Download Accepted so July I979 - Journal of General Virology

623
J. gen. Virol. 0979), 45, 623-630
Printed in Great Britain
Strain-specific Pathways of Cytological Change in Individual Chinese
Cabbage Protoplasts Infected with Turnip Yellow Mosaic Virus
By L. F R A S E R AND R. E. F. M A T T H E W S
Department of Cell Biology, University of Auckland, Auckland, New Zealand
(Accepted so July I979)
SUMMARY
Two mutually exclusive pathways of cytological change in the chloroplasts of
Chinese cabbage cells infected with turnip yellow mosaic virus have been defined by
light microscopic examination of individual infected protoplasts. In the first, all
the chloroplasts in a cell became rounded and clumped together. This was followed
by the development of a large vacuole, giving the chloroplast a sickled appearance.
In the second pathway the chloroplasts became angular in outline before clumping, and subsequently fragmented to yield small pieces of chloroplast. Both these
responses are controlled by the virus genome because some virus strains gave rise
to the sickling response, while others cause fragmentation. Photosynthetic activity
is a prerequisite for both pathways since they occur only when the protoplasts are
illuminated, and both are inhibited by an inhibitor of photosynthetic electron
transport.
INTRODUCTION
The pathogenesis of virus disease is a complex process, or series of processes, that is not
yet understood for any plant virus. A long-term research objective is to delineate the
biochemical steps by which the virus genome and the process of virus replication gives rise
to the diseased state. For certain diseases, studies using light microscopy provide a useful
bridge between macroscopic observations and electron microscopy and biochemical studies
of diseased cells.
Turnip yellow mosaic virus (TYMV) infection of Chinese cabbage is a particularly
favourable example for the use of light microscopy because the virus induces several
characteristic gross changes in the chloroplasts of infected cells in addition to biochemical
changes and structural changes seen only by electron microscopy. All strains of the virus
induce numerous, very small vesicles near the periphery of the chloroplasts which are sites
of virus RNA replication (Lafl6che & Boy6, I969, I97I; Ushiyama & Matthews, 197o).
For all strains of the virus that have been described, chloroplasts in infected cells became
rounded and subsequently clumped together. The rounding process occurs at an early stage
following infection, while clumping occurs later, soon after virus production has begun
(Hatta & Matthews, I974). Furthermore it is known that different strains of the virus may
differ in the other cytological effects they induce in the chloroplasts and that these changes
may be associated with diseases of varying severity. Additional abnormalities noted in
fresh sections from infected plants included angular plate-like chloroplasts, very large
vacuoles and fragmented chloroplasts (Chalcroft & Matthews, J967; Matthews, I973, I979).
These latter abnormalities became apparent late in infection after most virus replication
had already occurred.
Isolated protoplasts offer the possibility of studying the sequential development of
cytological changes. Thus Matthews & Sarkar (I976) showed that when protoplasts isolated
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L. F R A S E R
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R. E. F. M A T T H E W S
from TYMV-infected leaves were exposed to light, a proportion of the rounded and
clumped chloroplasts developed large clear vesicles, giving rise to a sickled appearance. The
transition from rounding to sickling was inhibited by 3-(3,4-dichlorophenyl)-I,I-dimethylurea (DCMU). These experiments were limited by the fact that observations were made
at various times on samples taken from a bulk culture of protoplasts.
In the work described here we have developed a method which permits the repeated
examination by light microscopy of the changes taking place in individual protoplasts.
Using this method, alternative and mutually exclusive pathways of disease development in
chloroplasts have been delineated which we have termed sickling and fragmentation. Both
processes are dependent on active photosynthetic electron transport and are dependent on
some strain-specific function of the virus genome.
METHODS
Phmts and viruses. The stock culture of TYMV used in this laboratory was maintained
in Chinese cabbage plants (Brassicapekinensis Rupr. c.v. Wong Bok) grown in the glasshouse.
Strains of the virus varying in the extent to which they affected chloroplast structure were
isolated by the method ofChalcroft & Matthews (t 967). Experimental plants were maintained
in the glasshouse at 21-+3 °C under natural lighting, supplemented during winter with
light from Philips HLRG 40o W lamps to give an 8 h dark period. TYMV was isolated from
infected plants by the method of Matthews (196o) and stored at 4 °C in 5o ~ glycerol.
Chemicals and enz)wws. Macerozyme Rio and cellulose 'Onozuka' R i o were from
Yakult Biochemical Co. Ltd, Nishinomiya, Japan. DCMU was from Serva, Heidelberg.
Mannitol was from E. Merck, Darmstadt.
Electron microscopy. Protoplasts in o'4 M-mannitol were fixed and post-fixed using the
method of Takebe et al. (1973), in half-strength Millonig's phosphate buffer, pH 7"5,
containing glucose (Pease, 1964). Protoplasts were then embedded in agar using the method
of Kellenberger et al. (I958) and passed through an acetone dehydrating series at room
temperature. Embedding, sectioning and staining procedures have been described previously
(Ushiyama & Matthews, I97o). Sections were examined with a Philips EM 3ol electron
microscope.
Protoplast isolation. Protoplasts were isolated from healthy or infected leaves by the
method of Matthews & Sarkar (1976). The final pellet of protoplasts was washed three times
with 0"4 M-mannitol and finally suspended in medium to give a protoplast density of
I × ~oa protoplasts//d. The medium used was that of Aoki & Takebe (t969), modified in
the following ways: o'4 M-mannitol, 5 x final concentration of Mg and half strength with
respect to other components.
A microdrop method for incubating protoplasts. Study by light microscopy of time-dependent processes in infected protoplasts should be facilitated by a system with the following
properties: (i) containment of a small number of protoplasts sufficiently immobilized in a
confined space for the repeated recognition of individual protoplasts; (ii) adequate culture
conditions for survival of protoplasts for a minimum period of I to 2 days; (iii) ready
observation using the light microscope at any magnification.
Trials with various procedures and methods for microculture led to the simple system
shown in Fig. t and 2 which was used in all subsequent work.
The following factors were found to be important for successful microculture. (i) Drop
size: if the diam. of the drop when spread on the slide was too large (e.g. several mm) cells
near the centre died within 24 h, presumably due to lack of oxygen. (ii) Shear:great care was
needed when transferring the protoplasts by syringe. A very slow transfer rate was essential
to avoid mechanical damage. (iii) Light intensity: survival was best in the light intensity
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T Y M V strains in protoplasts
0
0
0
0
625
0
Fig. i. The microdrop method. Strips of double-sided sellotape were laid on a clean microscope
slide as indicated to form a chamber between slide and coverslip. Five o.2/zl drops of medium
containing protoplasts were then dispensed on to the slide. A 22 × 6o mm coverslip was placed in
position and two large drops of o'4 M-mannitol allowed to flow in on each side under the eoverslip.
The edges were then immediately sealed with warm wax. The warmth of the wax effectively humidified the chamber enclosing the drops. IB, sellotape; [], culture medium.
Fig. 2. A single 0.2/zl drop containing about 200 Chinese cabbage protoplasts.
range 2000 to 2500 lux. (iv) Protoplast concentration: optimal protoplast concentration
was about 2oo/o.2/~1 drop (i0" cells/ml). (v) Season of year: satisfactory protoplasts could
be prepared at all seasons except the winter months
RESULTS
Rounding, clumping and sickling
Protoplasts were prepared from Chinese cabbage leaves at various times after these had
been inoculated and incubated in microdrops. At the time of isolation, the protoplasts were
in three states with respect to TYMV infection: (i) infected; (ii) apparently normal but
developing cytological signs of infection during the incubation period; (iii)apparently
healthy.
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L. FRASER AND R. E. F. MATTHEWS
Fig. 3. Two effects of TYMV infection on Chinese cabbage chloroplasts. A comparison of rounded
clumped chloroplasts (a) and angular clumped chloroplasts (e). In the light rounded clumping gives
rise to sickling (b) and angular clumping to fragmentation (d).
Observations on protoplasts which developed rounding and clumping during the period
o f observation showed that all the chloroplasts in a cell became involved in the process at an
early stage. Within a given cell, the time between the earliest signs of rounding and full
rounding and clumping was about 2 to 4 h.
By contrast, the development o f sickling within the chloroplasts of any particular cell
t o o k 4 to IO h and there was no detectable synchrony in the process for the chloroplasts
within a single cell. Sickling was observed to begin only in protoplasts in which rounding
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Fig. 4. Section through part of a TYMV-infected protoplast with a chloroplast showing sickling.
and clumping had fully developed. Chloroplasts retained a bright yellow-green colour
throughout the sickling process. In some preparations of protoplasts from healthy leaves
about o'5 to T.o % of cells showed or developed a contraction and clumping of the chloroplasts, but this was readily distinguished from the smooth rounding of chloroplasts in
TYMV-infected cells.
Observations on many individual protoplasts confirmed that the development of sickling
from clumped chloroplasts was dependent on light and inhibited by D C M U (e.g. Table I).
The effects of light intensity and the time course of development in a population of individual protoplasts were very similar to those obtained by Matthews & Sarkar (I976) using
flask cultures of protoplasts. Fig. 3(a) and (b) illustrate the sickling pathway.
Electron microscopic examination of protoplasts with sickled chloroplasts suggested
that the vacuole formation which leads to the sickled appearance may be accompanied by a
substantial swelling of the chloroplast, but we have no quantitative data on this point. The
vacuole is bounded by a lipid bilayer membrane. It is unlikely that this membrane arises
from the external chloroplast membrane since the latter in infected cells always contains
numerous small vesicles which have never been observed in the membrane of the large
vesicle. Observations on many large vesicles (not illustrated here) suggest that the membrane
arises from distortions of stroma lamellae within the sickled chloroplast. The interior of the
large vesicles is electron lucent (Fig. 4) except where the membrane has been ruptured
allowing an influx of cytoplasmic material.
Angular clumped chloroplasts and fragmentation
In protoplasts isolated from leaves inoculated with the stock culture of TYMV most
infected protoplasts showed rounded and clumped chloroplasts. However, in some, the
clumped chloroplasts were angular rather than round (Fig. 3c). The time taken for the
angular outline and clumping to develop was about the same as for rounded chloroplasts
(2 to 4 h).
The angular chloroplasts underwent a slow colour change over a period of hours from
a bright yellowish green to a grey-green colour. In the light such chloroplasts increased in
diam. and gradually became webbed with fine lines dividing each chloroplast into sections.
These sections rounded up (Fig. 3d) and became separated. The process was markedly
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L. FRASER AND R. E. F. MATTHEWS
Table
Inhibition of fragmentation and sickling by DCMU*
[.
DCMU
concentration
infected
protoplasts with
fragmented
chloroplasts
A
_
~ infected
protoplasts with
sickled
chloroplasts
_
~ infected
protoplasts showing
only rounding and
clumping
,~_
A
oh
24h
oh
24 h
oh
24 h
o.o
o
36
6
28
[ × Io -~ M
o
z
2
4
94
98
36
95
* Protoplasts were prepared from leaves inoculated with a 'yellow' culture of TYMV which gave both
fragmented and sickled cells. Slides were exposed to 23oo lux continuous illumination at 23 °C. Ten
microdrops were assayed for each condition immediately after isolation and after 24 h.
i
©
100
--
,~
i
I
!
Strain A
i
I
_
~=
60--
20--
-
I
I
I
--
~-
II
15
15
7
Time after inoculation (days)
Fig. 5. Influence of strain of TYMV on the proportion of protoplasts developing sickled or fragmented chloroplasts. Protoplasts were prepared from leaves at various times between 7 and I6
days after inoculation with a strain of TYMV (A or B). Microdrops were cultured for 24 h under
23oo lux at 23 °C. The percentages of infected cells showing sickling or fragmentation were then
recorded. Strain A: this developed mainly necrotic local lesions which increased in size and developed chlorotic haloes after about I4 days. Strain B : this gave only chlorotic local lesions until day
~4 when a few chlorotic local lesions developed necrotic centres. The day when necrotic local
lesions appeared is arrowed for each strain. ( 3 - - 0 , protoplasts with sickling; 0 - - 0 , protoplasts
with fragmentation.
7
ll
a s y n c h r o n o u s with respect to the chloroplasts in a single cell. The f r a g m e n t a t i o n process
did n o t occur in the dark. F r a g m e n t a t i o n was an alternative to sickling, since each whole
protoplast was committed to one or the other process. A n g u l a r c l u m p i n g and fragmentation of chloroplasts like that f o u n d in TYMV-infected cells were not observed in chloroplasts o f protoplasts derived from healthy plants.
Inhibition of fragmentation by DCMU
The experiment summarized in Table I confirms the observation of Matthews & Sarkar
(1976) that the sickling response is inhibited by D C M U and shows that the f r a g m e n t a t i o n
process is similarly inhibited.
Influence of virus strain on the proportion of sickled and fragmented chloroplasts
Six cultures of T Y M V isolated from the stock culture of mixed strains a n d showing various
local lesion types (and p r e d o m i n a n t systemic symptoms) were tested for the development of
the sickling and fragmentation responses. The response in vitro by the isolated protoplasts
was d e a r l y d e p e n d e n t on the strain of virus used to inoculate the leaves. This is illustrated
for two isolates in Fig. 5.
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TYMV
strains in protoplasts
629
In this e x p e r i m e n t and in others not described here there was a consistent correlation
between the a p p e a r a n c e o f necrosis in the inoculated leaves a n d the d e v e l o p m e n t o f the
f r a g m e n t a t i o n response in isolated protoplasts. However, there was no evidence that strains
p r o d u c i n g necrosis in the leaf caused increased d e a t h o f p r o t o p l a s t s in vitro. F o r example
8o to 9o % o f isolated p r o t o p l a s t s survived for 24 h at all leaf s a m p l i n g times for b o t h the
strains illustrated in Fig. 5.
DISCUSSION
The d e v e l o p m e n t o f a p r o c e d u r e for the repeated e x a m i n a t i o n o f individual p r o t o p l a s t s
has enabled us to delineate alternate a n d a p p a r e n t l y m u t u a l l y exclusive p a t h w a y s o f disease
induction by T Y M V in the c h l o r o p l a s t s o f infected cells. The usefulness o f the technique
will be limited o f course to those rather u n c o m m o n h o s t - v i r u s c o m b i n a t i o n s t h a t give rise
to cytological changes readily discernible by light microscopy.
Several biochemical changes associated with c h l o r o p l a s t activity have been r e p o r t e d for
T Y M V - i n f e c t e d tissue. Goffeau & Boy6 (I965) f o u n d that the Hill reaction a n d b o t h
cyclic and non-cyclic p h o t o p h o s p h o r y l a t i o n were increased in chloroplasts o f inoculated
leaves c o m p a r e d with uninfected leaves. B e d b r o o k & M a t t h e w s (t972) f o u n d that, in systemically infected leaves, there was a diversion o f the p r o d u c t s o f p h o t o s y n t h e t i c c a r b o n
fixation a w a y f r o m sugars and into organic acids a n d a m i n o acids.
The fact that D C M U inhibits sickling a n d f r a g m e n t a t i o n shows that b o t h processes
require active p h o t o s y n t h e t i c electron t r a n s p o r t , b u t the biochemical bases for the two
kinds o f disease process are not yet understood. A l t h o u g h b o t h processes are seen only at a
late stage o f infection, the initiating biochemical activity m a y well occur very soon after
infection. Since the sickling a n d f r a g m e n t a t i o n responses are m a r k e d l y s t r a i n - d e p e n d e n t
a n d since we have never observed the two responses in the same cell, it is likely that most
individual cells in a diseased p l a n t are infected entirely o r p r i m a r i l y by one strain o f the
virus (Matthews, I973).
O u r results show that in further w o r k a i m e d at exploring the biochemical basis for these
cytological effects it will be necessary to use single strains o f the virus or cultures c o n t a i n i n g
p r e d o m i n a n t l y a single strain. This m a y be a difficult objective, since it is k n o w n that single
lesion isolates f r o m the stock culture o f T Y M V r a p i d l y revert to a mixture o f strains.
W e t h a n k M r s J. Keeling for assistance with electron microscopy.
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