J. gen. Virol. (1989), 70, 1609-1612. Printed in Great Britain
1609
Key words: PSTV/TMV/transport function
Potato Spindle Tuber Viroid Does Not Complement Tobacco Mosaic Virus
Temperature-sensitive Transport Function
By O. A. K O N D A K O V A , 1 S. I. M A L Y S H E N K O , 1 K. A. M O Z H A E V A , 2
T. YA. V A S I L I E V A , - ' M. E. T A L I A N S K Y 1 AND J. G. A T A B E K O V 1.
1Department of Virology, Moscow State University, Moscow 119899 and
2Institute of Phytopathology, Moscow District 143050, U.S,S.R.
(Accepted 27 February 1989)
SUMMARY
In mixed infections, the potato spindle tuber viroid (PSTV) does not complement the
transport function of the tobacco mosaic virus (TMV) Lsl mutant, which has a
temperature-sensitive transport function. This appears to be due to the fundamentally
different mechanisms of virus and viroid transport. However, PSTV significantly
enhances the accumulation of temperature-resistant TMV in mixed infections at a
temperature non-permissive for Lsl.
When a plant is inoculated with a virus, the latter replicates in the primarily infected cells and
then moves into neighbouring healthy ones. The process of systemic spread of viral infection in a
plant is controlled by a special transport function (TF) encoded in the virus genome (for review,
see Atabekov & Dorokhov, 1984). Tobacco mosaic virus (TMV) mutants Ni2519 (Zimmern &
Hunter, 1983) and Lsl (Ohno et al., 1983), temperature-sensitive (ts) in systemic spread
(transport) through infected plants, possess mutations in the gene coding for the 30K protein
that is responsible for the TF (Deom et al., 1987; Meshi et aI., 1987). Viroids do not code for
transport proteins yet are also capable of systemic spread (Palukaitis, 1987), but it is evident that
movement of viruses and viroids through the plant occurs in fundamentally different ways.
The virus-encoded TF can be regarded as a factor controlling the resistance of a plant to a
virus (Taliansky et al., 1982c). In many virus-host combinations a virus is restricted to
replication only in primarily infected cells or in isolated protoplasts because TF is somehow
blocked (e.g. the transport protein is not functional in the given type of plant or it becomes nonfunctional at a non-permissive temperature). This type of resistance can be overcome by TF
complementation under conditions of mixed infection when transport of a virus that is deficient
in TF is provided by a helper virus. It is of note that complementation can occur not only
between related viruses but also between unrelated ones (Taliansky et al., 1982b, c; Carr & Kim,
1983; Atabekov et al., 1984; Barker, 1987; Malyshenko et al., 1987, 1988). Although the
molecular mechanisms of viral TF are still obscure, it can be suggested that the virus-specific
transport protein plays a significant role in this process.
In this connection the possibility of complementation in TF between viruses and viroids was
of interest. The possibility could not be ruled out that the viroid might promote the movement of
the virus, causing some general physiological effects in the plant cells. However in this work we
show that the systemic spread of TMV ts transport mutant Lsl cannot be complemented by the
potato spindle tuber viroid (PSTV).
Tomato plants (cv. Rutgers) were first infected with PSTV (isolate from Armenia); a total
preparation of low M~ R N A (1 mg/ml) from PSTV-infected Scopolia plants was used for
inoculations (Morris & Wright, 1975). Three to 4 weeks after the inoculation of tomato plants
with PSTV, the uninoculated leaves showing symptoms of PSTV disease from systemic
infection were superinfected with the TMV ts mutant Lsl (10 gg/ml) (the second inoculation)
0000-8752 © 1989 SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 07:10:55
1610
Short communication
Table 1. Accumulation o f the T M V ts mutant L s l in tomato plants preinoculated with P S T V
Analysis of virus progeny
h
Amount of TMV
Inoculum
(ng/g of
leaf tissue)t
1st
2nd
Temperature c~ "
~ r
inoculation
inoculation*
(°C)
I§
II
PSTV
PSTV
Lsl
Lsl
Lsl
Lsl
33
24
33
24
< 10
1200
1000
860
< 10
1000
490
1050
I
Infectivity
(results of TST)$
~
1I
,
HI
0
0
1 (1)
87 (s)
165 (s)
78 (s)
2 (s)+ 123 (1) 8(s)+ 142 (1) 7(s) + 108 (1)
105 (s)
132 (s) 90 (s)+ 1 (1)
* Concentration of Lsl inoculum was 10 ~tg/ml.
t Detected by ELISA.
The virus progeny was analysed under conditions of TST (24 °C ~ 33 °C --, 24 °C) (see text); (s) denotes small
lesions, (1) large lesions. The average numbers of local lesions per half-leaf of N. tabacum are shown.
§ Roman numerals indicate individual experiments.
and incubated at 24 °C or 33 °C. L s l was supplied by Dr N. Oshima and purified as described
earlier (Atabekov et al., 1970). Some plants were infected with Lsl in the absence of PSTV and
were used as a control. Four days later the leaves inoculated with Ls 1 were harvested and treated
with anti-TMV serum to remove any surface virus particles. After 15 min antiserum was
removed by washing the leaves with water. The leaves were then homogenized in 0.01 Mphosphate buffer p H 7.2. The virus content in the extracts was determined by the double
antibody sandwich method of E L I S A using serial dilutions of purified T M V as concentration
standards (Malyshenko et al., 1985). T M V infectivity was determined as the average number of
local lesions per half-leaf of Nicotiana tabacum cv. Samsun N N . Temperature sensitivity of the
virus in T F was determined by temperature shift treatment (TST) (Jockusch, 1968). The
inoculated leaves of N. tabacum were kept at 24 °C for 2 days to allow the growth of primary local
lesions and then transferred to 33 °C for 1 day. The formation of lesions was blocked at 33 °C
and the virus spread systemically only if it was not ts in transport. During the subsequent
transfer to 24 °C, the infected parts of the leaf became necrotic. As a result the T M V m u t a n t ts in
transport induces small lesions whereas temperature-resistant (tr) T M V produces large lesions
with a characteristic halo of necrotic tissue.
The T M V mutant Lsl (ts in TF) accumulated in very low amounts for 4 days in tomato plants
at the non-permissive (33 °C) temperature in the absence of the viroid (Table 1). Unexpectedly,
a significant amount of the virus progeny accumulated at the same temperature in L s l inoculated tomato plants preinfected with PSTV (Table 1). These results could have been
interpreted as evidence for the ability of PSTV to complement the L s l ts transport function.
However the data from TST show that the virus progeny in these experiments differed from the
initial T M V Lsl in that it was not ts but tr in T F (Table 1). W e propose two suggestions to
explain these unexpected results. Firstly, it is possible that the L s l preparations contain a low
level o f contamination of tr T M V and tr revertants may be produced; these m a y have spread
and accumulated selectively at the non-permissive high temperature. This suggestion is in
accordance with the fact l h a t upon prolonged incubation (8 to 10 days) in the control plants
inoculated with T M V ts mutants the gradual accumulation of tr T M V occurs frequently,
although it is not detectable in the usual 4 day experiments (Taliansky et al., 1982 a, b). A similar
effect has been detected by Bosch & Jockusch (1972) in work with Ni2519, another T M V
mutant ts in TF. It is clear, however, that the proportion, if any, of the tr contaminant in the
initial Lsl inoculum is extremely low according to two control tests the results of which are
shown in Table 1 (Lsl only, at 33 °C and 24 °C). Another suggestion is that upon mixed
infection, PSTV is capable of enhancing the reproduction of T M V particularly in the case of tr
T M V accumulation at the temperature non-permissive for Lsl. The phenomenon of virus
replication enhancement by a viroid has been demonstrated recently by K a r o s a w a & E h a r a
(1988) in mixed infections of cucumber mosaic virus and the hop stunt viroid.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 07:10:55
Short communication
1611
Table 2. Enhancement of reproduction of TMV tr strain vulgate by PSTV in mixedly infected
tomaW p&n~
Inoculum
r
~
~
2nd inoculation
1st inoculation
(TMV; ~g/ml)
PSTV
PSTV
PSTV
-
PSTV
10.0
10.0
10.0
10.0
0.1
0.1
0.1
0.1
Analysis of virus progeny
~
-~
A m o u n t of T M V
Infectivity
(ng/g of leaf tissue)*
(results of TST)~
I~
II
I
r
Temperature
(°C)
24
24
33
33
24
24
33
33
1400
1800
1600
1500
80
640
120
1400
1100
1300
1200
1300
50
800
87
112
105
94
5
42
(1)
(1)
(1)
(1)
(1)
(1)
9 (1)
64 (1)
* Detected by ELISA 4 days after inoculation with TMV.
t The virus progeny was analysed as described in the footnote of Table 1.
:~ R o m a n numerals indicate individual experiments.
A series of model experiments were performed to examine the second suggestion and to
evaluate the enhancement of TMV accumulation in the presence of PSTV. The standard TMV
tr strain vulgare (U 1) was used in these experiments because the real nature and the origin of the
accumulated tr TMV in plants mixedly infected with PSTV and Lsl at 33 °C (Table 1) was
obscure. Plants preinfected with PSTV were superinoculated with TMV vulgare at different
concentrations and temperatures (Table 2). PSTV significantly enhanced the accumulation o f t r
TMV (at 24 °C as well as at 33 °C) when the concentration of TMV in the inoculum was quite
low (0.1 ~tg/ml) (Table 2). When TMV concentration was increased (to 10pg/ml) the
enhancement was not detectable, which is probably due to the virus itself rapidly accumulating
in this case.
It seems probable that at the temperature non-permissive for Ls 1 transport, the conditions for
selective spread of TMV tr contaminants (and/or revertants) are established and PSTV
enhances their accumulation in mixedly infected tomato plants. It is important to note that
systemic movement of Lsl at 33 °C in the presence of viroid did not occur (Table 1), i.e. the TF
of TMV was not complemented by PSTV. This finding may be due to differences in the
mechanisms of TF between viruses and v~roids in infected plants.
REFERENCES
ATABEKOV, L O. & DOROKHOV, YU. L. (1984). Plant virus specific transport function and resistance of plants to
viruses. Advances in Virus Research 29, 313-364.
ATABEKOV,J. O., NOVIKOV,V. K., VISHNICHENKO,B. K. & KAFTANOVA,A. S. (1970). Some properties of hybrid viruses
reassembled in vitro. Virology 41, 519-532.
ATABEKOV,J. G., TALIANSKY, M. E., DRAMPYAN,A. H., KAPLAN, L B. & TURKA, I. E. (1984). Systemic infection by a
phloem-restricted virus in parenchyma cells in a mixed infection. Biologicheskie Nauki 10, 28 31 (in Russian).
BARKER, H. (1987). Invasion of non-phloem tissue in Nieotiana clevelandii by potato leafroll luteovirus is enhanced
in plants also infected with potato Y potyvirus. Journal of General Virology 68, 1223 1227.
BOSCH, F. X. & JOCKUSCH, H. (1972). Temperature-sensitive m u t a n t s of T M V : behaviour of a non-coat protein
m u t a n t in isolated tobacco cells. Molecular and General Genetics 116, 95-98.
CARR, R. J. & KIM, K. S. (1983). Evidence that bean golden mosaic virus invades non-phloem tissue in double
infections with tobacco mosaic virus. Journal of General Virology 64, 2489-2492.
DEOM, C. M., OLIVER, M. J. & BEACHY, R. N. (1987). The 30-kilodalton gene product of tobacco mosaic virus
potentiates virus movement. Science 237, 389-394.
JOCKUSCH, n. (1968). Two m u t a n t s of tobacco mosaic virus temperature-sensitive in two different functions.
Virology 35, 94-101.
KAROSAWA,A. & EHARA,Y. (1988). Double infection with hop stunt viroid and cucumber mosaic virus on cucumber
plants. Abstracts of 5th International Congress of Plant Pathology, Kyoto, Japan, p. 71.
MALYSHENKO,S. I., KAPLAN, I. B., MUSHEGYAN,A. R., TALIANSKY, M. E. & ATABEKOV,J. G. (1985). Stimulation of
potato virus X reproduction in the cells of susceptible and resistant plants. Izvestia Akademii Nauk SSSR,
Seria Biologicheskaya 3, 339 343 (in Russian).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 07:10:55
Short communication
1612
MALYSHENKO, S. I., TALIANSKY, M. E., KONDAKOVA, O. A., ULANOVA, E. F. & ATABEKOV, J. G. (1987). Plant virus-
specific transport function is a factor controlling virus host range. Izvestia Akademii Nauk SSSR, Seria
Biologicheskaya 5, 680-685 (in Russian).
MALYSHENKO,S. I., LAPCHIC, L. G., KONDAKOVA,O. A., KUZNETZOVA, L. L., TALIANSKY,M. E. & ATABEKOV,J. G. (1988).
Red clover mottle comovirus B-RNA spreads between cells in tobamovirus-infected tissues. Journal of
General Virology 69, 407-412.
MESHI, T., WATANABE,Y., SAITO, T., SUGIMOTO, A., MAEDA, T. & OKADA, Y. (1987). Functions of the 30 kd protein of
tobacco mosaic virus: involvement in cell-to-cell movement and dispensability for replication. EMBO
Journal 6, 2557-2563.
MORRIS, T. J. & WRIGHT, N. S. (1975). Detection on P A A G of a diagnostic nucleic acid from tissue infected with
potato spindle tuber viroid. American Potato Journal 52, 57-63.
OHNO, T., TAKAMATSU,N., MESHI, T., OKADA, Y., NISHIGUCHI, M. & KIHO, Y. (1983). Single amino acid substitution in
30K protein of TMV defective in virus transport function. Virology 131, 255 258.
PALUKAITIS,P. (1987). Potato spindle tuber viroid: investigation of the long-distance, intra-plant transport route.
Virology 158, 239 241.
TALIANSKY,M. E., ATABEKOVA,T. I., KAPLAN, I. B., MOROZOV, S. YU., MALYSHENKO,S. I. & ATABEKOV,J. G. (1982a). A
study of TMV ts mutant Ni2519. I. Complementation experiments. Virology 118, 301-308.
TALIANSKY,M. ~., MALYSHENKO,S. I., PSHENNXKOVA,E. S., KAPLAN,I. B., ULANOVA,E. F. & ATABEKOV,J. G. (1982b).
Plant virus-specific transport function. I. Virus genetic control required for systemic spread. Virology 122,
318-326.
TALIANSKY,M. E., MALYSHENKO,S. I., PSHENNIKOVA, E. S. & ATABEKOV,J. G. (1982c). Plant virus-specific transport
function. II. A factor controlling virus host range. Virology 122, 327-332.
ZIMMERN, D. & HUNTER, T. (1983). Point mutation in the 30K open reading frame of TMV implicated in
temperature-sensitive assembly and local lesion spreading of mutant Ni2519. EMBO Journal 2, 1893-1900.
(Received 2 November 1988)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 07:10:55