C)
t
o
o
o
30
30
O
2O
IO
o
I0
IO
IO
2,
Fluorescing
protoplastst
(~)
35
43
I7
27
69
23
* Protoplasts were inoculated with : # g / m l BMV R N A together with I # g / m l poly-L-ornithine in
Io mM-citrate buffer, pH 5"o (Expt. 0 , or inoculated as described in Methods (Expt. 2).
t Determined by fluorescent antibody staining after incubation for 43 h (Expt. I) or 24 h (Expt. 2).
I
I
I
I
102
-
100
g
r~
10
-
-
50
.~
m
-
?.
©
f
0
10
20
30
Time after inoculation (h)
40
Fig. 4. BMV multiplication in barley protoplasts inoculated with BMV RNA. For the inoculation
conditions, refer to Methods. BMV infectivity in protoplasts ( 0 - - 0 )
and percentage of
fluorescing protoplasts ( ( 3 - - ( 3 ) were determined by bioassay and fluorescent antibody staining,
respectively.
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Infection of protoplasts with B M V RNA
71
Table 8. Effect of ribonuclease treatment on infection of barley protoplasts
with BMV RNA*
Temperature
(°C)
,
o
2o
Relative time when
ribonuclease added
(rain)
Treatment
no.
I
-- I0
2
0
-
0"~
0
2
3
2
I2
--
4
5
6
7
8
5
8
IO
I5
Not added
28
29
33
56
7o
6
4
I5
23
* Protoplasts were inoculated as described in Methods. Ribonuclease (RNase) was added to make a final
concentration of 5/Jg/ml at the start of pre-inoculation incubation (treatment no. I) or during inoculation
(no. 2 to 7). Inoculated protoplasts were washed with mannitol solution containing 5 #g/ml RNase (treatments 5 to 7) or with RNase-free solution (other treatments). All protoplasts were then incubated in
RNase-free medium.
Figures are % fluorescing protoplasts determined by fluorescent antibody staining after incubation
for 24 h.
I
I
I
J
I
I
|
I
I
I
8°I
60
r~
o
40
o
20
O
0
~
5
Time of addition (rnin)
10
Fig. 5. Effect of CaCI2 ( O - - Q ) and pH shift ( O - - © ) on infection of barley protoplasts with
BMV. CaC12 and phospbate buffer, pH 7"2, were added to inoculation mixture at different times
after mixing protoplasts with BMV to make final concentrations 1o and 2 raM, respectively.
Infection was determined by fluorescent antibody staining after incubation for 44 h.
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72
T. OKUNO AND I. FURUSAWA
Fig. 6. An electron micrograph of a section of a barley protoplast inoculated with BMV. Protoplasts isolated in 0"5 M-mannitol were inoculated in 0"7 M-mannitol. For the other conditions of
inoculation, refer to the legend of Fig. 2.
Temperature and pre-inoculation incubation
The temperature that is optimal for infection of protoplasts with virus R N A differs in
different experimental conditions (Aoki & Takebe, I969; Motoyoshi et al. ~973; Sarkar
et al. r974; Beier & Bruening, I976). In the barley protoplast-BMV R N A system described
here, protoplasts were more efficiently infected by inoculation at o °C than at 2o or 3o °C
(Table 7). In contrast, infection with BMV was hardly affected by temperature during
inoculation (data not shown). Infection with BMV R N A was slightly increased by the
pre-inoculation incubation of BMV R N A and poly-L-ornithine at either o or 3o °C
(Table 7).
Virus growth curve
Fig. 4 shows that the infectivity of protoplast extracts and the proportion of fluorescing
protoplasts increased with increasing interval after inoculation. Fluorescing protoplasts
(1%) and infectivity were first detectable 7 and 12 h after inoculation, respectively, and
increased rapidly until 26 h after inoculation and thereafter more slowly.
Establishment of infection of barley protoplasts with B M V and B M V RNA
To investigate the effect of exogenous ribonuclease (RNase) on the infection of barley
protoplasts with BMV RNA, pancreatic RNase A was added at different times before,
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Infection oJ protoplasts with B M V R N A
73
during and after inoculation and the mixtures incubated for Io to 3o'min at o or 30 °C.
Table 8 shows that infection efficiency increased with increase in the delay before adding
RNase. Infection at o or 3o °C was hardly affected by exogenous RNase added at postwashing (data not shown).
Infection with BMV was similarly affected by adding CaC12 or by changing the pH to
7"0 (Fig. 5)- Barley protoplasts, inoculated with 2o/zg/ml BMV in low ionic strength
medium, are not infected in the presence of ~o mM-CaCI2 or when the pH is above 6-6
(Furusawa & Okuno, I978; Okuno & Furusawa, I978a). Fig. 5 shows, however, that such
factors no longer affected infection 5 to IO min after mixing protoplasts with BMV.
The mechanism of infection of barley pro toplasts with B M V
The effect of osmotic shock on the ultrastructure of BMV-inoculated barley protoplasts
was investigated by electron microscopy. Protoplasts were inoculated with BMV and an
osmotic shock was applied by either increase or decrease of mannitol concentration immediately before inoculation. After exposure to the inoculum for IO rain, protoplasts were
collected and fixed. Invaginations suggestive of pinocytosis and the resulting vesicles containing virus-like particles, were seen only in protoplasts inoculated after an increase of
mannitol concentration, and 86 % of these protoplasts became infected (Fig. 6). No such
structures were observed in protoplasts inoculated after a decrease of mannitol concentration and only 3 % of these protoplasts became infected. In neither sample were the
membrane lesions reported by Burgess et al. (I 973) observed.
DISCUSSION
It was demonstrated in this paper that up to 90 % of barley protoplasts became infected
after inoculation with BMV RNA (0"5 #g/ml). This infection efficiency is nearly a hundred
times greater than that reported by Beier & Bruening (1976) in the cowpea protoplastcowpea mosaic virus (CPMV) RNA system and also superior to that reported by Sarkar
et al. (I974) in the tobacco protoplast-TMV RNA system. Most infection was obtained
when protoplasts were isolated and inoculated in medium of low osmotic strength, and
infection efficiency decreased when the osmotic strength of the medium was increased
during isolation and/or inoculation. These effects may be related to findings that medium
of high osmotic strength triggers de novo synthesis of RNase in tobacco protoplasts and
that RNase activity increases with increasing osmotic strength of the incubation medium
(Premecz et aL t977). Indeed the effects of mannitol concentration, cycloheximide and
L-lysine on infection of barley protoplasts with BMV RNA all suggest that low RNase
activity may be one of the main requirements for infection to occur in this system. Furthermore, these factors can reasonably explain the discrepancy between the effects of osmotic
shock on infection of barley protoplasts with BMV or with BMV RNA and also the
relatively small amount of infection produced by inoculating tobacco protoplasts with
several different kinds of virus RNA using medium containing relatively high concentrations
of mannitol (o'7 or o.8 M) (Aoki & Takebe, 1969; Motoyoshi & Hull, 1974; Motoyoshi
et al. I974a, b). Indeed, Beier & Bruening (1976) obtained a relatively high level of infection
(65%) in the cowpea protoplast-CPMV RNA system using medium containing 0.45 Umannitol throughout protoplast isolation and inoculation.
The effects of several other factors on infection with BMV RNA were investigated in
the experiments described in this paper. Infection was most efficient when barley protoplasts were inoculated as described in Methods. The effect of RNA concentration on
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74
T. O K U N O
A N D I. F U R U S A W A
infection suggests that electrostatic properties of BMV RNA-poly-L-ornithine complexes
are also of great importance in infection as are those of complexes of BMV particles and
poly-L-ornithine (Okuno & Furusawa, I978a). Infection with BMV R N A probably also
reflects the stability of R N A molecules against RNase attack. This is suggested by the low
temperature requirement for infection with BMV RNA. A change in one factor seemed to
alter the optimum levels of other factors. When phosphate buffer was used instead of
citrate, the optimum concentration of BMV R N A is increased, athough concentrations
greater than 9 #g/ml BMV R N A were not tested; similar results are reported by Beier &
Bruening (1976) in the cowpea protoplast-CPMV R N A system. The optimum concentration of citrate buffer depended on the concentration of BMV R N A used (Table 4,
Expt. 4). The effect of divalent cations on infection is complicated because they affect not
only the configuration of R N A molecules, and hence the interaction of R N A with poly-Lornithine and its stability against RNase attack, but also the stabilization of the protoplast
plasma membrane. The difference between the effect of divalent cations on infection with
BMV and its RNA may reflect the difference between the charge on BMV particles and
complexes of BMV R N A and poly-L-ornithine.
The finding that infection with BMV R N A was enhanced by increasing the osmotic
strength of the medium at the stage where BMV R N A was converted to a RNase-insensitive state suggest that the incorporation mechanism may be similar for BMV and its RNA.
Two different mechanisms have been suggested to explain infection of protoplasts with
virus. One is endocytosis, an energy-requiring physiological process and the other involves
membrane lesions and is a non-energy-requiring process in which there is a passive influx
of virus (see Takebe, ~975)- The results presented in this paper demonstrate two important
phenomena: (I) that infection of protoplasts with BMV and its RNA occurs at nonphysiological temperatures and (2) that invaginations suggestive of pinocytosis and vesicles
containing virus-like particles were observed only in samples of protoplasts, the majority
of which became infected with BMV, but not in samples in which few protoplasts became
infected, and no membrane lesions of the type reported by Burgess et al. (I973) were
observed in either sample. Thus it might be concluded that the invaginations and resulting
vesicles were responsible for infection, and that the formation of invaginations was stimulated by the osmotic shock produced by increase of osmotic strength of the medium.
Burgess et al. 0973) reported in the tobacco protoplast-pea enation mosaic virus system
that endocytotic vesicles involving virus particles could be observed in each protoplast
section only in the infection system in which up to 4o % of protopIasts became infected
without poly-L-ornithine. Therefore it is interesting to speculate that osmotic shock may
also stimulate infection by acting at a stage following virus entry into cells.
We thank Professor K. Takeuci, Laboratory of Cytology, Faculty of Science, Kyoto
University, for his help with fluorescence microscopy.
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