Resistance to Tobacco Mosaic Virus in Tomato - Journal of General ...

J. gen. ViraL 0980), 48, 87-96 87
Priated in Great Britain
Resistance to Tobacco Mosaic Virus in Tomato:
Effects of the Tin-1 Gene on Virus Multiplication
By R. S. S. FRASER AND S. A. R. LOUGHLIN
Biochemistry Section, National Vegetable Research Station,
Wellesbourne, Warwick CV35 9EF, U.K.
(Accepted I I December ~979)
SUMMARY
The gene Tin-1 in tomato plants is dominant for suppression of mosaic symp-
toms caused by tobacco mosaic virus isolates designated as tomato strain o. Virus
multiplication (measured either by virus RNA content or by virus coat protein
content) was inhibited in plants containing Tm-l. Inhibition was greater in hosts
homozygous for Tin-1 (90 to 95 %) than in hosts heterozygous for Tin-1 (65 to
75 %). Thus, inhibition of tobacco mosaic virus multiplication is Tin-1 gene dosage-
dependent; suppression of visible symptoms is not.
Inhibition of TMV RNA accumulation occurred in both directly inoculated and
systemically-infected leaves of Tin-l-containing hosts, and was consistent from
experiment to experiment. Tin-1 inhibited accumulation of RNA of different
isolates of strain o to different extents: two isolates causing yellow mosaic symptoms
on susceptible plants were especially strongly inhibited.
In Tm-I hosts, there was a delay between inoculation and the first detection of
virus multiplication, measured either as increase in virus RNA content or recover-
able infectivity. The delay was about 8 days in heterozygous Tm-1 hosts and about
t 6 days in homozygous Tin-1 hosts. The implications of these results for the mode
of action of the Tin-1 gene resistance are discussed.
INTRODUCTION
Tobacco mosaic virus (TMV) causes a serious disease of tomato, but good control has
been achieved by use of host resistance. Several sources of resistance have been found
(reviewed by Pelham, 1966; Walter, I967; Alexander, 1971; Fletcher, 1973). Of these, the
resistance genes designated Tm-1, Tin-2 and Tm-22 have been studied most. However, we
do not yet know the biochemical mechanisms by which these genes prevent virus multi-
plication or symptom development.
The Tin-1 gene suppresses the visible mosaic symptoms caused by TMV, but detectable
virus multiplication occurs (Clayberg, I96O; P&aut, T962, I964; Pelham, I966, I972). We
have studied the effects of the Tm-1 gene on the amount and pattern of TMV multiplication,
as a first approach to understanding how the gene works.
METHODS
Plants and viruses. Near-isogenic lines of tomato (Lycopersicon esculentum Mill. cv.
Craigella) were obtained from Dr T. J. Hall, Glasshouse Crops Research Institute, Little-
hampton, Sussex, U.K. The susceptible line contained no genes for resistance to TMV. The
two resistant lines had the Tin-1 gene in either heterozygous or homozygous form (Table I).
OO22-1317/80/0OO0-3955 $02.00 ~) 1980 SGM

88 R.S.S. FRASER AND S.A.R. LOUGHLIN
Table I. Symptoms and changes in chlorophyll content resulting from infection of three
tomato lines of different TMV-resistant genotypes with TMV strain o*
Symptoms Chlorophyll content (mg/g fresh wt)
produced by , --~ ,
Host line Genotype TMV infection Healthy TMV-infected
GCR z6 +/+ Systemic mosaic 4"o2+0-02 2"~9+_o'I t
J 484 Tin-I~ + None 3"74 -+ o.ol 3"55 + o'o8
GCR z37 Tm-1/Tm-1 None 3"53 --+ 0"05 3"59 + o'o I
* Plants were inoculated with TMV at a concentration of 2 #g/ml. Eighteen days later, symptoms were
observed and chlorophyll concentration determined on four replicate samples of upper, systemically-
infected leaves. Values are means + standard errors.
The plants were grown in I2. 5 cm diam. pots in Levington compost/sand (3:Q and
transferred to larger pots as necessary. Plants were kept in a glasshouse at I6 °C at night;
during the day the temperature rose to between 23 and 3 ° °C, depending on the season.
Natural lighting was supplemented in the winter months with morning and evening illumi-
nation from mercury vapour lamps giving an irradiance of 5o W/m e. Day length was a
minimum of I4 h.
Isolates of TMV tomato strains o and ~ (Pelham, I972) and a strain o isolate producing
yellow symptoms, designated as strain or, were obtained from Dr Hall, GCRI. The tomato
strainflavum (Melchers, I94o) and the tobacco strain vulgare were originally from the Max
Planck Institut for Biologie, Ttibingen, West Germany. All TMV strains were multiplied in
tobacco (Nicotiana tabacum L. cv. Samsun) and purified by the method of Mundry (I957).
Plants of uniform height were selected for each experiment; over all experiments the
plants ranged from to to 3o cm tall at the time of inoculation. Between 3 and 7 replicate
plants were used for each treatment or host genotype. Plants were inoculated on two upper,
expanded leaves by dusting with 4oo-mesh Carborundum and rubbing by hand with a
suspension of purified TMV at 2 to 5 O/zg/ml in 5o raM-sodium phosphate buffer, pH 7.
Leaves were washed with running tap water immediately after inoculation.
Measurement of TM V RNA. At each sampling time, leaflets were taken from all leaves
inoculated for each treatment. Samples were also taken from the 4th and 5th leaves above
the inoculated leaves. Midribs were removed and the laminas chopped roughly and mixed.
Three sub-samples of 0"5 or I'o g were then taken from each sample. Nucleic acids were
extracted as described by Fraser & Whenham 0978) and fractionated by electrophoresis
on 2.i % polyacrylamide gels (Loening, 1969) at 4 V/cm, 2"5 mA/gel, for 6 h. TMV RNA
concentration per g fresh weight of leaf was calculated from the peak area of the TMV RNA
on the u.v. absorption scan of each gel (Fraser, J97t).
Slab gel electrophoresis of proteins. Samples of o'5 g leaf were homogenized in 5 ml,
pH 8-6, buffer (4o mM-boric acid; 4I m~i-tris; 4%, v/v, 2-mercaptoethanol; 1o%, w/v,
sucrose ; o'75 %, w/v, sodium dodecyl sulphate) and heated in a boiling water bath for 3 rain
to extract and denature proteins. Debris was removed by centrifugation at 12 ooo g for 3 rain
and the protein extract stored at -2o °C. Samples of up to too/zl extract were fractionated
on slab polyacrylamide gels 560 mm wide and I mm thick, comprising a stacking gel (5 %
acrylamide), lo mm long, and a separating gel, t5o mm long, which consisted of a linear
gradient of lo to 3o% acrylamide. Gel preparation and the discontinuous buffer system
used were as described by Neville (I971). After electrophoresis for 12 h at lo mA constant
current per gel, the gels were stained with Page Blue G9o (CI 42655) and destained as
described by Laemmli (197o).
Chlorophyll content. Chlorophyll was extracted from sub-samples of 0"5 g leaf from

TMV resistance in tomato 89
systemically-infected leaves using 8o % acetone and was estimated spectrophotometrically
(Vernon, 196o).
Infectivity bioassay. Leaflets were harvested from inoculated/eaves at various times after
inoculation. The midribs were removed and weighed samples were stored at -2o °C until
samples from all harvest times were to hand. Each leaf sample was ground with 3 vol. of
5o raM-sodium phosphate buffer, pH 7, using a pestle and mortar. The extract was filtered
through muslin and used to inoculate Iz half-leaves of the local lesion host N. tabacum
L. cv. Xanthi-nc. The plants had been trimmed to four expanded leaves each, r day before
inoculation. The different inocula were distributed at random through the test plants, but
each inoculum was applied to the same number of lowest, second lowest, etc. leaves. Leaves
were dusted with Carborundum before inoculation and washed with running tap water after
inoculation. Lesions were counted 5 to 7 days after inoculation.
Measurement of TMV particle concentration. Virus was purified from samples of 5o to
200 g systemically-infected leaves by polyethylene glycol precipitations (Gooding & Hebert,
1967) and further purified by three cycles of differential centrifugation (Mundry, I957). Virus
concentration of the final solution was measured by u.v. absorption spectrophotometry,
taking an absorbance at z6o nm of I.o to equal o'37 mg TMV/ml.
RESULTS
Effects of the Tm-1 gene on symptoms and TMV multiplication
In confirmation of the results &Pelham (1972), we found that the Tm-1 gene is dominant
for suppression of visible mosaic symptoms caused by TMV (Table I). In either the
heterozygous or homozygous form, Tm-1 completely prevented the loss of chlorophyll in
systemically-infected leaves normally resulting from TMV infection (Table I).
We measured the amounts of TMV produced in susceptible and resistant plants by
estimating their contents of TMV RNA and coat protein. Ultraviolet absorption scans of
gels of nucleic acids showed that, in susceptible plants, TMV RNA accumulated to form a
very large peak and became the commonest nucleic acid species in the leaf. The Tm-l/+
host showed a much smaller TMV RNA peak, while even less TMV RNA accumulated in
Tm-1/Tm-1 plants. There was no evidence that TMV RNA in resistant hosts was suffering
from any partial degradation, which would have shown as a skewing of the peak to the more
mobile side.
Fig. I shows time courses ofTMV RNA accumulation in susceptible and resistant plants.
In the inoculated leaves of +/+ plants, TMV RNA concentration rose to a maximum at
2o days after inoculation, then declined. The decline was probably not due to degradation
ofTMV RNA, but to continued leaf growth diluting out the TMV RNA after accumulation
had slowed or stopped: leaf fresh weight continued to increase after 2o days when maximum
TMV RNA concentration was reached. The pattern of TMV RNA accumulation in
systemically-infected leaves of the susceptible host was similar, and not detectably later than
in inoculated leaves with the sampling frequency used.
In inoculated leaves of the resistant hosts, TMV RNA accumulation was much reduced
and the extent of reduction was Tm-I gene dosage-dependent (Fig. I). The heterozygous host
showed a TMV RNA content 3o % of that in the + / + control at 2o days after inoculation;
in the homozygous resistant host the TMV RNA concentration was only 5 % of that in the
control.
The patterns of accumulation of TMV RNA in inoculated and systemically-infected
leaves of resistant plants were very similar, though inhibition of TMV RNA accumulation
by Tm-1/+ or Tm-1/Tm.1 tended to be a little less in systemically-infected leaves than in

90
400
R.S.S. FRASER AND S.A.R. LOUGHLIN
. . . . . . I I I I I
(a) o
200 ~ o ~
o ~ '~'--t-D 1 I I I
~ soo o) o~
400 300 i) _
20O
,oo
o
0 ~0
0 10 20 30 40 50
Time after inoculation (days)
-
Fig. [. Changes in TMV RNA concentration in TMV-infected tomato leaves. The host genotypes
were: O--O, +/+ ; A--~, Tm-I/+ and U3--U], Tm-l/Tm-l. (a) Changes in leaves which became
infected by systemic spread of virus; (b) changes in leaves directly inoculated with strain o at
I o gg/ml.
inoculated leaves. These results suggest that the resistance does not operate against systemic
spread of the virus as such. In that case the multiplication of TMV in systemically-infected
leaves of Tin-1 plants would have been inhibited more than multiplication in inoculated
leaves.
The inhibition ofTMV RNA accumulation by Tm-I was long lasting: up to 56 days after
inoculation, when the experiment ended, the TMV RNA concentration in the homozygous
resistant host was still very much lower than in the control. Both resistant hosts showed a
fairly steady concentration of TMV RNA from 20 to 56 days. As the leaves were visibly
expanding during this time, it is probable that further virus synthesis was occurring, albeit
at a low rate.
Fig. 2 shows that susceptible plants contained large amounts of coat protein. The intensity
of the coat protein band was considerably less in the Tin-l~+ host, and coat protein was
barely detectable in the homozygous resistant host. The effects of the Tin-1 gene on accumu-
lation of coat protein were thus similar to the effects on accumulation of TMV RNA.
In systemically-infected leaves of Tm-1/Tm-1 plants which had a TMV RNA concentra-
tion I5 % of that in comparable leaves of + / + plants, the concentration of assembled TMV
was also found to be [ 5 % of the + / + level. Therefore it is likely that the low amounts of

TMV resistance in tomato
(a) (b) (c)
Fig. 2. Polyacrylamide slab gel electrophoresis of proteins from TMV-infected tomato leaves of
genotypes (a) +/+ ; (b) Tm-1/+ and (c) Tm-1/Tm-1. The TMV coat protein band is indicated by
arrows. Proteins were extracted from inoculated leaves z5 days after inoculation with TMV strain
o at Io #g/ml.
TMV RNA and coat protein present in resistant plants were assembled into intact virus
particles.
Nature of the TMV accumulated in resistant hosts
In considering our results showing a small accumulation of TMV RNA in leaves of Tm-1
hosts, it was necessary to ask whether this represented strain o virus which had achieved
some multiplication despite the presence of Tm-1 resistance, or whether it resulted from
chance occurrence of strain I (Pelham, 1972 ). This arises either by mutation or by contami-
nation of the initial strain o inoculum (Dawson, I965, t967; Pelham et al. I97o; Cirulli &
Ciccarese, I975; Dawson et al. I979) and is able to overcome the Tin-1 resistance.
No systemic mosaic symptoms were detected in any strain o-infected Tm-1/+ or Tm-l/
9I

92 R.S.S. FRASER AND S. A. R. LOUGHLIN
Tm-I hosts, even in plants kept until 96 days after inoculation. There was therefore no sign
of selection of strain t virus by this time, even though systemic spread of TMV was detectable
in such hosts by lo to 2o days after inoculation.
As a more stringent test, we also took symptomless, systemically-infected leaves from
Tm-1/+ and Tm-1/Tm-I plants at times after inoculation with strain o when gel electro-
phoresis showed that they did contain TMV RNA. These leaves were homogenized in
phosphate buffer and the diluted sap used to inoculate fresh + / +, Tm-1/+ and Tm-1/Tm-I
plants.
Sap from all Tin-l-containing first hosts caused symptoms on all +/+ second hosts,
confirming the presence of infectious TMV in the inocula. Inoculum from two out of eight
Tm-1/+ first hosts, and from one out of I I Tm-l/Tm-1 first hosts caused mosaic symptoms
on the Tm-1/+ and Tm-1/Tm-1 second hosts, thus showing strain I characteristics. The
symptoms produced by these inocula were visibly different from those produced by the
GCRI isolate of strain i and from each other. It is thus likely that these variants originated
by mutation or selection during multiplication of the strain o inoculum in either first or
second host.
In the remaining cases (the overwhelming majority of plants tested), inoculum from first
hosts containing the Tm-I gene caused no symptoms on second hosts containing this gene,
even by 4o days after inoculation. Symptoms produced by purified virus of strain o on + / +
plants and by strain I on all three types of host, were well developed by I5 days after
inoculation. These results therefore suggest that the virus multiplying in Tm-1 plants
continued to behave as strain o. We were also unable to detect any increased ability of TMV
to multiply in the second Tin-1 host. We conclude that the limited amount of TMV multi-
plication observed in Tm-1 plants was principally because Tm-1 resistance permits a
reduced amount of multiplication of strain o and was not significantly due to selection of
TMV strains able to overcome the inhibition of multiplication.
The early part of TMV multiplication in Tin-1 hosts
A notable feature of both resistant hosts was that the first detection of TMV RNA after
inoculation was much later than in susceptible hosts (Fig. 0- No TMV RNA could be
detected in Tm-1/+ plants 6 days after inoculation, or in the Tm-1/Tm-1 plants at 13 days.
Thus during this 'eclipse' period, TMV RNA concentration remained below the limit of
detection by this method, which is about o.I #g/g fresh weight.
To study the pattern of virus multiplication in the early part of infection of resistant hosts,
we followed changes in infectivity. This method is just sensitive enough to detect TMV
continuously after infection, provided that the tomato plants are inoculated with a high
concentration of TMV. As a different batch of Xanthi-nc test plants was used to follow
development of infectivity in each host, no inference can be drawn from differences in the
absolute numbers of lesions produced by inoculum from different tomato hosts.
In +/+ hosts, there was a very rapid increase in infectivity after inoculation (Fig. 3).
Both resistant hosts initially showed a decline in the level of recoverable infectivity, then an
increase. The effect was clearest in the Tm-1/Tm-1 plants, where infectivity declined for
I5 days before beginning to increase.
The infectivity results are consistent with the assays of TMV RNA in showing a delay
which is dependent on Tm-1 gene dosage before the onset of detectable virus accumulation.
Part of the infectivity recoverable in the early part of the experiment, before detectable
increase in infectivity, may have been from virus remaining on the surface of the leaf and
was unlikely ever to initiate infection. Such residual surface virus should not, however,
interfere with the above interpretation of the timing of eventual increase in infectivity in
resistant hosts.

100
10
TMV resistance in tomato 93
! !
/
1 -
0.ll I !
0 10 20 30
Time after inoculation (days)
Fig. 3- Changes in infectivity recovered from tomato leaves with time after inoculation with TMV
strain o at 5o #g/ml. The host genotypes were: O--O, +/+ ; z~--~, Tm-I/+ and I-i--[], Tm-1/
Tm-l. Values are means + standard errors.
Dawson (I965) found a delay between inoculation and measurable increase in infectivity
in tomatoes of resistant line CStMWIS. This line was originally reported to have three
recessive genes for resistance to TMV (Walter, J956), but has also been reported to contain
Tin-1 (P&aut, 1964; Pelham, I972).
Reproducibility of inhibition of TMV multiplication by Tin-1
Table 2 shows that in several experiments, the multiplication of TMV strain o was
inhibited to similar extents by gene Tm-l. Inhibition was consistently more effective in
homozygous than in heterozygous Tm-I hosts. The extent of inhibition was not affected by
the time of year at which the experiment was done, nor by the size of plant at the time of
inoculation.
To test whether the Tm-I inhibition of virus multiplication was equally effective against
other isolates of strain o, plants were inoculated with three other isolates. Table 3 shows
that none of these isolates multiplied quite as well in directly inoculated leaves of +/+
tomato as did the GCR1 isolate of strain o. However, only strain oY multiplied less well
than strain o in systemically-infected leaves.
Tm-1 resistance was effective against a!l strains, but not to the same extent. Multiplication
of the two strains producing yellow-green mosaic symptoms (oY andflavum) was especially

94 R. S. S. FRASER AND S. A. R. LOUGHLIN
Table 2. Effects of the Tm-x gene on multiplication of TMV strain o "in inoculated and
systemically-infected leaves of tomato hosts of different TMV-resistance genotypes
Month
July
Sept.
Nov.
April
TMV RNA content of resistant hosts*
Height of : ~ ,
plants at Sampling Tm-1/+ Tm-l/Tm-I
time of time , ~ , , A •
inoculation (days after Inoculated Systemic Inoculated Systemic
(cm) inoculation) leaves leaves leaves leaves
30 28 18 26 -- --
- - 49 I 2 - - - - - -
15 21 25 34 2"2 15
15 18 29 32 6'1 15
IO I9 3I -- 3'8 --
-- 28 -- 32 -- I'4
* Expressed as percentage of TMV RNA content of comparable leaves on the +/+ susceptible host.
Table 3- Concentrations of four type o isolates of TMV in inoculated and
systemically-infected leaves of susceptible and resistant tomato plants*
Host genotype
r+ / + Tin-l~+
Inoculated Systemic Inoculated Systemic
TMV isolate leaves leaves leaves leaves
o 222 i66 4I (i8 %) 43 (26%)
oY 57 119 2"5 (4%) o (0%)
flavum 59 169 1'5 (2"5 %) 5'I (3 %)
vulgare I 18 166 7"3 (6.2 %) 43 (26 %)
* Values are/*g TMV RNA/g fresh weight, measured 28 days after inoculation with each strain of virus at
lo p,g/ml. Figures in parentheses are TMV RNA concentrations in the Tm-l/+ hosts expressed as a per-
centage of the concentration in comparable leaves of the + / + host.
severely inhibited in Tm-I plants. Strain vulgate multiplication was inhibited to about the
same extent as strain o.
DISCUSSION
Previous studies, using infectivity bioassay of TMV concentration, have suggested that
the Tin-1 gene either inhibits TMV multiplication (Pelham, I972; Arroya & Selman, I977;
Motoyoshi & Oshima, 1977), has no effect on it (Clayberg, I96O; Pelham, 1972 ) or even
stimulates it (Pelham, I972). None of these reports gave a complete picture of virus multi-
plication in resistant plants. In our investigation, we chose not to use infectivity bioassay for
two reasons. Firstly, unless very large numbers of test plants are used, infectivity bioassay is
unable to distinguish between samples having small but possibly important differences in
virus concentration (Bawden, t95o). Secondly, we have found that the specific infectivity
of TMV multiplied in +/+ hosts is 5 to I5 times that of TMV multiplied in Tm-I/Tm-I
hosts (R. S. S. Fraser & S. A. R. Loughlin, unpublished data). Thus comparison of TMV
concentrations in different hosts by infectivity is likely to be unreliable.
Our results, based on direct measurements ofTMV RNA and coat protein concentrations,
show that the Tm-1 gene consistently inhibited accumulation of both components of the
virus. The differences in virus RNA content between +/+ and Tm-1/+ plants, and
between Tin-l/+ and Tm-1/Tm-1 plants, were of a size which would be difficult to detect
by infectivity bioassay. But as TMV quickly came to form the predominant nucleic acid and

TMV resistance in tomato 95
protein components of susceptible plants, its synthesis must have utilized a large proportion
of host anabolic capacity. Reduction of TMV accumulation to 3o % (Tm-1/+) or Io %
(Tm-1/Tm-1) of the amount in +/+ plants would significantly reduce this drain on host
synthetic capacity, and thus have less effect on growth.
From our results, certain suggestions can be made about the mode of action of the Tm-1
gene. Measurements of virus concentration in resistant plants suggested that the limited
amounts of TMV RNA and coat protein present were assembled into intact particles. This
shows only that assembly can occur in Tm-1 plants; it does not exclude the possibility that
Tm-1 resistance might act on the assembly process, with non-assembled TMV RNA and
coat protein being degraded.
The greater inhibition of TMV accumulation in Tm-I/Tm-I plants than in Tm-I/+
plants suggests that the extent of inhibition is dependent on the concentration of gene
product within the plant. In plants homozygous for Tm-1, both gene copies are presumably
active, leading to a higher concentration of gene product than in the heterozygote with only
one copy.
The pleiotropic effects of the gene on symptom suppression and inhibition of TMV
multiplication give rise to an apparent paradox: the gene is dominant for symptom sup-
pression, but inhibition of TMV multiplication is gene-dosage dependent. Various explana-
tions are possible. A single active product of the gene might have the ability to suppress
symptom formation at lower concentrations than those effective against virus multiplica-
tion; or the pathway between gene and active products could be branched, with separate
products inhibiting symptom formation and multiplication. The third possibility is that
the amount of virus accumulated in Tm-1/+ plants might be below some threshold con-
centration required for production of mosaic symptoms. However, if plants are grown at
a constant 33 °C, TMV multiplies as well in Tm-1 hosts as in +/+ hosts, but visible
symptoms are still suppressed by Tm-1 (our unpublished results). This suggests that the two
effects of the Tm-1 gene are due to partially separable mechanisms and that symptom
formation is not solely controlled by amount of virus accumulated.
From the time courses of development of infectivity and accumulation of TMV RNA
after inoculation of resistant hosts, it appears that one action of the antiviral gene is to delay
the onset of detectable multiplication. The decline in infectivity recoverable from Tm-1/Tm-1
plants from inoculation until I5 days later suggests that the resistance agent is not produced
in response to an initial phase of virus multiplication,~but is likely to be present in the plant
before inoculation. In this feature Tm- 1 differs from the N gene for TMV resistance in
tobacco (Holmes, I938), where an initial phase of virus multiplication occurs before the
resistance mechanism becomes effective (Taniguchi, I963; Fraser, I979).
The patterns of TMV RNA accumulation and infectivity changes after inoculation of
Tm-1/TmJ plants further suggest that the stage of virus multiplication which is inhibited
is an early one in the establishment of infection in the plant. If the susceptible stage were
a comparatively late one, such as cell-to-celI spread of virus, then some initial rise in
infectivity would be expected.
Having characterized some of the effects of Tm-1 on TMV multiplication and symptom
suppression, we are now in a position to ask more specific questions about which stage of
virus multiplication is the target of resistance, and about the nature of the resistance gene
product.
We thank Dr T. J. Hall, Glasshouse Crops Research Institute, for useful discussions and
for supplies of tomato seeds and virus strains. We thank Jane Connor and David Brown for
capable technical assistance.
4 VIR 4 8

96 R.S.S. FRASER AND S. A. R. LOUGHLIN
REFERENCES
ALEXANDER, L. J. (1971). Host-pathogen dynamics of tobacco mosaic virus on tomato. Phytopathology 62,
6t 1-617.
ARROYA, A. & SELMAN, I. W. (I977). The effects of rootstock and scion on tobacco mosaic virus infection in
susceptible, tolerant and immune cultivars of tomato. Annals of Applied Biology 85, 249-256.
BAWDEN, F. C. (1950). In Plant Viruses and Virus Diseases. Waltham, Mass: Chronica Botanica Company.
CIRULLI, M. & ClCCARESE, F. (1975). Interactions between TMV isolates, temperature, allelic conditions and
combination of the Tm resistance genes in tomato. Phytopathologica Mediterranea 24, Ioo-~o5.
CLAYSERG, C. D. (1960). Relative resistance of Tm-z and Tm-2 to tobacco mosaic virus. Tomato Genetics
Cooperative Report io, 13-14.
DAWSON, J. R. O. (1965). Contrasting effects of resistant and susceptible tomato plants on tomato mosaic
virus multiplication. Annals" of Applied Biology 56, 485-49I.
DAWSON, J. R. O. 0967). The adaptation of tomato mosaic virus to resistant tomato plants. Annals" of Applied
Biology 60, 2o9-214.
DAWSON, J. R. O., REES, M. W. & SHORT, M. N. (I979). Lack of correlation between the coat protein composition
of tobacco mosaic virus isolates and their ability to infect resistant tomato plants. Annals of Applied
Biology 91, 353-358.
FLETCHER, J. T. (1973). Tomato mosaic. In The U.K. Tomato Manual, pp. I96-2o8. Edited by H. G. Kingham.
London: Grower Books.
ERASER, g. S. S. (J97I). Extraction and assay of TMV RNA. Virology 45, 8o4-8o7.
FRASER, R. S. S. 0979)- Systemic consequences of the local lesion reaction to tobacco mosaic virus in a tobacco
variety lacking the N gene for hypersensitivity. Physiological Plant Pathology x4, 383 394.
ERASER, g. S. S. & WHENHAM, R. J. 0978). Chemotherapy of plant virus disease with methyl benzimidazol-
2yl-carbamate: effects on plant growth and multiplication of tobacco mosaic virus. Physiological Plant
Pathology x3, 51-64.
GOODING, G. V. & HEBERT, T. T. 0967). A simple technique for purification of tobacco mosaic virus in large
quantities. Phytopathology 57, 1285.
HOLMES, F. O. (1938). Inheritance of resistance to tobacco mosaic disease in tobacco. Phytopathology 28, 553-
561.
LAEMMLI, U. K. 097O). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature, London 2~'7, 68o-685.
LOENIN6, U. E. 0969). The determination of the molecular weight of ribonucleic acid by polyacrylamide gel
electrophoresis. Biochemical Journal IX3, 13 I-138.
MELCHE~',S, G. (t940). Die biologische Untersuchung des 'Tomatenvirus DahIem t94o". Biologisches Zentrat-
blatt 60, 527-537.
MOTOVOSH1, I. & OSHIMA, N. (I977). Expression of genetically controlled resistance to tobacco mosaic virus
infection in isolated tomato leaf mesophyll protoplasts. Journal of General Virology 34, 499-5 °6.
MUNORV, K. W. 0957). Die AbhAngigkeit des Auftretens neuer Virusst~imme vonder Kulturtemperatur der
Wirtspflanzen. Zeitschrift f~r induktive Abstammungs- lind Vererbungslehre 88, 4o7-426.
NEVILLE, D. M. (I97Q. Molecular weight determination of protein dodecyl sulphate complexes by gel electro-
phoresis in a discontinuous buffer system. Journal of Biological Chemistry 246, 6328-6334.
P~CAt~T, P. 0964). R6sistance ~ la mosaique du tabac. Report of Station d'Amdlioration des Plantes Mara-
ichOres (INRA) I96L 17-18.
P~CAOT, P. (I964). Tomate: s61ection pour la r6sistance aux maladies. Report of Station d'Amdlioration des
Plantes ]~araichdres (INRA) 1963, 51-55.
PELHAM, J. 0966). Resistance in tomato to tobacco mosaic virus. Euphytiea 15, 258-267.
PELHAM, J. (t972). Strain-genotype interaction of tobacco mosaic virus in tomato. Annals of Applied Biology
7x, 219-228.
PELHAM, J., FLETCHER, J. T. & HAWKINS, J. H. (I970). The establishment of a new strain of tobacco mosaic virus
resulting from the use of resistant varieties of tomato. Annals" of Applied Biology 65, 293-297.
TANIGVCHI, T. 0963). Similarity in the accumulation of tobacco mosaic virus in systemic and local necrotic
infection. Virology 29, 237-238.
VERNON, L. P. 0960). Spectrophotometric determination of chlorophylls and pheophytins in plant extracts.
Analyt&al Chemistry 32, 546-553.
WALTER, J. M. (1956). Hereditary resistance to tobacco mosaic virus in tomato. Phytopathology 46, 513-516.
WALTER, S. M. 0967). Hereditary resistance to disease in tomato. Annual Review of Phytopathology 5, 13 I-162.
(Received 20 September I979)