209
J. gen. Virol. (1981), 54, 209-212
Printed in Great Britain
The Role of the Epidermis in Virus-induced Local Lesions on Cowpea
and Tobacco Leaves
(Accepted 13 January 1981)
SUMMARY
Removal of the lower epidermis from cowpea and tobacco leaves inoculated with
tobacco mosaic virus (TMV) or tobacco necrosis virus (TNV) resulted in reduction
of local lesion numbers. The reduction was time-dependent and was greatly
influenced by darkening the plants 24 h before inoculation. In both darkened and
non-darkened plants the period of contact between epidermis and mesophyll required
for lesion formation to occur differed markedly. For cowpea inoculated with TMV
these periods were 1.5 and 8 h respectively, and in the combination cowpea-TNV
they were 1 and 6 h respectively. For Xanthi nc tobacco inoculated with TMV these
periods were 1 and 8.5 h respectively, and in the tobacco-TNV system they were 1
and 6 h respectively. More plasmodesmata were observed in superficial cells of
dark-treated plants than in those of non-dark-treated plants. The significance of this
observation in relation to the passage of infectious virus units from the epidermis into
the mesophyll is discussed.
When transmitted by sap inoculation, plant viruses must pass through the epidermis of a
leaf before they multiply in the mesophyll. Ectodesmata, as portals of entry for virus, are
possibly involved only in the initiation of virus infection (Brants, 1965). Thomas & Fulton
(1968) also concluded that ectodesmata function as infection sites. The hypersensitive
reaction of plants to viruses has been shown to be perturbed by treatments carried out within
24 h after inoculation (Shimomura, 1977; Kalpagam et al., 1977). The epidermis also appears
to have a r61e in the formation of necrotic lesions on leaves by tobacco mosaic virus (TMV).
For example, TMV replicates without lesion formation in the inoculated isolated lower
epidermis of hypersensitive tobacco (Dijkstra, 1962; Kasamo & Shimomura, 1977).
Some of the early events of infection in leaves inoculated with virus have been elucidated by
stripping the epidermal layer from leaves at different times after inoculation and incubating
the leaf as well as the isolated epidermal strips for a few days (Courts, 1980). The time
required for the virus to pass through the epidermis varies with temperature. The rate of virus
multiplication and transport through plasmodesmata could be limiting factors in these
processes.
Coutts (1980) noted that, in cowpea, tobacco necrosis virus (TNV) or TNV RNA passes
rapidly into the mesophyll from the epidermis. Removal of the TNV-inoculated lower
epidermis from cowpea leaves which had been shaded 24 h before inoculation significantly
reduced lesion number and extractable virus from the leaves only if stripping was carried out
not later than 1 h after inoculatioh. Courts proposed that the epidermis has an active r61e in
the infection process of cowpea leaves by TNV.
Light intensity often affects symptom expression and pre-inoculation darkening or shading
is used routinely to increase the susceptibility of test plants. The explanation of the effect of
shading is still obscure (Loebenstein, 1972).
In studying the r61e of the epidermis in the formation of virus-induced lesions the spread of
TMV and TNV from the epidermis of darkened and non-darkened leaves has been examined.
This paper reports on experiments designed to determine the length of time after inoculation
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that the epidermis needs to be attached to the mesophyll in order to produce lesions in the
mesophyll.
Cowpea plants (Vigna unguiculata L.) Walp. cv. Blackeye Early Ramshorn was grown
under greenhouse conditions at 22 + 3 °C. Fully expanded primary leaves from 10-to 13-dayold plants were used for virus inoculation. Virus suspensions obtained from cowpea
leaves infected with TNV strain D or from Nicotiana tabacum L. cv. Samsun leaves
systematically infected with TMV cowpea strain prepared as described by Wieringa-Brants
(1977) served as inocula and were adjusted to elicit about 50 local lesions on a cowpea leaf or
75 to 100 lesions on a tobacco leaf. The plants were not shaded before inoculation. The lower
epidermis of half leaves was inoculated and then removed using a fine forceps. At least five
half leaves per treatment were stripped, each treatment being carried out at time intervals of
15 rain, starting 15 min after inoculation. The epidermal strips of one treatment (about 500
mg) were kept on 2% water agar in closed Petri dishes at 22 _+ 3 °C under continuous
fluorescent light of 4000 lux for 5 days. Local lesions were never observed in the isolated
strips. The strips were collected, frozen with liquid nitrogen, homogenized in a mortar and
then treated ultrasonically with a Kerry Vibrason (100 W at 20 kHz for 3 min) to disrupt the
cell walls. Control virus suspensions retained their infectivity after such a treatment. The
homogenates were tested on five half leaves of N. tabacum L. cv. Xanthi nc (Xanthi) for virus
infectivity.
The cowpea leaves with or without their lower inoculated epidermis Were kept in closed
plastic boxes on moist filter paper under continuous fluorescent light of 4000 lux at 22 +
3 °C. Local lesions were counted 3 days after inoculation.
Similar experiments were done with leaves from Xanthi plants grown under greenhouse
conditions at 22 + 3 ° C. Two fully expanded leaves of 6-week-old plants were used for virus
inoculation. The virus inocula were the same as those used in the experiments with cowpea.
For each combination the minimum contact period between epidermis and mesophyll
required for lesion formation was determined in six experiments.
In the cowpea-TMV system local lesions were formed in the mesophyll only when the
epidermis was attached for a period of at least 8 h post-inoculation (Table 1). In the
combination cowpea-TNV a period of at least 6 h was needed. In Xanthi leaves TMV lesions
appeared only if the epidermis was not removed until 8.5 h after inoculation. In the
combination Xanthi-TNV a period of at least 6 h was required.
The virus activity tests of the homogenates of epidermal strips revealed that TMV and TNV
had multiplied in the isolated cowpea epidermis during the 5 days incubation on agar and that
for both viruses the activity in Xanthi epidermis was higher (Table 1).
No significant differences were found in virus activity in the isolated epidermal strips
obtained from inoculated darkened and non-darkened leaves (Table 1).
When peeled half cowpea leaves were inoculated with TNV or TNV RNA no lesions were
formed. No virus activity could be detected when the inoculated mesophyll was homogenized
and tested on Xarithi leaves. However, the TNV and TNV RNA suspensions did produce
local lesions in the unpeeled cowpea half leaves. The same effect was shown in the
cowpea-TMV system (Table 1).
When peeled Xanthi leaves were inoculated with TMV, TMV RNA, TNV or TNV RNA,
lesions were induced in the mesophyll (Table 1).
The time required for the virus to pass through the epidermis as determined in our
experiments differed significantly from the time interval measured by Coutts (1980). As
shading increases the susceptibility of test plants markedly, the experiments were repeated
with leaves from cowpea and Xanthi which had been in complete darkness for 24 h before
inoculation, at the same temperature as the non-darkened plants.
In the cowpea-TNV system the first lesions appeared when the epidermis had been in
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211
Table 1. Effect of removal of the lower epidermis on lesion formation in eowpea and Xanthi
nc tobacco leaves, non-darkened or kept in darkness 24 h before inoculation with TMV or
TNV
Plant
Light
conditions*
Virus
Minimum
contact
period (h)
Cowpea
Cowpea
Cowpea
Cowpea
Xanthi
Xanthi
Xanthi
Xanthi
+
+
+
-+
TMV
TMV
TNV
TNV
TMV
TMV
TNV
TNV
8
1.5
6
1
8.5
1
6
1
Virus activity
in homogenate
of about 500 mg
epidermal stripst
20.20
23-00
15.20
16.70
45.10
44.60
40.00
42.35
+_ 6.53
+_ 8.10
_+ 4.15
+ 7.70
4-_5-39
+ 4.70
+ 7.52
+ 6.60
Local lesions
in mesophyll$
A
Virus
RNA
0
0
0
0
35.10+7.81
11.20_+5.39
28.20 + 5.40
9.20 _+ 5.24
* --, Leaves not darkened; +, leaves kept in darkness for 24 h.
t Number of lesions/half leaf _+ standard error. Mean of 10 experiments.
:~ Number of lesions/half leaf _+ standard error in stripped leaves. Mean of 10 experiments.
contact with the mesophyU for at least 1 h. In the combination cowpea-TMV this period was
at least 1.5 h. For darkened Xanthi leaves inoculated with TMV or TNV at least 1 h was
required (Table 1).
Therefore, transferring hypersensitive reacting plants to complete darkness 24 h before
inoculation drastically reduced the time needed for infectious virus to enter the mesophyll
tissue from the inoculated epidermal cells. It is possible that shading stimulates the contact
between epidermis and mesophyll cells by increasing the number of plasmodesmata.
Darkened and non-darkened leaves of cowpea and Xanthi were therefore examined for the
presence of plasmodesmata using the procedure of Johansen (1940). Fresh leaf sections were
cut 2 5 / l m thick with a Lab-line/Hooker plant microtome and stained. Examination of 100
mm cell wall between epidermis and mesophyll showed an average of 3.2 plasmodesmata per
mm cell wall in darkened cowpea leaves, whereas in non-darkened leaves an average of 0.8
plasmodesmata per mm cell wall occurred. For darkened and non-darkened Xanthi leaves an
average of 7.6 and 1.6 plasmodesmata per mm cell wall respectively, was found.
Since viruses have no mobility of their own, cytoplasmic streaming probably plays an
important part in the process by which infective material moves from cell to cell or from
epidermis into mesophyll. Within 1 h after inoculation it is unlikely that the rate of virus
multiplication in the epidermis is high. The fact that in darkened leaves a strongly reduced
epidermal passage time coincides with a significantly increased number of plasmodesmata
points to a limiting r61e of plasmodesmata in the epidermal passage of infectious units in
non-darkened leaves.
Thanks are due to Mrs C. P. Schot, B. C. Spiele and Mr G. Meeuwissen for their participation in this
work.
Phytopathological Laboratory
'Willie Commelin Scholten'
Javalaan 20, 3742 CP Baarn
The Netherlands
D . H . WIERINGA-BRANTS
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