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Plasmalemma deposits in tissues infected with lettuce infectious yellows virus
JOURNAL
OF ULTRASTRUCTURE
AND
MOLECULAR
STRUCTURE
RESEARCH
100,
245-254 (1988)
Plasmalemma Deposits in Tissues Infected with Lettuce Infectious Yellows Virus ROBIN U.S.
Department
L. PINTO, of Agriculture,
LYNN
L. HOEFERT,
AND GAIL
Agricultural Research Station, Salinas, California 93905 Received
November
L. FAIL
1636 East
Alisal
Street.
3, 1988
Lettuce infectious yellows virus (LIYV) is a phloem-associated virus that is whiteflytransmitted. The physical characteristics and cytopathology of this virus are similar to those of other members of the Closterovirus group. One unique ultrastructural effect of the infection is the formation of conical deposits on the plasmalemmae of phloem parenchyma cells. The electrondense deposits are osmiophilic stacks of membrane lamellae spaced at 7 nm. Flexuous virions extend between these deposits from the cytoplasm through plasmodesmata and into adjacent sieve elements. We hypothesize that these plasmalemma deposits may facilitate movement of LIYV into other phloem parenchyma cells for further replication or into sieve elements for rapid transport throughout the host. o 1988 Academic press. IK.
Lettuce infectious yellows virus (LIYV) is one of five whitefly-transmitted viruses with possible relationships to the Closterovirus group (Duffus, 1987; Hoefert et al., 1988). Most Closteroviruses are transmitted by aphids and are generally confined to the phloem of the host. Purified preparations of LIYV show virions to be approximately 2000 nm long and 13-14 nm wide, with a flexuous profile (Duffus et al., 1986). LIYV infects economically important crops such as cantaloupe, carrot, cucumber, casaba, honeydew, lettuce, safflower, squash, sugarbeet, sunflower, and watermelon (Duffus and Flock, 1982; Nameth et al., 1986), as well as common weed species. We have examined the development of this disease and its ultrastructural effects in a variety of hosts (Houk and Hoefert, 1983; Hoefert et al., 1988; Hoefert, 1988). Ultrastructural symptoms, such as fibrilcontaining vesicles, long, flexuous virus particles, and inclusion bodies, are similar to those of other Closteroviruses (Lister and Bar-Joseph, 1981). A unique ultrastructural effect of this virus disease is the deposition of electron-dense material on the plasmalemma of infected leaf and root tissue. These deposits lie adjacent to plas-
modesmata of phloem parenchyma cells. The deposits have been observed in all LIYV-infected plants, including members of the families Malvaceae, Asteraceae, Chenopodiaceae, Brassicaceae, and Solanaceae. In this study we present the results of examinations of tissues of Nicotinna clevelandii L., Chenopodiun murale 1,. , and Lactuca sativa L. We hypothesize that plasmalemma deposits may influence the transport of virus particles throughout the phloem parenchyma. MATERIALS
AND METHODS
Collections of leaf and root tissue for electron microscopy were made from greenhouse-grown plants. The whitefly vector, Bemisia tabaci Genn., was used to infect N. clevelandii L., C. murale L., and L. sativa L. with LIYV. Young leaves, middle-aged leaves, and roots were taken from the plants at various stages after inoculation from as early as 5 days to as late as 2 months depending on the rate of spread of the virus infection within each species. Uninoculated, healthy controls of a similar age were collected at the same time. Samples were fixed for 2-3 hr in a 3% paraformaldehyde/3% glutaraldehyde solution in 0.05 M phosphate buffer at pH 6.8 in an ice bath and under vacuum. The tissue was postfixed overnight at 4°C in 2% osmium tetroxide in the same buffer. The tissue was then dehydrated in an acetone and finally a propylene oxide series. Samples were embedded in an Epon 812 equivalent resin. Sections were cut with di245 0889-1605188 $3.00 Copyright 0 1988 by Academic Press, lnc All rights of reproduction in any form rescrwed
246
PINTO,
HOEFERT,
AND
FAIL
DEPOSITS
IN TISSUES
INFECTED
WITH
LIYV
247
248
PINTO,
HOEFERT,
AND
FAIL
DEPOSITS IN TISSUES INFECTED amond knives and stained with uranyl acetate and lead tartrate (Millonig, 1961). RESULTS
Electron-dense deposits are found on the plasmalemmae of phloem parenchyma cells in plants infected with LIYV, but not in healthy tissue. Figure 1 illustrates a number of the deposits in two phloem parenchyma cells of C. murule. Plasmalemma deposits (PDs) are seen only in cells that show other cellular effects of the virus disease. These effects include lipid bodies, fibrilcontaining vesicles, and viroplasm. Membranes of chloroplasts, mitochondria, and nuclei often appear distorted. Aggregates of virus particles also are visible in the phloem parenchyma cells. An intervening sieve element has been filled by virus particles. Figure 2 shows another view of similar cells at higher magnification. In longitudinal section (Figs. 3 and 4) the PDs appear conical in shape. They are tightly appressed to the plasmalemma and
WITH LIYV
249
are located near the plasmodesmata. The PDs are composed of 6- to 7-nm layers of membranous material lying parallel to the plasmalemma (Fig. 3). When sectioned in a plane that is not perpendicular to the plasmalemma, the layers are not always visible; TEM examination with a tilting stage confirms that the layers are present. The entire plasmalemma deposit is not bounded by any discernible membrane and, therefore, is not separated from the rest of the cytoplasm. The PDs are found adhering to pit fields (the parenchyma side of plasmodesmata which connect to sieve elements and other parenchyma cells), but never to lateral sieve areas (plasmodesmatal connections on the sieve element side). As many as ten PDs in a closely clustered group may be visible in these pit fields (Figs. 1, 3, and 4). Figure 5 is an oblique cut through areas of deposit and parenchyma cell wall. The deposits appear as a broad expanse of electron-dense material overlying the cell wall. Virus particles in infected tissues often
l-5. Phloem tissue in leaves of C. murale infected with LIYV 8 weeks past inoculation. FIG. 1. Phloem parenchyma cells (PP) contain typical virus symptoms of LIYV: vesicles (Ve), regions of viroplasm (VP), virus particles (V), lipid droplets (L), and distorted chloroplast (C) and mitochondrial (M) membranes. Plasmalemma deposits (PD) are found in the phloem parenchyma cells, but not in the sieve element (SE). Note that deposits are clustered around the plasmodesmata (P) in the pit field and not in the lateral sieve area. The sieve element is completely filled with virus particles. X 26 000. FIG. 2. Sieve element (SE) tilled with virus particles. The adjoining phloem parenchyma cell (PP) contains plasmalemma deposits (PD). Virus particles (arrow) near the deposits are oriented perpendicular to the cell wall and between the deposits. Note virus particles (arrow) in cross section in the median nodule of a plasmodesma (P). x 30 ooo. FIG. 3. Plasmalemma deposits (PD) composed of membrane-like material adhere to the plasmalemma (PI) of a phloem parenchyma cell. The deposits border the plasmodesmatal pores (P). Virus particles (arrows) are visible extending from the cell cytoplasm into the plasmodesmata. Note that no desmotubule is apparent. x 110000. FIG. 4. Virions (arrows) extend from the plasmodesma of a phloem parenchyma cell into the lumen of the adjacent sieve element (SE). Note that no desmotubules are apparent. Plasmalemma deposits (PD) abut the pores of the plasmodesmata (P). Membranous layers approximately 7 nm thick are clearly visible. Virions are evident in the cytoplasm. x 112 000. FIG. 5. An oblique section through an expanse of deposit material (PD) and cell wall (CW). Virus particles are reoriented away from virus aggregates (V) in the cytoplasm and toward plasmalemma deposits and plasmodesmata. Virions (arrows) in cross section are visible as they penetrate the plasmalemma deposit and the plasmodesmata. Note that the deposit lies adjacent to the common walls of a sieve element (SE) and another phloem parenchyma cell (PP). Virus particles fill the sieve element and also are present in the parenchyma cell. x 35ooo. FIG. 6. Virus particles in the phloem parenchyma of L. sativa 3.5 days after inoculation. Virus particles (arrows) near plasmalemma deposits appear to be reoriented away from other virus aggregates (V) and toward a plasmodesma (P). A chloroplast (C) and mitochondrion (M) are also visible in the cell. X 63 000. FIGS.
250
PINTO, HOEFERT,
are seen in close association with the PDs. In Figs. 1, 2, and 3, virus particles in the cytoplasm are oriented between the PDs and in toward plasmodesmata. In Fig. 4, virions extend into the adjoining sieve element through the plasmodesma. In cross section (Fig. 5), virions appear in small clusters surrounded by regions of plasmalemma deposit. Virions associated with PDs often appear to be reoriented away from large aggregates of virus particles in the cytoplasm and toward the plasmodes-
AND FAIL
mata (Figs. 1, 5, and 6). Desmotubules are not evident in either cross or longitudinal sections of plasmodesmata that contain vir-us particles. A schematic diagram (Fig. 7) illustrates our interpretation of the shape and structure of the plasmalemma deposits and their spatial relationship to other host and virus-induced structures in typical phloem parenchyma cells infected with LIYV. Both the LIYV particles and PDs are limited to phloem parenchyma cells of all pre-
W
SE
;
FIG. 7. A schematic diagram of a sieve element (SE) and two phloem parenchyma (PP) cells infected with LIYV. Virus symptoms typical of a Closterovirus disease, such as vesicles (Ve), virus particles (V), viroplasm (VP), and lipid droplets (L) are visible in the phloem parenchyma cells. Mitochondria (M) and chloroplasts (C) contain distorted membranes. Plasmalemma deposits (PD) abut plasmodesmata (P) in pit fields that connect the phloem parenchyma cell to the sieve element and adjacent parenchyma cell. Virus particles can be seen in the cytoplasm of the phloem parenchyma cell, the lumen of the sieve element, and in the pit fields of the two parenchyma cells. Not to scale.
DEPOSITS
IN TISSUES
viously examined tissue, except in N. clevelandii where border parenchyma cells also contain PDs. In these cells, the deposits are situated only on the phloem parenchyma cell side of border parenchyma (Fig. 8) and not on the mesophyll side. In root tissue, plasmalemma deposits are generally smaller in size and less frequent than in leaf tissue. Figure 9 shows virus particles in root tissue of L. sativa directed toward a plasmodesma and between PDs. The deposits are evident in infected tissues as soon as other symptoms, such as virus vesicles, are detectable. Small PDs, in leaves, of Lactuca are visible as early as 5 days after inoculation (Fig. 10). DISCUSSION
Many ultrastructural studies have implicated plasmodesmata in the transport of virus particles between cells. Virus particles, isometric and rod-shaped, from many different groups have been seen in plasmodesmata. Examples of isometric viruses include dahlia mosaic virus (DMV; Kitajima and Lauritis, 1969), beet western yellows virus (BWYV; Esau and Hoefert, 1972a, b), cauliflower mosaic virus (CaMV; Bassi et al., 1974), and cowpea mosaic virus (CMV; Kim and Fulton, 1975). Some of the flexuous and stiff rods detected in plasmodesmata include beet yellows virus (BYV; Esau and Hoefert, 1971, potato virus Y (PVY; Weintraub et al., 1974), and tobacco etch and tobacco mosaic virus (TEV and TMV; Weintraub et al., 1976). In our survey of LIYV in a variety of hosts, we have not observed desmotubules in any plasmodesma that also contains virus particles. Modified or displaced desmotubules have been identified in plasmodesmata with PVY particles (Weintraub et al., 1974; McMullen and Gardner, 1980) or TEV (Weintraub et al., 1976). Desmotubules were not evident in tissue infected with BYV (Esau and Hoefert, 1971) or BWYV (Esau and Hoefert, 1972b). These authors concluded that the absence of the desmotubule was necessary to permit the
INFECTED
WITH
LIYV
251
passage of the virus through the plasmodesmata. Kitajima and Lauritis (1969) stated that the “plasmodesma is not a static but a dynamic structure”; it seems that this flexibility of structure, including the desmotubule, may be important to facilitate virus transport. Many virus infections induce the formation of various kinds of wall anomalies or deposits. Often these deposits have been associated with plasmodesmata. Bassi et al. (1974) identified the tubule component of wall protrusions caused by CaMV as a plasmodesma-like material. Similar wall protrusions were typical of Como- and Nepovirus infections (Kim and Fulton, 1975). Virus particles were seen penetrating cell walls or wall overgrowths (Jones et al., 1973; Van der Scheer and Groenewegen, 1971). In early infections of potyviruses, cylindrical or bundle inclusions appeared to be attached to and, in some cases, to have penetrated plasmodesmata (Weintraub ef al., 1976; McMullen and Gardner, 1980). Viruses were associated with cylindrical inclusions at the plasmodesmata on either side of cell walls. Langenberg (1986) suggested that these inclusions might be responsible for either alignment or active transport of flexuous PVY particles into plasmodesmata. Raine et al. (1979) described plasmalemma deposits in Prunus leaves infected with little cherry disease (LCD). LCD is caused by a phloem-limited virus of probable Ciosterovirus affinity. Cylindrical deposits, or tubules, in phloem parenchyma cells contained hollow centers positioned over plasmodesmatal pores. Like those in tissues infected with LIYV, the LCD deposits were composed of lamellae 7 nm thick. Most virus particles in infected cherry tissue were found in aggregates oriented along the long axis of the cell. Viruses near the tubules appeared to be directed away from the majority of particles and reoriented into the plasmodesmata in a manner identical to that seen in LIYV-infected tissue (Figs. 1, 5, and 7). LCD virus particles extended
252
PINTO,
HOEFERT,
AND
FAIL
DEPOSITS IN TISSUES INFECTED
through these tubules and plasmodesmata from the companion cell cytoplasm into the sieve element lumen (Raine et al., 1979, Fig. 2). Molecular research using temperaturesensitive isolates of TMV has identified a transport factor associated with plasmodesmata which may influence the spread of plant viruses. Under elevated temperatures, these TMV isolates replicate in epidermal cells of infected tissue but do not move beyond the site of initial infection to other cells. Comparisons of protein structures synthesized by healthy and temperature-restricted isolates of TMV showed a mutation in one protein, p30 (Leonard and Zaitlin, 1982). Leonard and Zaitlin (1982) suggested that the alteration of p30 was correlated with the lack of cell-to-cell movement of the virus from the initial infection site. Furthermore, Shalla et al. (1982) showed a statistically significant reduction in the number of plasmodesmata in mesophyll cells of plants infected with the temperature-sensitive isolate. Thus, the presence of this protein appeared to be necessary for replicated virions to invade other cells beyond the initial infection site. The specific function of the LIYV plasmalemma deposits is, as yet, undetermined. One possibility is that the deposits are a defensive reaction by the host plant to inhibit the transport of the virus and thereby limit the spread of the infection. This function seems improbable because no deposit has been seen occluding any plasmodesmatal pore, and because intact virus particles have been seen within plasmodesmata surrounded by those deposits. In ad-
WITH LIYV
33
dition, Pinto et al. (1988) have shown that infected tissue of L. sativa contained PDs and virions at the earliest stages of virus development (5 days after inoculation), whereas cell wall overgrowths, which have been considered a possible plant defense reaction to virus invasion (Esau, 1967), did not form until 14 days past inoculation (Hoefert et al., 1988). LIYV causes a common alteration of the desmotubules within the plasmodesmata of infected phloem parenchyma cells. This alteration of the desmotubule appears to facilitate the movement of vu-ions through the plasmodesmata. We postulate that the presence of the plasmalemma deposits also may assist the virus in cell-to-cell transport through the host plant. Reorientation of virions toward and into the plasmodesmata suggests that the conical PDs may act to either direct or attract these virions to the pores. The deposits are found on pit fields which specifically connect phloem parenchyma cells to sieve elements or other phloem parenchyma cells. This association with these plasmodesmata suggests that the PDs influence virus movement into the two different cell types. These cells might be opportunistic sites for the virus for replication in the case of the phloem parenchyma cells or for distribution into other areas of the host plant in the case of the sieve elcments (Hoefert et al., 1988). The mechanism of virus transport is critical in the cytopathology of a disease. Many viruses, both spherical and rod-shaped, are transported throughout a host during an infection. A virus-induced transport factor has now been identified (Leonard and Zait-
FIG. 8. A border parenchyma cell (BP) in N. clevelandii contains virus particles (arrows), vesicles (Ve), and plasmalemma deposits (PD). Plasmodesmata (P) in cross section are visible in a common wall between phloem and border parenchyma cells. x 25 000. Fro. 9. A phloem parenchyma cell in L. sntiva root tissue contains plasmalemma deposits (PD) and virus particles (arrows). Virus particles are oriented toward the plasmodesma (P) that connects this phloem cell to the adjacent sieve element (SE). x 57 000. FIG. 10. Early development of virus infection (5 days after inoculation) in phloem parenchyma of L. sutivn. Virus vesicles (Ve) are beginning to aggregate; mitochondria (M) look normal. Small plasmalemma deposits (PD) abut plasmodesmatal pores (P). A few virions (arrows) are visible in the cytoplasm and are oriented toward the olasmodesmata. x 41 500.
254
PINTO,
HOEFERT,
lin, 1982), but its mode of action is still poorly understood. Evidence that plasmodesmata participate in cell-to-cell virus transport is accumulating (Kitajima and Lauritis, 1969; Esau and Hoefert, 1972a, b; Weintraub et al., 1976; Shalla et al., 1982; and Langenberg, 1986); it seems logical that a virus-induced transport factor might operate on those plasmodesmata. The formation of the LIYV-induced plasmalemma deposits may reflect the activity of such a transport factor. REFERENCES BASSI, M., FAVALI, M. A., AND CONTI, G. G. (1974) Virology 60, 353-358. DUFFUS, J. E., (1987) in HARRIS, K. F. (Ed.), Current Topics in Vector Research, Vol. 4, pp. 73-91, Springer-Verlag, New York/Berlin. DUFFUS, J. E., AND FLOCK, R. A. (1982) Calif. Agric. 36,4-6. DUFFUS, J. E. LARSEN, R. C., AND LIU, H. Y. (1986) Phytopathology 76, 97-100. ESAU, K. E. (1967) Annu. Rev. Phytopathol. 5,45-70. ESAU, K. E., AND HOEFERT, L. L. (1971) Protoplasma 72, 45W76. ESAU, K. E., AND HOEFERT, L. L. (1972a) J. Ultrustrut. Res. 40, 556-571. ESAU, K. E., AND HOEFERT, L. L. (1972b) Virology 48, 724-738. HOUK, M. S., AND HOEFERT, L. L. (1983) Phytopathology 73, 7%. [Abstract] HOEFERT, L. L. (1988) Amer. J. Bat. 75(6), 57. [Abstract]
AND
FAIL
HOEFERT, L. L., PINTO, R. L., AND FAIL, G. L. (1988) J. Ultrastruct. Mol. Struct. Res. 98, 243-253. JONES, A. T., KINNINMONTH, A. M., AND ROBERTS, I. M. (1973) J. Gen. Virol. 18, 61-64. KIM, K. S., AND FULTON, J. P. (1975) Virology 64, 560-565. KITAJIMA, E. W., AND LAURITIS, J. A. (1%9) Virology 37, 681-684. LANGENBERG, W. G. (1986) J. Gen. Virol. 67, 11611168. LEONARD, D. A. AND ZAITLIN, M. (1982) Virology 117,416-424. LISTER, R. M., AND BAR-JOSEPH, M. (1981) in KURSTAK, E. (Ed.), Handbook of Plant Virus Infections and Comparative Diagnosis, pp. 809-844, Elsevier/North-Holland Biomedical Press, New York/Amsterdam. MCMULLEN, C. R., AND GARDNER, W. S. (1980) J. Ultrastruct Res. 72, 65-75. MILLONIG, G. (1961) .I. Biophys. Biochem. Cytol. 11, 736-739. NAMETH, S. T., DODDS, J. A., PAULUS, A. O., AND LAEMMLEN, F. F. (1986) Plant Dis. 70, 8-11. PINTO, R. L., HOEFERT, L. L., AND FAIL, G. L. (1988) Amer. J. Bot. 75(6), 58. [Abstract] RAINE, J., WEINTRAUB, M., AND SCHROEDER, B. (1979) J. Ultrastruct. Res. 67, K&l 16. SHALLA, T. A., PETERSEN, L. J., AND ZAITLIN, M. (1982) J. Gen. Virol. 60, 355-358. VAN DER SCHEER, C., AND GROENEWEGEN, J. (1971) Virology 46, 493497. WEINTRAUB, M., RAGETLI, H. W. J., AND Lo, (1974) J. Ultrastruct. Res. 46, 131-148. WEINTRAUB, M., RAGETLI, H. W. J., AND LEUNG, (1976) J. Ultrastruct. Res. 56, 351-364.
E. E.
OF ULTRASTRUCTURE
AND
MOLECULAR
STRUCTURE
RESEARCH
100,
245-254 (1988)
Plasmalemma Deposits in Tissues Infected with Lettuce Infectious Yellows Virus ROBIN U.S.
Department
L. PINTO, of Agriculture,
LYNN
L. HOEFERT,
AND GAIL
Agricultural Research Station, Salinas, California 93905 Received
November
L. FAIL
1636 East
Alisal
Street.
3, 1988
Lettuce infectious yellows virus (LIYV) is a phloem-associated virus that is whiteflytransmitted. The physical characteristics and cytopathology of this virus are similar to those of other members of the Closterovirus group. One unique ultrastructural effect of the infection is the formation of conical deposits on the plasmalemmae of phloem parenchyma cells. The electrondense deposits are osmiophilic stacks of membrane lamellae spaced at 7 nm. Flexuous virions extend between these deposits from the cytoplasm through plasmodesmata and into adjacent sieve elements. We hypothesize that these plasmalemma deposits may facilitate movement of LIYV into other phloem parenchyma cells for further replication or into sieve elements for rapid transport throughout the host. o 1988 Academic press. IK.
Lettuce infectious yellows virus (LIYV) is one of five whitefly-transmitted viruses with possible relationships to the Closterovirus group (Duffus, 1987; Hoefert et al., 1988). Most Closteroviruses are transmitted by aphids and are generally confined to the phloem of the host. Purified preparations of LIYV show virions to be approximately 2000 nm long and 13-14 nm wide, with a flexuous profile (Duffus et al., 1986). LIYV infects economically important crops such as cantaloupe, carrot, cucumber, casaba, honeydew, lettuce, safflower, squash, sugarbeet, sunflower, and watermelon (Duffus and Flock, 1982; Nameth et al., 1986), as well as common weed species. We have examined the development of this disease and its ultrastructural effects in a variety of hosts (Houk and Hoefert, 1983; Hoefert et al., 1988; Hoefert, 1988). Ultrastructural symptoms, such as fibrilcontaining vesicles, long, flexuous virus particles, and inclusion bodies, are similar to those of other Closteroviruses (Lister and Bar-Joseph, 1981). A unique ultrastructural effect of this virus disease is the deposition of electron-dense material on the plasmalemma of infected leaf and root tissue. These deposits lie adjacent to plas-
modesmata of phloem parenchyma cells. The deposits have been observed in all LIYV-infected plants, including members of the families Malvaceae, Asteraceae, Chenopodiaceae, Brassicaceae, and Solanaceae. In this study we present the results of examinations of tissues of Nicotinna clevelandii L., Chenopodiun murale 1,. , and Lactuca sativa L. We hypothesize that plasmalemma deposits may influence the transport of virus particles throughout the phloem parenchyma. MATERIALS
AND METHODS
Collections of leaf and root tissue for electron microscopy were made from greenhouse-grown plants. The whitefly vector, Bemisia tabaci Genn., was used to infect N. clevelandii L., C. murale L., and L. sativa L. with LIYV. Young leaves, middle-aged leaves, and roots were taken from the plants at various stages after inoculation from as early as 5 days to as late as 2 months depending on the rate of spread of the virus infection within each species. Uninoculated, healthy controls of a similar age were collected at the same time. Samples were fixed for 2-3 hr in a 3% paraformaldehyde/3% glutaraldehyde solution in 0.05 M phosphate buffer at pH 6.8 in an ice bath and under vacuum. The tissue was postfixed overnight at 4°C in 2% osmium tetroxide in the same buffer. The tissue was then dehydrated in an acetone and finally a propylene oxide series. Samples were embedded in an Epon 812 equivalent resin. Sections were cut with di245 0889-1605188 $3.00 Copyright 0 1988 by Academic Press, lnc All rights of reproduction in any form rescrwed
246
PINTO,
HOEFERT,
AND
FAIL
DEPOSITS
IN TISSUES
INFECTED
WITH
LIYV
247
248
PINTO,
HOEFERT,
AND
FAIL
DEPOSITS IN TISSUES INFECTED amond knives and stained with uranyl acetate and lead tartrate (Millonig, 1961). RESULTS
Electron-dense deposits are found on the plasmalemmae of phloem parenchyma cells in plants infected with LIYV, but not in healthy tissue. Figure 1 illustrates a number of the deposits in two phloem parenchyma cells of C. murule. Plasmalemma deposits (PDs) are seen only in cells that show other cellular effects of the virus disease. These effects include lipid bodies, fibrilcontaining vesicles, and viroplasm. Membranes of chloroplasts, mitochondria, and nuclei often appear distorted. Aggregates of virus particles also are visible in the phloem parenchyma cells. An intervening sieve element has been filled by virus particles. Figure 2 shows another view of similar cells at higher magnification. In longitudinal section (Figs. 3 and 4) the PDs appear conical in shape. They are tightly appressed to the plasmalemma and
WITH LIYV
249
are located near the plasmodesmata. The PDs are composed of 6- to 7-nm layers of membranous material lying parallel to the plasmalemma (Fig. 3). When sectioned in a plane that is not perpendicular to the plasmalemma, the layers are not always visible; TEM examination with a tilting stage confirms that the layers are present. The entire plasmalemma deposit is not bounded by any discernible membrane and, therefore, is not separated from the rest of the cytoplasm. The PDs are found adhering to pit fields (the parenchyma side of plasmodesmata which connect to sieve elements and other parenchyma cells), but never to lateral sieve areas (plasmodesmatal connections on the sieve element side). As many as ten PDs in a closely clustered group may be visible in these pit fields (Figs. 1, 3, and 4). Figure 5 is an oblique cut through areas of deposit and parenchyma cell wall. The deposits appear as a broad expanse of electron-dense material overlying the cell wall. Virus particles in infected tissues often
l-5. Phloem tissue in leaves of C. murale infected with LIYV 8 weeks past inoculation. FIG. 1. Phloem parenchyma cells (PP) contain typical virus symptoms of LIYV: vesicles (Ve), regions of viroplasm (VP), virus particles (V), lipid droplets (L), and distorted chloroplast (C) and mitochondrial (M) membranes. Plasmalemma deposits (PD) are found in the phloem parenchyma cells, but not in the sieve element (SE). Note that deposits are clustered around the plasmodesmata (P) in the pit field and not in the lateral sieve area. The sieve element is completely filled with virus particles. X 26 000. FIG. 2. Sieve element (SE) tilled with virus particles. The adjoining phloem parenchyma cell (PP) contains plasmalemma deposits (PD). Virus particles (arrow) near the deposits are oriented perpendicular to the cell wall and between the deposits. Note virus particles (arrow) in cross section in the median nodule of a plasmodesma (P). x 30 ooo. FIG. 3. Plasmalemma deposits (PD) composed of membrane-like material adhere to the plasmalemma (PI) of a phloem parenchyma cell. The deposits border the plasmodesmatal pores (P). Virus particles (arrows) are visible extending from the cell cytoplasm into the plasmodesmata. Note that no desmotubule is apparent. x 110000. FIG. 4. Virions (arrows) extend from the plasmodesma of a phloem parenchyma cell into the lumen of the adjacent sieve element (SE). Note that no desmotubules are apparent. Plasmalemma deposits (PD) abut the pores of the plasmodesmata (P). Membranous layers approximately 7 nm thick are clearly visible. Virions are evident in the cytoplasm. x 112 000. FIG. 5. An oblique section through an expanse of deposit material (PD) and cell wall (CW). Virus particles are reoriented away from virus aggregates (V) in the cytoplasm and toward plasmalemma deposits and plasmodesmata. Virions (arrows) in cross section are visible as they penetrate the plasmalemma deposit and the plasmodesmata. Note that the deposit lies adjacent to the common walls of a sieve element (SE) and another phloem parenchyma cell (PP). Virus particles fill the sieve element and also are present in the parenchyma cell. x 35ooo. FIG. 6. Virus particles in the phloem parenchyma of L. sativa 3.5 days after inoculation. Virus particles (arrows) near plasmalemma deposits appear to be reoriented away from other virus aggregates (V) and toward a plasmodesma (P). A chloroplast (C) and mitochondrion (M) are also visible in the cell. X 63 000. FIGS.
250
PINTO, HOEFERT,
are seen in close association with the PDs. In Figs. 1, 2, and 3, virus particles in the cytoplasm are oriented between the PDs and in toward plasmodesmata. In Fig. 4, virions extend into the adjoining sieve element through the plasmodesma. In cross section (Fig. 5), virions appear in small clusters surrounded by regions of plasmalemma deposit. Virions associated with PDs often appear to be reoriented away from large aggregates of virus particles in the cytoplasm and toward the plasmodes-
AND FAIL
mata (Figs. 1, 5, and 6). Desmotubules are not evident in either cross or longitudinal sections of plasmodesmata that contain vir-us particles. A schematic diagram (Fig. 7) illustrates our interpretation of the shape and structure of the plasmalemma deposits and their spatial relationship to other host and virus-induced structures in typical phloem parenchyma cells infected with LIYV. Both the LIYV particles and PDs are limited to phloem parenchyma cells of all pre-
W
SE
;
FIG. 7. A schematic diagram of a sieve element (SE) and two phloem parenchyma (PP) cells infected with LIYV. Virus symptoms typical of a Closterovirus disease, such as vesicles (Ve), virus particles (V), viroplasm (VP), and lipid droplets (L) are visible in the phloem parenchyma cells. Mitochondria (M) and chloroplasts (C) contain distorted membranes. Plasmalemma deposits (PD) abut plasmodesmata (P) in pit fields that connect the phloem parenchyma cell to the sieve element and adjacent parenchyma cell. Virus particles can be seen in the cytoplasm of the phloem parenchyma cell, the lumen of the sieve element, and in the pit fields of the two parenchyma cells. Not to scale.
DEPOSITS
IN TISSUES
viously examined tissue, except in N. clevelandii where border parenchyma cells also contain PDs. In these cells, the deposits are situated only on the phloem parenchyma cell side of border parenchyma (Fig. 8) and not on the mesophyll side. In root tissue, plasmalemma deposits are generally smaller in size and less frequent than in leaf tissue. Figure 9 shows virus particles in root tissue of L. sativa directed toward a plasmodesma and between PDs. The deposits are evident in infected tissues as soon as other symptoms, such as virus vesicles, are detectable. Small PDs, in leaves, of Lactuca are visible as early as 5 days after inoculation (Fig. 10). DISCUSSION
Many ultrastructural studies have implicated plasmodesmata in the transport of virus particles between cells. Virus particles, isometric and rod-shaped, from many different groups have been seen in plasmodesmata. Examples of isometric viruses include dahlia mosaic virus (DMV; Kitajima and Lauritis, 1969), beet western yellows virus (BWYV; Esau and Hoefert, 1972a, b), cauliflower mosaic virus (CaMV; Bassi et al., 1974), and cowpea mosaic virus (CMV; Kim and Fulton, 1975). Some of the flexuous and stiff rods detected in plasmodesmata include beet yellows virus (BYV; Esau and Hoefert, 1971, potato virus Y (PVY; Weintraub et al., 1974), and tobacco etch and tobacco mosaic virus (TEV and TMV; Weintraub et al., 1976). In our survey of LIYV in a variety of hosts, we have not observed desmotubules in any plasmodesma that also contains virus particles. Modified or displaced desmotubules have been identified in plasmodesmata with PVY particles (Weintraub et al., 1974; McMullen and Gardner, 1980) or TEV (Weintraub et al., 1976). Desmotubules were not evident in tissue infected with BYV (Esau and Hoefert, 1971) or BWYV (Esau and Hoefert, 1972b). These authors concluded that the absence of the desmotubule was necessary to permit the
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passage of the virus through the plasmodesmata. Kitajima and Lauritis (1969) stated that the “plasmodesma is not a static but a dynamic structure”; it seems that this flexibility of structure, including the desmotubule, may be important to facilitate virus transport. Many virus infections induce the formation of various kinds of wall anomalies or deposits. Often these deposits have been associated with plasmodesmata. Bassi et al. (1974) identified the tubule component of wall protrusions caused by CaMV as a plasmodesma-like material. Similar wall protrusions were typical of Como- and Nepovirus infections (Kim and Fulton, 1975). Virus particles were seen penetrating cell walls or wall overgrowths (Jones et al., 1973; Van der Scheer and Groenewegen, 1971). In early infections of potyviruses, cylindrical or bundle inclusions appeared to be attached to and, in some cases, to have penetrated plasmodesmata (Weintraub ef al., 1976; McMullen and Gardner, 1980). Viruses were associated with cylindrical inclusions at the plasmodesmata on either side of cell walls. Langenberg (1986) suggested that these inclusions might be responsible for either alignment or active transport of flexuous PVY particles into plasmodesmata. Raine et al. (1979) described plasmalemma deposits in Prunus leaves infected with little cherry disease (LCD). LCD is caused by a phloem-limited virus of probable Ciosterovirus affinity. Cylindrical deposits, or tubules, in phloem parenchyma cells contained hollow centers positioned over plasmodesmatal pores. Like those in tissues infected with LIYV, the LCD deposits were composed of lamellae 7 nm thick. Most virus particles in infected cherry tissue were found in aggregates oriented along the long axis of the cell. Viruses near the tubules appeared to be directed away from the majority of particles and reoriented into the plasmodesmata in a manner identical to that seen in LIYV-infected tissue (Figs. 1, 5, and 7). LCD virus particles extended
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through these tubules and plasmodesmata from the companion cell cytoplasm into the sieve element lumen (Raine et al., 1979, Fig. 2). Molecular research using temperaturesensitive isolates of TMV has identified a transport factor associated with plasmodesmata which may influence the spread of plant viruses. Under elevated temperatures, these TMV isolates replicate in epidermal cells of infected tissue but do not move beyond the site of initial infection to other cells. Comparisons of protein structures synthesized by healthy and temperature-restricted isolates of TMV showed a mutation in one protein, p30 (Leonard and Zaitlin, 1982). Leonard and Zaitlin (1982) suggested that the alteration of p30 was correlated with the lack of cell-to-cell movement of the virus from the initial infection site. Furthermore, Shalla et al. (1982) showed a statistically significant reduction in the number of plasmodesmata in mesophyll cells of plants infected with the temperature-sensitive isolate. Thus, the presence of this protein appeared to be necessary for replicated virions to invade other cells beyond the initial infection site. The specific function of the LIYV plasmalemma deposits is, as yet, undetermined. One possibility is that the deposits are a defensive reaction by the host plant to inhibit the transport of the virus and thereby limit the spread of the infection. This function seems improbable because no deposit has been seen occluding any plasmodesmatal pore, and because intact virus particles have been seen within plasmodesmata surrounded by those deposits. In ad-
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dition, Pinto et al. (1988) have shown that infected tissue of L. sativa contained PDs and virions at the earliest stages of virus development (5 days after inoculation), whereas cell wall overgrowths, which have been considered a possible plant defense reaction to virus invasion (Esau, 1967), did not form until 14 days past inoculation (Hoefert et al., 1988). LIYV causes a common alteration of the desmotubules within the plasmodesmata of infected phloem parenchyma cells. This alteration of the desmotubule appears to facilitate the movement of vu-ions through the plasmodesmata. We postulate that the presence of the plasmalemma deposits also may assist the virus in cell-to-cell transport through the host plant. Reorientation of virions toward and into the plasmodesmata suggests that the conical PDs may act to either direct or attract these virions to the pores. The deposits are found on pit fields which specifically connect phloem parenchyma cells to sieve elements or other phloem parenchyma cells. This association with these plasmodesmata suggests that the PDs influence virus movement into the two different cell types. These cells might be opportunistic sites for the virus for replication in the case of the phloem parenchyma cells or for distribution into other areas of the host plant in the case of the sieve elcments (Hoefert et al., 1988). The mechanism of virus transport is critical in the cytopathology of a disease. Many viruses, both spherical and rod-shaped, are transported throughout a host during an infection. A virus-induced transport factor has now been identified (Leonard and Zait-
FIG. 8. A border parenchyma cell (BP) in N. clevelandii contains virus particles (arrows), vesicles (Ve), and plasmalemma deposits (PD). Plasmodesmata (P) in cross section are visible in a common wall between phloem and border parenchyma cells. x 25 000. Fro. 9. A phloem parenchyma cell in L. sntiva root tissue contains plasmalemma deposits (PD) and virus particles (arrows). Virus particles are oriented toward the plasmodesma (P) that connects this phloem cell to the adjacent sieve element (SE). x 57 000. FIG. 10. Early development of virus infection (5 days after inoculation) in phloem parenchyma of L. sutivn. Virus vesicles (Ve) are beginning to aggregate; mitochondria (M) look normal. Small plasmalemma deposits (PD) abut plasmodesmatal pores (P). A few virions (arrows) are visible in the cytoplasm and are oriented toward the olasmodesmata. x 41 500.
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lin, 1982), but its mode of action is still poorly understood. Evidence that plasmodesmata participate in cell-to-cell virus transport is accumulating (Kitajima and Lauritis, 1969; Esau and Hoefert, 1972a, b; Weintraub et al., 1976; Shalla et al., 1982; and Langenberg, 1986); it seems logical that a virus-induced transport factor might operate on those plasmodesmata. The formation of the LIYV-induced plasmalemma deposits may reflect the activity of such a transport factor. REFERENCES BASSI, M., FAVALI, M. A., AND CONTI, G. G. (1974) Virology 60, 353-358. DUFFUS, J. E., (1987) in HARRIS, K. F. (Ed.), Current Topics in Vector Research, Vol. 4, pp. 73-91, Springer-Verlag, New York/Berlin. DUFFUS, J. E., AND FLOCK, R. A. (1982) Calif. Agric. 36,4-6. DUFFUS, J. E. LARSEN, R. C., AND LIU, H. Y. (1986) Phytopathology 76, 97-100. ESAU, K. E. (1967) Annu. Rev. Phytopathol. 5,45-70. ESAU, K. E., AND HOEFERT, L. L. (1971) Protoplasma 72, 45W76. ESAU, K. E., AND HOEFERT, L. L. (1972a) J. Ultrustrut. Res. 40, 556-571. ESAU, K. E., AND HOEFERT, L. L. (1972b) Virology 48, 724-738. HOUK, M. S., AND HOEFERT, L. L. (1983) Phytopathology 73, 7%. [Abstract] HOEFERT, L. L. (1988) Amer. J. Bat. 75(6), 57. [Abstract]
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HOEFERT, L. L., PINTO, R. L., AND FAIL, G. L. (1988) J. Ultrastruct. Mol. Struct. Res. 98, 243-253. JONES, A. T., KINNINMONTH, A. M., AND ROBERTS, I. M. (1973) J. Gen. Virol. 18, 61-64. KIM, K. S., AND FULTON, J. P. (1975) Virology 64, 560-565. KITAJIMA, E. W., AND LAURITIS, J. A. (1%9) Virology 37, 681-684. LANGENBERG, W. G. (1986) J. Gen. Virol. 67, 11611168. LEONARD, D. A. AND ZAITLIN, M. (1982) Virology 117,416-424. LISTER, R. M., AND BAR-JOSEPH, M. (1981) in KURSTAK, E. (Ed.), Handbook of Plant Virus Infections and Comparative Diagnosis, pp. 809-844, Elsevier/North-Holland Biomedical Press, New York/Amsterdam. MCMULLEN, C. R., AND GARDNER, W. S. (1980) J. Ultrastruct Res. 72, 65-75. MILLONIG, G. (1961) .I. Biophys. Biochem. Cytol. 11, 736-739. NAMETH, S. T., DODDS, J. A., PAULUS, A. O., AND LAEMMLEN, F. F. (1986) Plant Dis. 70, 8-11. PINTO, R. L., HOEFERT, L. L., AND FAIL, G. L. (1988) Amer. J. Bot. 75(6), 58. [Abstract] RAINE, J., WEINTRAUB, M., AND SCHROEDER, B. (1979) J. Ultrastruct. Res. 67, K&l 16. SHALLA, T. A., PETERSEN, L. J., AND ZAITLIN, M. (1982) J. Gen. Virol. 60, 355-358. VAN DER SCHEER, C., AND GROENEWEGEN, J. (1971) Virology 46, 493497. WEINTRAUB, M., RAGETLI, H. W. J., AND Lo, (1974) J. Ultrastruct. Res. 46, 131-148. WEINTRAUB, M., RAGETLI, H. W. J., AND LEUNG, (1976) J. Ultrastruct. Res. 56, 351-364.
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