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Infection of leaf epidermis by wheat striate mosaic virus
JOURNAL OF ULTRASTRUCTURE RESEARCH
52, 227-234 (1975)
Infection of Leaf Epidermis by Wheat Striate Mosaic Virus 1 AMPARO VELA 2 AND PETER E . LEE
Department of Biology, Carleton University, Ottawa, Canada Received October 15, 1974, and in revised form December I2, 1974 Wheat striate mosaic virus (WSMV) systemically infects epidermal cells. The virus on rare occasions was found in guard cells. Although infection of subsidiary cells was common, guard cells bordering such subsidiary cells were generally free of virions. From a study of the relationship of plasmodesmata to mature cells of the wheat leaf epidermis, it appears that cytoplasmic connections are retained between subsidiary and adjacent epidermal cells after differentiation, but these connections are aborted between subsidiary and guard cells when the latter are fully differentiated. Thus, guard cell infection via plasmodesmata must occur at an early stage during cell differentiation. INTRODUCTION
Wheat striate mosaic virus (WSMV) causes a systemic infection of wheat plants (Ramsey Durum variety), and virus can be found located in parenchyma and phloem companion cells of leaves as well as in root cells (4, 5). This leafhopper transmitted virus is most probably inoculated to and acquired from phloem cells during the insect feeding process. Movement of the infectious principle from an infected cell to adjacent healthy cells must be via plasmodesmata, and it is unlikely that the mature virions can move through plasmodesmata due to their size, since the cytoplasmic connections between wheat cells range in diameter from 30 nm to 50 nm and the virion size from 200 x 80 nm to 250 × 80 nm. In the present study, the single layer of cells comprising the epidermis was studied in relation to WSMV infection. The morphology of the epidermis of some plants has been studied by Esau (2) and the structures of corn leaf and onion stomata have been investigated by the electron microscope (1, 6). In onion, plasmodesmata were not found between subsidiary and guard cells, nor were these cytoplasmic connections present 1This study supported in part by National Research Council Operating Grant (A2911) and Capital Grant (E-2134). Postdoctoral Fellow supported by Grant (A2911). 227 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
in fully differentiated corn leaves. Our observations show that the epidermis of wheat can be infected by WSMV, and that although plasmodesmata have not been found between fully differentiated guard and subsidiary cells, nonetheless WSMV on a few occasions was found in guard cells. MATERIALS AND METHODS Ventral strips of epidermis from wheat leaves showing virus symptoms 2-3 weeks after a 2-day inoculation period by viruliferous leafhoppers of 10day old wheat were removed with the aid of a fine pair of forceps and fixed in 6% glutaraldehyde in 0.1 M phosphate buffer pH 7.2 for 6 hr. After glutaraldehyde fixation, the strips were washed in buffer overnight, then postfixed in 2% osmic acid for 4 hr. The procedure was conducted at 4°C. Doubly-fixed strips were washed in H20, dehydrated in an ethyl alcohol series and fiat-embedded in Epon-Araldite. Flatembedding was accomplished by spraying an even layer of release coating (Acheson Colloids Ltd., Brantford, Ontario) in a small glass Petri dish and wiping the sprayed area throughly. To this treated glass surface, complete resin was added to form a thin layer approximately 3 mm deep and strips of epidermis already in complete resin were transferred to the Petri dish. Under a dissecting microscope strips were oriented with a needle so that they were paradermal with respect to the bottom of the dish. After resin polymerization at 80°C for 2 days, the flat strips were cut out with a coping saw and mounted paradermally with epoxy glue to resin blocks formed in 00 gelatin capsules. Infected leaf pieces were also processed for electron microscopy and thin sections were cut serially to study cytoplasmic connections between subsidiary and guard cells and adjacent epidermal ones with
FIc. 1. Paradermal section of infected wheat epidermis showing a pair of guard cells, Note folds in the walls of subsidiary and guard cells indicated by arrows. Guard cell (gc); subsidiary cell (sc); nucleus (n). 228
WSMV INFECTION OF LEAF EPIDERMIS
229
Fro. 2. Area of guard cells enclosed in circle of Fig. 1. Virions in nuclear region, either in between nuclear m e m b r a n e s or distributed in masses among heterochromatin. Virus (v).
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Fro. 3. Area of guard cells enclosed in rectangle of Fig. 1. Virus particles free in the cytoplasm, membrane bond in the cytoplasm or located between the nuclear membranes indicated by arrows. Cytoplasm with virus particles (cv); virions between unit nuclear membranes (vn).
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FIG. 4. Nuclear region of guard cells from infected epidermis. Micrograph shows well demarcated pore in the anticlinal wall (aw) with a unit membrane bound heterochromatin knob (ck) traversing the pore, and numerous virions unit membrane bound associated with both nuclei.
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subsidiary cells. Healthy epidermis and leaf pieces were prepared simultaneously for comparison. All sections were cut paradermally and were doubly stained in uranyl acetate and lead citrate. RESULTS W S M V was readily found in a large n u m b e r of e p i d e r m a l cells. Virus particles were also found in subsidiary cells b u t only infrequently in guard cells (Figs. 1-5). T h e localization of virus within infected epiderm a l cells was similar to t h a t in other leaf cells infected with W S M V : i.e., virions were found in m a s s e s in the c y t o p l a s m , at the nuclear m e m b r a n e , as c y t o p l a s m i c invaginations within the nucleus, or free in the n u c l e o p l a s m (Figs. 2, 3, and 5). Although guard cell infection was not a c o m m o n occurrence, as the m i c r o g r a p h s illustrate (Figs. 1-4), infection of these cells was authentic. W h e a t contains elon-
gated guard cells (Fig. 1) as do other grasses. T h e nuclei of these cells are similarly s h a p e d with c y t o p l a s m i c m a s s e s at either apex of each cell and peripheral c y t o p l a s m d e m a r c a t i n g the nucleus along either cell wall in a longitudinal plane. Figs. 2 and 3 show t h a t virions are found in the c y t o p l a s m , or between the unit m e m branes of the nucleus where the inner nuclear m e m b r a n e was in some locations extended into the n u c l e o p l a s m with large n u m b e r s of virus particles. On occasion a well-defined pore was present in the anticlinal wall between a pair of guard cells. Fig. 4 shows such a pore with heteroc h r o m a t i n from one nucleus extending over to the a d j a c e n t one, b u t in this connection the h e t e r o c h r o m a t i n knob is b o u n d e d by a membrane. A p a r t from large viral inclusions associated with the nuclei, some e p i d e r m a l cells
Fla. 5. Picnotic nucleus of adjacent epidermis cell. In addition to the massive clumping of the heterochromatin (c), note the alignment of several virions at the inner nuclear membrane (arrows) and at the circular unit membrane bound inclusions.
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Fro. 6. Epidermal serial section, showing plasmodesmata (p) between subsidiary (sc) and adjacent cells (ac). FIa. 7. Aborted plasmodesmata (ap) between subsidiary and guard cells (gc). Note plasmodesmata absent in guard cell wall (gw) abutting subsidiary cell wall (sw). Section from serial series. contained picnotic nuclei (Fig. 5) and some contained fibrillar inclusions, which were restricted to infected cells as reported in other infected wheat leaf tissue (see 7, Fig. 8). Not all infected cells, with virus associated with the nucleus, contained, picnotic nuclei, but the dense clumping of heteroc h r o m a t i n was never observed in comparative healthy samples. No p l a s m o d e s m a t a were observed be-
tween guard and subsidiary cells in samples from epidermis strips. Although it has been reported t h a t fully differentiated corn leaves do not have p l a s m o d e s m a t a between these two cell types (6), nevertheless we felt t h a t if cytoplasmic connections were present in guard/subsidiary cells, the destruction of such connections might occur when the epidermis was stripped off. To test this possibility, serial sections were
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cut paradermally from small leaf pieces and examined for plasmodesmata. Plasmodesmata were readily found between subsidiary and adjacent cells (Fig. 6), but these connections were absent between guard and subsidiary cells. Fig. 7 shows plasmodesmata that are aborted and noncontinuous between a guard and subsidiary cells. In cutting paradermal sections of the epidermis, it was difficult to obtain sections without folds in the cell walls. These should not be misconstrued as plasmodesmata; Figs. I and 3 show examples of cell wall folds. DISCUSSION Although the incidence of virions in guard cells was low, we present evidence that such cells can be infected by WSMV. The main question is: How do guard cells become infected if there are no continuous cytoplasmic connections between guard and subsidiary cells, when the cells are fully differentiated? Our observations support the idea that during early differentiation and growth of the wheat leaf, continuous cytoplasmic connections do exist between guard and subsidiary cells. At maturity, although plasmodesmata are retained between subsidiary and adjacent cells (Fig 6), such cytoplasmic connections are aborted between guard and subsidiary cells (Fig. 7). This infers that if such connec-
tions are the only means to initiate infection from one cell to another, infection must occur prior to cell maturation when cytoplasmic connections are intact. Infection via plasmodesmata is based on the assumption that the intact virion is too large to move through the plasmodesmata and infection is accomplished by the fibrillar inner component of the virion (7) whose diameter permits movement through cytoplasmic connections between cells. In a study of stomata development in Avena sativa, plasmodesmata were found to be present between guard and subsidiary cells during early development (3). This is taken as further evidence to substantiate the view that guard cells of wheat can only become infected during early development. However, this statement is speculative and should not be considered to the exclusion of other possibilities. REFERENCES 1. ALLAWAY,W. G., AND SETTERFIELD,G., Canad. J. Bot. 50, 1405 (1972). 2. ESAU, K., Plant Anatomy, 2nd ed. John Wiley and Sons, New York, 1965. 3. KAUFMAN,P. B., PETERING, L. B., YOCUM, C. S., ANDBArn, D., Amer. J. Bot. 57, 33 (1970). 4. LEE, P. E., Virology 33, 84 (1967). 5. SINHA,R. C., Virology 44, 342 (1971). 6. SRIVASTAVA,L. M., ANDSINGH, A. P., J. Ultrastruct. Res. 39, 345 (1972). 7. VELA, A., AND LEE, P. E., J. Ultrastruct. Res. 47, 169 (1974).
52, 227-234 (1975)
Infection of Leaf Epidermis by Wheat Striate Mosaic Virus 1 AMPARO VELA 2 AND PETER E . LEE
Department of Biology, Carleton University, Ottawa, Canada Received October 15, 1974, and in revised form December I2, 1974 Wheat striate mosaic virus (WSMV) systemically infects epidermal cells. The virus on rare occasions was found in guard cells. Although infection of subsidiary cells was common, guard cells bordering such subsidiary cells were generally free of virions. From a study of the relationship of plasmodesmata to mature cells of the wheat leaf epidermis, it appears that cytoplasmic connections are retained between subsidiary and adjacent epidermal cells after differentiation, but these connections are aborted between subsidiary and guard cells when the latter are fully differentiated. Thus, guard cell infection via plasmodesmata must occur at an early stage during cell differentiation. INTRODUCTION
Wheat striate mosaic virus (WSMV) causes a systemic infection of wheat plants (Ramsey Durum variety), and virus can be found located in parenchyma and phloem companion cells of leaves as well as in root cells (4, 5). This leafhopper transmitted virus is most probably inoculated to and acquired from phloem cells during the insect feeding process. Movement of the infectious principle from an infected cell to adjacent healthy cells must be via plasmodesmata, and it is unlikely that the mature virions can move through plasmodesmata due to their size, since the cytoplasmic connections between wheat cells range in diameter from 30 nm to 50 nm and the virion size from 200 x 80 nm to 250 × 80 nm. In the present study, the single layer of cells comprising the epidermis was studied in relation to WSMV infection. The morphology of the epidermis of some plants has been studied by Esau (2) and the structures of corn leaf and onion stomata have been investigated by the electron microscope (1, 6). In onion, plasmodesmata were not found between subsidiary and guard cells, nor were these cytoplasmic connections present 1This study supported in part by National Research Council Operating Grant (A2911) and Capital Grant (E-2134). Postdoctoral Fellow supported by Grant (A2911). 227 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
in fully differentiated corn leaves. Our observations show that the epidermis of wheat can be infected by WSMV, and that although plasmodesmata have not been found between fully differentiated guard and subsidiary cells, nonetheless WSMV on a few occasions was found in guard cells. MATERIALS AND METHODS Ventral strips of epidermis from wheat leaves showing virus symptoms 2-3 weeks after a 2-day inoculation period by viruliferous leafhoppers of 10day old wheat were removed with the aid of a fine pair of forceps and fixed in 6% glutaraldehyde in 0.1 M phosphate buffer pH 7.2 for 6 hr. After glutaraldehyde fixation, the strips were washed in buffer overnight, then postfixed in 2% osmic acid for 4 hr. The procedure was conducted at 4°C. Doubly-fixed strips were washed in H20, dehydrated in an ethyl alcohol series and fiat-embedded in Epon-Araldite. Flatembedding was accomplished by spraying an even layer of release coating (Acheson Colloids Ltd., Brantford, Ontario) in a small glass Petri dish and wiping the sprayed area throughly. To this treated glass surface, complete resin was added to form a thin layer approximately 3 mm deep and strips of epidermis already in complete resin were transferred to the Petri dish. Under a dissecting microscope strips were oriented with a needle so that they were paradermal with respect to the bottom of the dish. After resin polymerization at 80°C for 2 days, the flat strips were cut out with a coping saw and mounted paradermally with epoxy glue to resin blocks formed in 00 gelatin capsules. Infected leaf pieces were also processed for electron microscopy and thin sections were cut serially to study cytoplasmic connections between subsidiary and guard cells and adjacent epidermal ones with
FIc. 1. Paradermal section of infected wheat epidermis showing a pair of guard cells, Note folds in the walls of subsidiary and guard cells indicated by arrows. Guard cell (gc); subsidiary cell (sc); nucleus (n). 228
WSMV INFECTION OF LEAF EPIDERMIS
229
Fro. 2. Area of guard cells enclosed in circle of Fig. 1. Virions in nuclear region, either in between nuclear m e m b r a n e s or distributed in masses among heterochromatin. Virus (v).
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VELA AND LEE
Fro. 3. Area of guard cells enclosed in rectangle of Fig. 1. Virus particles free in the cytoplasm, membrane bond in the cytoplasm or located between the nuclear membranes indicated by arrows. Cytoplasm with virus particles (cv); virions between unit nuclear membranes (vn).
WSMV INFECTION OF LEAF EPIDERMIS
231
FIG. 4. Nuclear region of guard cells from infected epidermis. Micrograph shows well demarcated pore in the anticlinal wall (aw) with a unit membrane bound heterochromatin knob (ck) traversing the pore, and numerous virions unit membrane bound associated with both nuclei.
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subsidiary cells. Healthy epidermis and leaf pieces were prepared simultaneously for comparison. All sections were cut paradermally and were doubly stained in uranyl acetate and lead citrate. RESULTS W S M V was readily found in a large n u m b e r of e p i d e r m a l cells. Virus particles were also found in subsidiary cells b u t only infrequently in guard cells (Figs. 1-5). T h e localization of virus within infected epiderm a l cells was similar to t h a t in other leaf cells infected with W S M V : i.e., virions were found in m a s s e s in the c y t o p l a s m , at the nuclear m e m b r a n e , as c y t o p l a s m i c invaginations within the nucleus, or free in the n u c l e o p l a s m (Figs. 2, 3, and 5). Although guard cell infection was not a c o m m o n occurrence, as the m i c r o g r a p h s illustrate (Figs. 1-4), infection of these cells was authentic. W h e a t contains elon-
gated guard cells (Fig. 1) as do other grasses. T h e nuclei of these cells are similarly s h a p e d with c y t o p l a s m i c m a s s e s at either apex of each cell and peripheral c y t o p l a s m d e m a r c a t i n g the nucleus along either cell wall in a longitudinal plane. Figs. 2 and 3 show t h a t virions are found in the c y t o p l a s m , or between the unit m e m branes of the nucleus where the inner nuclear m e m b r a n e was in some locations extended into the n u c l e o p l a s m with large n u m b e r s of virus particles. On occasion a well-defined pore was present in the anticlinal wall between a pair of guard cells. Fig. 4 shows such a pore with heteroc h r o m a t i n from one nucleus extending over to the a d j a c e n t one, b u t in this connection the h e t e r o c h r o m a t i n knob is b o u n d e d by a membrane. A p a r t from large viral inclusions associated with the nuclei, some e p i d e r m a l cells
Fla. 5. Picnotic nucleus of adjacent epidermis cell. In addition to the massive clumping of the heterochromatin (c), note the alignment of several virions at the inner nuclear membrane (arrows) and at the circular unit membrane bound inclusions.
WSMV INFECTION OF LEAF EPIDERMIS
233
Fro. 6. Epidermal serial section, showing plasmodesmata (p) between subsidiary (sc) and adjacent cells (ac). FIa. 7. Aborted plasmodesmata (ap) between subsidiary and guard cells (gc). Note plasmodesmata absent in guard cell wall (gw) abutting subsidiary cell wall (sw). Section from serial series. contained picnotic nuclei (Fig. 5) and some contained fibrillar inclusions, which were restricted to infected cells as reported in other infected wheat leaf tissue (see 7, Fig. 8). Not all infected cells, with virus associated with the nucleus, contained, picnotic nuclei, but the dense clumping of heteroc h r o m a t i n was never observed in comparative healthy samples. No p l a s m o d e s m a t a were observed be-
tween guard and subsidiary cells in samples from epidermis strips. Although it has been reported t h a t fully differentiated corn leaves do not have p l a s m o d e s m a t a between these two cell types (6), nevertheless we felt t h a t if cytoplasmic connections were present in guard/subsidiary cells, the destruction of such connections might occur when the epidermis was stripped off. To test this possibility, serial sections were
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cut paradermally from small leaf pieces and examined for plasmodesmata. Plasmodesmata were readily found between subsidiary and adjacent cells (Fig. 6), but these connections were absent between guard and subsidiary cells. Fig. 7 shows plasmodesmata that are aborted and noncontinuous between a guard and subsidiary cells. In cutting paradermal sections of the epidermis, it was difficult to obtain sections without folds in the cell walls. These should not be misconstrued as plasmodesmata; Figs. I and 3 show examples of cell wall folds. DISCUSSION Although the incidence of virions in guard cells was low, we present evidence that such cells can be infected by WSMV. The main question is: How do guard cells become infected if there are no continuous cytoplasmic connections between guard and subsidiary cells, when the cells are fully differentiated? Our observations support the idea that during early differentiation and growth of the wheat leaf, continuous cytoplasmic connections do exist between guard and subsidiary cells. At maturity, although plasmodesmata are retained between subsidiary and adjacent cells (Fig 6), such cytoplasmic connections are aborted between guard and subsidiary cells (Fig. 7). This infers that if such connec-
tions are the only means to initiate infection from one cell to another, infection must occur prior to cell maturation when cytoplasmic connections are intact. Infection via plasmodesmata is based on the assumption that the intact virion is too large to move through the plasmodesmata and infection is accomplished by the fibrillar inner component of the virion (7) whose diameter permits movement through cytoplasmic connections between cells. In a study of stomata development in Avena sativa, plasmodesmata were found to be present between guard and subsidiary cells during early development (3). This is taken as further evidence to substantiate the view that guard cells of wheat can only become infected during early development. However, this statement is speculative and should not be considered to the exclusion of other possibilities. REFERENCES 1. ALLAWAY,W. G., AND SETTERFIELD,G., Canad. J. Bot. 50, 1405 (1972). 2. ESAU, K., Plant Anatomy, 2nd ed. John Wiley and Sons, New York, 1965. 3. KAUFMAN,P. B., PETERING, L. B., YOCUM, C. S., ANDBArn, D., Amer. J. Bot. 57, 33 (1970). 4. LEE, P. E., Virology 33, 84 (1967). 5. SINHA,R. C., Virology 44, 342 (1971). 6. SRIVASTAVA,L. M., ANDSINGH, A. P., J. Ultrastruct. Res. 39, 345 (1972). 7. VELA, A., AND LEE, P. E., J. Ultrastruct. Res. 47, 169 (1974).