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Fine structure of plant cells infected with bean pod mottle virus
WROLOGY49, 112-121 (1972)
Fine Structure of Plant Cells Infected with Bean Pod Mottle Virus' KYUNG SOO K I M AND J. P. FULTON Virology and Bio-Control Laboratory, Department of Plant Pathology, University of Arkansas, Fayetteville, Arkansas 72701 Accepted March 31, 1972 Bean pod mottle virus (BPMV) was present in epidermal cells, mesophyll cells, and all component cells of the vascular bundle including matured sieve and tracheal elements. Virions were present in the ground cytoplasm and central vacuole within infected cells. Honeycomblike bundles of spheres bounded by a membrane were also present in vacuoles, and four types of structural changes of mitochondria were noted in infected cells of a systemic host. Proliferation of the membrane system was an early effect of infection. This occurred in the plasmalemma, endoplasmic reticulum, and mitochondria to produce vesicles, concentric membrane aggregates, loose membranes and tubules. BPMV also induced filamentous inclusions in cytoplasm of both hosts. In the systemically infected host striking changes in ultrastrueture were evident at an early stage of infection.
INTRODUCTION Virus particles within tubules in bean leaf cells infected with bean pod mottle virus (BPMV) have been described by Kim and Fulton (1971). The tubules with virus particles were located between the plasmalemma and the cell wall and, in the systemically infected bean, embedded within cell wall proliferations. Since a great deal of abnormal cellular activity appeared to be associated with BPMV infection, studies were extended to determine all possible sites of virus occurrence and effects of virus infection upon cellular uRrastructure in a local lesion host and a systemic host. MATERIALS AND METHODS
Greenhouse-grown bean plants (Phaseolus vulgaris L., cvs. 'Cherokee Wax' and 'Pinto') were used throughout this study. BPMV produced local lesions in Pinto bean and systemic mottling in Cherokee Wax bean. The first true leaves (first node above cotyledons) of both varieties of bean plants were mechanically inoculated with sap extracted from infected Cherokee Wax bean plants. 1Supported ia part by Cooperative States Research Services Grant No. 816-15-16.
Specimens were prepared for electron microscopy as described previously (Kim and Fulton, 1971). For the study of local lesion infection, pieces of lesions 1-2 mm ~ were taken from the inoculated primary leaves of Pinto bean. The first recognizable local lesions appeared 3 days after inoculation. For each sequential study, lesions were taken from the same inoculated leaf 3, 4, 5, 6, and 7 days after inoculation. For the study of systemic infection, similar samples were taken from the central leaflet of the first trifoliolate (second node above cotyledons) leaf of Cherokee Wax bean 6, 7, 8, 9, 10, 11, 12, 13, and 24 days after inoculation. Seven days after inoculation, the first trifoliolate leaves showed vein clearing, followed by mottling 9 days after inoculation. In all cases, appropriate healthy control leaves were sampled at the same times as infected leaves. Four separate samplings were made during this study. BPhIV was purified by a method of Bancroft (1962), and top, middle, and bottom components were separated by sucrose gradient eentrifugation. The bottom component was uRracentrifuged to produce a compact pellet, which was then treated in
112 Copyright © 1972 by Academic Press, Inc. All rit~'htsof reproduction in any form reserved.
BPMV-INFECTED PLANT CELL ULTRASTRUCTURE a manner similar to leaf tissue in preparing thin sections. RESULTS Location of Virus Particles
In cells of the systemically infected Cherokee Wax bean leaves, virus particles were observed 6 days after inoculation and appeared to increase in amount up to 8 days after inoculation. This coincided with the first evidence of vein:clearing in the trifoliolate leaves. Mottling appeared later; fewer virus particles were observed in cells of all samples taken more than 8 days after inoculation. Virus particles in the ground cytoplasm were difficult to distinguish from ribosomes, especially if they were scattered randomly.
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Spherical particles resembling ribosomes were, however, often densely packed and were surrounded by a loose membrane. These were interpreted as BPFIV since no such structures were observed in the cytoplasm of uninfected leaf cells. In 7-day and older samples, virus particles corresponding in size (25-28 nm) to the particles of a purified, embedded, and stained preparation (Fig. 1B) were observed in the central vacuole, either scattered or contained within a membranous sac (Fig. 1A). Some membranous sacs contained honeycomblike bundles of spheres (13-16 nm) with empty centers in addition to normally stained particles (Fig. 1A). Although the honeycomblike bundles and virus particles occurred in the same sac they were not intermingled. In
Key to figure abbreviations: CH, chloroplast ; Cr, cristae; CW, cell wall; ER, endoplasmic reticulum ; F1, filamentous inclusion; HS, honeycomblike spheres; M, membrane; Mi, mitochondria; N, nucleus; ST, sieve tube; SW, secondary cell wall; Ve, membranous vesicles; VP, virus particles. FIG. 1. (A) Two honeyeomblike bundles of spheres bounded by a membrane in the central vacuole of a Cherokee Wax bean leaf cell 6 days after inoculation. Virus particles are not intermingled with honeycomblike spheres even though they occur in the same sac. (B) BPMV particles in purified, embedded, and sectioned preparation.
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KIM ARrD FULTON addition to the occurrence within the cells, virus particles were also noted in intercellular spaces. Virus particles were present in all types of cells: epidermal, mesophyll, and all the component cells of vascular bundles. In matured sieve elements, particles were either closely packed and enclosed in loose membranes or in crystals (Fig. 2). In xylem tracheal elements, particles were not enclosed by a membrane but were closely associated with the surfaces of primary and secondary cell walls (Fig. 3). The lumen of the matured sieve elements in systemically infected Cherokee Wax bean leaves was the only place where cryslals of virus particles were observed. In the local~lesion host, Pinto bean, the location of virus particles was similar to the systemically infected host except that tubules with particles were not embedded in the cell wall (Kim and Fulton, 1971). The honeyeomblike masses of spheres were not evident in the vacuole. Three days after inoculation virus particles were not observed in the vacuole, but in all later stages of infection, ~they were observed in the central vacuole in a heavily aggregated form. There was no evidence of virus particles in nuclei, mitoehondria, or chloroplasts of locally or systemically infected hosts. Effects on Cell Ultrastrueture The most noticeable evidence of abnormal activity in infected cells of both systemically infected and local lesion hosts was an area of dense cytoplasm in the vicinity of the nucleus. In most cases this area bulged into the central vacuole. The area was composed of a great number of spherical vesicles of various sizes (Fig. 4A, B). Only one area of this type was observed in any cell. Such areas were bordered by several long segments of rough endoplasmic retieuhim (Fig. 4A) and were FIG. 2. Virus particles in crystalline form in sieve element of Cherokee Wax bean leaf 6 days after inoculation. FIG. 3. BPMV particles are closely associated with the surface of primary and secondary cell walls (arrows) of a xylem tracheal element in Cherokee Wax bean leaf 7 days after inoculatiom
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FIG. 5. (A) The formation of concentric membrane aggregate presumably from smooth endoplasmic reticuhm in Cherokee Wax bean leaf cells 7 days after inoculatiou with BPMV. (B) An increase of rough endoplasmic reticuhm with a deep circular bending which results in a ring. Transversely sectioned tubules with virus particles between the plasmalemma and the cell wall are also showing (arrows). From a locally infected Pinto bean leaf cell.
devoid of larger organelles, such as mitochondria or chloroplasts. In other areas of the cytoplasm of systemically infected cells, smooth endoplasmic reticulum increased in amount, and concentrically arranged membrane aggregates were often noted (Fig. 5A). In cells of the local lesion host, an increase of rough endoplasmic reticulum was very evident in the cytoplasm. The cisternae of this endoplasmic reticulum were bent into a ring containing some cytoplasmic components (Fig. 5B). A filamentous inclusion was found in the cytoplasm of all types of cells in both systemic and local lesion hosts. In longitudinal section i~ appeared as bundles of fibers or sectioned sheets (Fig. 6A) and in transverse section as a central area of sectioned fibers surrounded by concentrically arranged sheets (Fig. 6B). Rough endoplasmic reticulure was often closely associated with or aligned between the bundles of the filamentous inclusion (Fig. 6A). Individual filaments 100-120 A in diameter and sheets of comparable thickness were not as electron dense as virus particles. Four types of modified mitochondria were observed in systemically infected cells: (1) Some were pleomorphic and usually in the shape of a horseshoe or arch (Fig. 7). (2) The mitochondrial matrix contained concentrically arranged, thin, membranous laminae which appeared to have originated from either the inner or both the inner and outer mitochondrial membranes (Fig. 8A, B). Dark granular material was often contained in the center of the membrane aggregate. (3) Osmiophllic globules, similar to cytoplasmic lipid globules and varying in size, shape, and number, occurred in the matrix of this type of mitochondrion (Fig. 9). (4) Occasionally mitochondria were noted containing an electron opaque body bound by a membrane (Fig. I0). In addition to these four types of abnormal mitochondria, apparently normal mitochondria were present. Like those observed in healthy cells, they were either spherical or rod shaped, bounded by typical double unit membranes and containing tubulelike cristae without fixed arrangements and intramitochondrial granules. The greatest number of abnormal mito-
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FIG. 6. (A) Bundles of filamentous inclusion bodies sectioned somewhat longitudinally in the vicinity of a vesieulated area. Strands of rough endoplasmie retieulum are closely associated with or between the bundles of the filamentous inclusion. (B) Somewhat transversely sectioned filamentous inclusions which appear as a central area of sectioned fibers surrounded by concentrically arranged sheets. Both A and B are from Cherokee Wax bean leaf cells 8 days after inoculation with BPMV. ehondria was noted 6-8 days after inoculation. A t this stage, approximately 50 % of the mitochondria were of the four abnormal types. The relative n u m b e r of abnormal mitochondria decreased with time of sampiing and were only occasionally noted 24 days after inoculation. There was a striking increase in the number of mitochondria in the local-lesion host, Pinto bean. Five days after inoculation, infected cells averaged approximately twice as m a n y as control cells. Only a few abnormal mitochondria were noted. These were identical to the fourth type mentioned above or were of a fifth type identified b y the occurrence of thin, curved osmiophilie bands in the matrices (Fig. 11). DISCUSSION I t seems logical to assume t h a t virus particles in matured xylem tracheal elements
or phloem sieve tubes were synthesized during the i m m a t u r e stages of these cells. The abundance of B P M V particles in undifferentiated phloem cells m a y support this possibility. However, this does not explain the virus particles occurring in the intercellular spaces. I t would seem t h a t virus particles could accumulate in such an area only b y active m o v e m e n t from living ceils, probably through plasmadesmata. A similar phenomenon m a y account for the particles in the matured sieve element, but not for those in the tracheal elements, since there are no plasmadesmata between matured trachea] elements and p a r e n c h y m a cells. Paliwal (1970) found a large n u m b e r of the particles of bromegrass mosaic virus in the intercelhrlar spaces of mesophyll cells and the vascular bundle sheath. I-Ie discussed the possibility t h a t virions might be assembled there.
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FIG. 7. Horseshoe or arch-shaped mitochondria in a Cherokee Wax bean leaf cell 7 days afterinoculation with BPMV. Intramitochondrial granules are also present (arrows). FIG. 8. (A) Concentrically arranged membranous lamina in a mitochondrion which appear to have originated from the mitochondrial envelope (see arrows). Dark granular materials are present in the center of the lamina. Cristae are sparsely distributed or grouped creating the translucent area. (B) Concentric membrane layers appear detached from the mitochondrial envelope and are in the center of the matrix. Both figures are from Cherokee Wax bean leaf cell 7 days after inoculation with BPMV.
It is of interest to note that the crystals of BPMV particles occurred only in matured sieve elements and were completely absent in any other cell type. Some other icosahedral viruses such as cowpea strain of southern bean mosaic virus (Weintraub and IIagetli,
1970) a n d bromegrass mosaic v i r u s (Paliwal, 1970), were f o u n d readily i n crystal form in t h e c y t o p l a s m of a n y infected cell. T h e particles of B P M V seem to be c o n c e n t r a t e d e n o u g h to form crystals i n c y t o p l a s m a n d vacuoles if the s a t u r a t i o n of these areas with
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~'I~. 9. (A) Present in a mitoehondrial matrix is an osmiophilie globule which is similar to the cytoplasmic lipid globules (see arrows). FI~. 10. An electron-opaque granular body bounded by a membrane from a Cherokee Wax bean leaf cell 8 days after inoculation with BPM¥. ~'Ia. 11. Two mitoehondria containing a thin, curved, osmiophilie band in their matrices in Pinto bean leaf cell 3 days after inoculation. particles is a factor in crystalline arrangement of virions (Paliwal, 1970). De Zoeten and Gaard (1969) observed that tomato ringspot virus particles were found as paracrystalline inclusions in infected Datura stramo'nium L. leaf cells, but not in Cucumis sativus L. They suggested that there is a
virus--host interaction which is a requirement for crystal formation. The occurrence of crystals of B P M V only in matured sieve elements m a y also suggest that there is a certain intracellular environment which favors the formation of virus crystals. The occurrence of the honeycomblike
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K I M AND F U L T O N
masses of spheres in the vacuoles of cells of the systemically infected host may be noninfective protein shells or "top component" (Bancroft, 1962). Their close association with virions within the membranous sac supports this view, but their size is somewhat smaIler, unless the fixation, embedding, and staining can account for the size difference. The dense area of cytoplasm with a large amount of vesicles appears to be the same area that has been termed the "inclusion body" (Roberts and Harrison, 1970; van der Scheer and Groenewegen, 1971). It would seem desirable to reserve the term inclusion to refer to materials which accumulate as a result of virus infection, e.g., the filamentous inclusion. The dense area is composed of cytoplasmic components modified so that the membrane system is increased greatly and vesicles are evident. These features suggest that this may be a metabolically active area in the cell which is analogous to the area which has been termed the "viro~ plasm" (Gaylord and NIelnick, 1953) or "x-bodies" (Fujisawa et al., 1967) even though no accumulation of virus particles was associated with this area. The various structural changes which were recorded in this study emphasize the abnormal activity associated with virus infection. Proliferation of the membrane system both in the vesiculated area and in increased amounts of smooth and rough endoplasmic reticuhim suggest increased cellular activity. The increased number of tubules with and/or without virus particles (Kim and Fulton, 1971) as well as some changes in mitochondria also indicate the proliferation of the membrane system in infected cells. The mitochondrial changes are of interest. In only a few cases have mitoehondrial changes been related to virus infection. Weintraub and Ragetli (1971) found hypertrophied mitochondria associated with an apple virus. Weintraub et al. (1964) also recorded an increase in number of mitochondria in cells of the local lesion host, Nicotiana glutinosa, infected with tobacco mosaic virus but no increase in systemically infected cells of Nicotiana tabacum. A similar reaction of local lesion and systemic hosts was recorded in this study. The fourth type of abnormal mitochondria recorded in this paper was
observed by Weintraub and Ragetli (1970) in eowpea leaf cells infected with southern bean mosaic virus. The same type of abnormal mitochondria is evident in Fig. 3 of a paper by Harrison and Roberts (1971) although no mention is made of this. This suggests that this type of abnormal mitochondria may be more commonly associated with virus infection than has been noted. The filamentous inclusions found in this study differ from P-protein and X-tubules described by Esau and Cronshaw (1967) in morphology, size~ and cell type. P-proteins and X-tubules found in TMV infected tobacco leaves were 230 A_ and 280 A in diameter, respectively, while o the filaments in this study were 100-120 A. The individual filaments are not tubular as in P-protein and X-tubules. P-proteins occur only in sieve elements and phloem-related parenchyma cells while the BPMV-induced filamentous inclusion occurs in all types of infected cells. Filaments were not as electron dense as virus particles suggesting they are composed mainly of protein. Endoplasmic reticulum associated with filaments suggests that it may be involved in the synthesis of the inclusion. The material presented in this paper as well as other studies which have appeared recently (Kim and Fulton, 1971; Roberts and Harrison, 1970; van der Scheer and Groenewegen, 1971) dispels the impression that small, polyhedral plant viruses (25-30 nm) cause only limited cytological changes in infected cells (Matsui and Yamag~chi, 1966). In this study the striking changes were most evident at a very early stage of infection in a systemically infected host. Cells seemed to recover from these changes at later stages of infection even though the tissue showed good symptoms of disease. REFERENCES BA~CaOFrr, J. B. (1962). Purification and properties of bean pod mottle virus and associated centrifugal and eleetrophoretie components. Virology 16, 4!9-427. DE ZOETEN, G. A., and GAAItD, G., (1969). Possibilities for inter- and intracelhflar t r a n s l o e a t i o n of some icosahedral p l a n t viruses. J. Ceil Biol. 40,814-823.
BPMV-INFECTED PLANT CELL ULTRASTRUCTURE ESAU, K. and CRONSI-IAW,J. (1967). Tubular components in cells of healthy and tobacco mosaic virus-infected Nicotiana. Virology 33, 26-35.
FUJISAWA, I., RUBIO-HUERTOS,M., ~/IATSUI, C., and YAMAGUCHI,A. (1967). Intracellular appearance of cauliflower mosaic virus particle. Phytopathology 57, 1130-1132. GAYLORD, W. I-I.,and MELNICK, J. L. (1953). Intracellular forms of poxviruses as shown by the electron microscope (Vaccinia, ectromelia, molluscum contagiosum). J. Exp. Med. 98, 157-172. HARRISON, B. D., and ROBERTS, I. M. (1971). Pinwheels and crystalline structures induced by Atropa mild mosaic virus, a plant virus with particles 925 nm. long. J. Gen. Virol. 10, 71-78. KIM, K. S., and FULTON, J. P. (1971). Tubules with viruslike particles in leaf cells infected with bean pod mottle virus. Virology 43,329-337. MATSUI, C., and YAMAGUCHI, A. (1966). Some aspects of plant viruses in situ. Advan. Virus Res. 12, 127-174. PALIWAL, Y. C. (1970). Electron microscopy of
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bromegrass mosaic virus in infected leaves. J. Ultrastruct. Res. 39, 491-502. ROBERTS, I. M., and HARRISON, B. D. (1970). Inclusion bodies and tubular structures in Chenopodium amaranticolor plants infected with strawberry latent ringspot virus. J. Gen. Virol. 7, 47-54. VAN DER SCHEER, C., and GROENEWEOEN, J. (1971). Structures in cells of Vigna unguiculata infected with cowpea mosaic virus. Virology 46, 493-497. WEINTRAUB, ~V~., and I=~AGETLI,H. W. J. (1970). Electron microscopy of the bean and cowpea strains of southern bean mosaic virus within leaf cells. J. Ultrastruct. Res. 32,167-189. W~INTnAUB,M., and I~AGETLI,H. W. J. (1971). A mitochondrial disease of leaf cells infected with an apple virus. J. Ultraslruct. Res. 36,669-693. WEINTRAUB, M., RAGETLI, H. W. J., and I)WURAZNA,M. M. (1964). Studies on the metabolism of leaves with localized virus infections. Mitochondrial activity in TMV-infected Nicoliana glutinosa L. Can. Y. Bot. 42,541-545.
Fine Structure of Plant Cells Infected with Bean Pod Mottle Virus' KYUNG SOO K I M AND J. P. FULTON Virology and Bio-Control Laboratory, Department of Plant Pathology, University of Arkansas, Fayetteville, Arkansas 72701 Accepted March 31, 1972 Bean pod mottle virus (BPMV) was present in epidermal cells, mesophyll cells, and all component cells of the vascular bundle including matured sieve and tracheal elements. Virions were present in the ground cytoplasm and central vacuole within infected cells. Honeycomblike bundles of spheres bounded by a membrane were also present in vacuoles, and four types of structural changes of mitochondria were noted in infected cells of a systemic host. Proliferation of the membrane system was an early effect of infection. This occurred in the plasmalemma, endoplasmic reticulum, and mitochondria to produce vesicles, concentric membrane aggregates, loose membranes and tubules. BPMV also induced filamentous inclusions in cytoplasm of both hosts. In the systemically infected host striking changes in ultrastrueture were evident at an early stage of infection.
INTRODUCTION Virus particles within tubules in bean leaf cells infected with bean pod mottle virus (BPMV) have been described by Kim and Fulton (1971). The tubules with virus particles were located between the plasmalemma and the cell wall and, in the systemically infected bean, embedded within cell wall proliferations. Since a great deal of abnormal cellular activity appeared to be associated with BPMV infection, studies were extended to determine all possible sites of virus occurrence and effects of virus infection upon cellular uRrastructure in a local lesion host and a systemic host. MATERIALS AND METHODS
Greenhouse-grown bean plants (Phaseolus vulgaris L., cvs. 'Cherokee Wax' and 'Pinto') were used throughout this study. BPMV produced local lesions in Pinto bean and systemic mottling in Cherokee Wax bean. The first true leaves (first node above cotyledons) of both varieties of bean plants were mechanically inoculated with sap extracted from infected Cherokee Wax bean plants. 1Supported ia part by Cooperative States Research Services Grant No. 816-15-16.
Specimens were prepared for electron microscopy as described previously (Kim and Fulton, 1971). For the study of local lesion infection, pieces of lesions 1-2 mm ~ were taken from the inoculated primary leaves of Pinto bean. The first recognizable local lesions appeared 3 days after inoculation. For each sequential study, lesions were taken from the same inoculated leaf 3, 4, 5, 6, and 7 days after inoculation. For the study of systemic infection, similar samples were taken from the central leaflet of the first trifoliolate (second node above cotyledons) leaf of Cherokee Wax bean 6, 7, 8, 9, 10, 11, 12, 13, and 24 days after inoculation. Seven days after inoculation, the first trifoliolate leaves showed vein clearing, followed by mottling 9 days after inoculation. In all cases, appropriate healthy control leaves were sampled at the same times as infected leaves. Four separate samplings were made during this study. BPhIV was purified by a method of Bancroft (1962), and top, middle, and bottom components were separated by sucrose gradient eentrifugation. The bottom component was uRracentrifuged to produce a compact pellet, which was then treated in
112 Copyright © 1972 by Academic Press, Inc. All rit~'htsof reproduction in any form reserved.
BPMV-INFECTED PLANT CELL ULTRASTRUCTURE a manner similar to leaf tissue in preparing thin sections. RESULTS Location of Virus Particles
In cells of the systemically infected Cherokee Wax bean leaves, virus particles were observed 6 days after inoculation and appeared to increase in amount up to 8 days after inoculation. This coincided with the first evidence of vein:clearing in the trifoliolate leaves. Mottling appeared later; fewer virus particles were observed in cells of all samples taken more than 8 days after inoculation. Virus particles in the ground cytoplasm were difficult to distinguish from ribosomes, especially if they were scattered randomly.
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Spherical particles resembling ribosomes were, however, often densely packed and were surrounded by a loose membrane. These were interpreted as BPFIV since no such structures were observed in the cytoplasm of uninfected leaf cells. In 7-day and older samples, virus particles corresponding in size (25-28 nm) to the particles of a purified, embedded, and stained preparation (Fig. 1B) were observed in the central vacuole, either scattered or contained within a membranous sac (Fig. 1A). Some membranous sacs contained honeycomblike bundles of spheres (13-16 nm) with empty centers in addition to normally stained particles (Fig. 1A). Although the honeycomblike bundles and virus particles occurred in the same sac they were not intermingled. In
Key to figure abbreviations: CH, chloroplast ; Cr, cristae; CW, cell wall; ER, endoplasmic reticulum ; F1, filamentous inclusion; HS, honeycomblike spheres; M, membrane; Mi, mitochondria; N, nucleus; ST, sieve tube; SW, secondary cell wall; Ve, membranous vesicles; VP, virus particles. FIG. 1. (A) Two honeyeomblike bundles of spheres bounded by a membrane in the central vacuole of a Cherokee Wax bean leaf cell 6 days after inoculation. Virus particles are not intermingled with honeycomblike spheres even though they occur in the same sac. (B) BPMV particles in purified, embedded, and sectioned preparation.
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KIM ARrD FULTON addition to the occurrence within the cells, virus particles were also noted in intercellular spaces. Virus particles were present in all types of cells: epidermal, mesophyll, and all the component cells of vascular bundles. In matured sieve elements, particles were either closely packed and enclosed in loose membranes or in crystals (Fig. 2). In xylem tracheal elements, particles were not enclosed by a membrane but were closely associated with the surfaces of primary and secondary cell walls (Fig. 3). The lumen of the matured sieve elements in systemically infected Cherokee Wax bean leaves was the only place where cryslals of virus particles were observed. In the local~lesion host, Pinto bean, the location of virus particles was similar to the systemically infected host except that tubules with particles were not embedded in the cell wall (Kim and Fulton, 1971). The honeyeomblike masses of spheres were not evident in the vacuole. Three days after inoculation virus particles were not observed in the vacuole, but in all later stages of infection, ~they were observed in the central vacuole in a heavily aggregated form. There was no evidence of virus particles in nuclei, mitoehondria, or chloroplasts of locally or systemically infected hosts. Effects on Cell Ultrastrueture The most noticeable evidence of abnormal activity in infected cells of both systemically infected and local lesion hosts was an area of dense cytoplasm in the vicinity of the nucleus. In most cases this area bulged into the central vacuole. The area was composed of a great number of spherical vesicles of various sizes (Fig. 4A, B). Only one area of this type was observed in any cell. Such areas were bordered by several long segments of rough endoplasmic retieuhim (Fig. 4A) and were FIG. 2. Virus particles in crystalline form in sieve element of Cherokee Wax bean leaf 6 days after inoculation. FIG. 3. BPMV particles are closely associated with the surface of primary and secondary cell walls (arrows) of a xylem tracheal element in Cherokee Wax bean leaf 7 days after inoculatiom
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FIG. 5. (A) The formation of concentric membrane aggregate presumably from smooth endoplasmic reticuhm in Cherokee Wax bean leaf cells 7 days after inoculatiou with BPMV. (B) An increase of rough endoplasmic reticuhm with a deep circular bending which results in a ring. Transversely sectioned tubules with virus particles between the plasmalemma and the cell wall are also showing (arrows). From a locally infected Pinto bean leaf cell.
devoid of larger organelles, such as mitochondria or chloroplasts. In other areas of the cytoplasm of systemically infected cells, smooth endoplasmic reticulum increased in amount, and concentrically arranged membrane aggregates were often noted (Fig. 5A). In cells of the local lesion host, an increase of rough endoplasmic reticulum was very evident in the cytoplasm. The cisternae of this endoplasmic reticulum were bent into a ring containing some cytoplasmic components (Fig. 5B). A filamentous inclusion was found in the cytoplasm of all types of cells in both systemic and local lesion hosts. In longitudinal section i~ appeared as bundles of fibers or sectioned sheets (Fig. 6A) and in transverse section as a central area of sectioned fibers surrounded by concentrically arranged sheets (Fig. 6B). Rough endoplasmic reticulure was often closely associated with or aligned between the bundles of the filamentous inclusion (Fig. 6A). Individual filaments 100-120 A in diameter and sheets of comparable thickness were not as electron dense as virus particles. Four types of modified mitochondria were observed in systemically infected cells: (1) Some were pleomorphic and usually in the shape of a horseshoe or arch (Fig. 7). (2) The mitochondrial matrix contained concentrically arranged, thin, membranous laminae which appeared to have originated from either the inner or both the inner and outer mitochondrial membranes (Fig. 8A, B). Dark granular material was often contained in the center of the membrane aggregate. (3) Osmiophllic globules, similar to cytoplasmic lipid globules and varying in size, shape, and number, occurred in the matrix of this type of mitochondrion (Fig. 9). (4) Occasionally mitochondria were noted containing an electron opaque body bound by a membrane (Fig. I0). In addition to these four types of abnormal mitochondria, apparently normal mitochondria were present. Like those observed in healthy cells, they were either spherical or rod shaped, bounded by typical double unit membranes and containing tubulelike cristae without fixed arrangements and intramitochondrial granules. The greatest number of abnormal mito-
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FIG. 6. (A) Bundles of filamentous inclusion bodies sectioned somewhat longitudinally in the vicinity of a vesieulated area. Strands of rough endoplasmie retieulum are closely associated with or between the bundles of the filamentous inclusion. (B) Somewhat transversely sectioned filamentous inclusions which appear as a central area of sectioned fibers surrounded by concentrically arranged sheets. Both A and B are from Cherokee Wax bean leaf cells 8 days after inoculation with BPMV. ehondria was noted 6-8 days after inoculation. A t this stage, approximately 50 % of the mitochondria were of the four abnormal types. The relative n u m b e r of abnormal mitochondria decreased with time of sampiing and were only occasionally noted 24 days after inoculation. There was a striking increase in the number of mitochondria in the local-lesion host, Pinto bean. Five days after inoculation, infected cells averaged approximately twice as m a n y as control cells. Only a few abnormal mitochondria were noted. These were identical to the fourth type mentioned above or were of a fifth type identified b y the occurrence of thin, curved osmiophilie bands in the matrices (Fig. 11). DISCUSSION I t seems logical to assume t h a t virus particles in matured xylem tracheal elements
or phloem sieve tubes were synthesized during the i m m a t u r e stages of these cells. The abundance of B P M V particles in undifferentiated phloem cells m a y support this possibility. However, this does not explain the virus particles occurring in the intercellular spaces. I t would seem t h a t virus particles could accumulate in such an area only b y active m o v e m e n t from living ceils, probably through plasmadesmata. A similar phenomenon m a y account for the particles in the matured sieve element, but not for those in the tracheal elements, since there are no plasmadesmata between matured trachea] elements and p a r e n c h y m a cells. Paliwal (1970) found a large n u m b e r of the particles of bromegrass mosaic virus in the intercelhrlar spaces of mesophyll cells and the vascular bundle sheath. I-Ie discussed the possibility t h a t virions might be assembled there.
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FIG. 7. Horseshoe or arch-shaped mitochondria in a Cherokee Wax bean leaf cell 7 days afterinoculation with BPMV. Intramitochondrial granules are also present (arrows). FIG. 8. (A) Concentrically arranged membranous lamina in a mitochondrion which appear to have originated from the mitochondrial envelope (see arrows). Dark granular materials are present in the center of the lamina. Cristae are sparsely distributed or grouped creating the translucent area. (B) Concentric membrane layers appear detached from the mitochondrial envelope and are in the center of the matrix. Both figures are from Cherokee Wax bean leaf cell 7 days after inoculation with BPMV.
It is of interest to note that the crystals of BPMV particles occurred only in matured sieve elements and were completely absent in any other cell type. Some other icosahedral viruses such as cowpea strain of southern bean mosaic virus (Weintraub and IIagetli,
1970) a n d bromegrass mosaic v i r u s (Paliwal, 1970), were f o u n d readily i n crystal form in t h e c y t o p l a s m of a n y infected cell. T h e particles of B P M V seem to be c o n c e n t r a t e d e n o u g h to form crystals i n c y t o p l a s m a n d vacuoles if the s a t u r a t i o n of these areas with
BPMV-INFECTED PLANT CELL ULTRASTI~UCTURE
119
~'I~. 9. (A) Present in a mitoehondrial matrix is an osmiophilie globule which is similar to the cytoplasmic lipid globules (see arrows). FI~. 10. An electron-opaque granular body bounded by a membrane from a Cherokee Wax bean leaf cell 8 days after inoculation with BPM¥. ~'Ia. 11. Two mitoehondria containing a thin, curved, osmiophilie band in their matrices in Pinto bean leaf cell 3 days after inoculation. particles is a factor in crystalline arrangement of virions (Paliwal, 1970). De Zoeten and Gaard (1969) observed that tomato ringspot virus particles were found as paracrystalline inclusions in infected Datura stramo'nium L. leaf cells, but not in Cucumis sativus L. They suggested that there is a
virus--host interaction which is a requirement for crystal formation. The occurrence of crystals of B P M V only in matured sieve elements m a y also suggest that there is a certain intracellular environment which favors the formation of virus crystals. The occurrence of the honeycomblike
120
K I M AND F U L T O N
masses of spheres in the vacuoles of cells of the systemically infected host may be noninfective protein shells or "top component" (Bancroft, 1962). Their close association with virions within the membranous sac supports this view, but their size is somewhat smaIler, unless the fixation, embedding, and staining can account for the size difference. The dense area of cytoplasm with a large amount of vesicles appears to be the same area that has been termed the "inclusion body" (Roberts and Harrison, 1970; van der Scheer and Groenewegen, 1971). It would seem desirable to reserve the term inclusion to refer to materials which accumulate as a result of virus infection, e.g., the filamentous inclusion. The dense area is composed of cytoplasmic components modified so that the membrane system is increased greatly and vesicles are evident. These features suggest that this may be a metabolically active area in the cell which is analogous to the area which has been termed the "viro~ plasm" (Gaylord and NIelnick, 1953) or "x-bodies" (Fujisawa et al., 1967) even though no accumulation of virus particles was associated with this area. The various structural changes which were recorded in this study emphasize the abnormal activity associated with virus infection. Proliferation of the membrane system both in the vesiculated area and in increased amounts of smooth and rough endoplasmic reticuhim suggest increased cellular activity. The increased number of tubules with and/or without virus particles (Kim and Fulton, 1971) as well as some changes in mitochondria also indicate the proliferation of the membrane system in infected cells. The mitochondrial changes are of interest. In only a few cases have mitoehondrial changes been related to virus infection. Weintraub and Ragetli (1971) found hypertrophied mitochondria associated with an apple virus. Weintraub et al. (1964) also recorded an increase in number of mitochondria in cells of the local lesion host, Nicotiana glutinosa, infected with tobacco mosaic virus but no increase in systemically infected cells of Nicotiana tabacum. A similar reaction of local lesion and systemic hosts was recorded in this study. The fourth type of abnormal mitochondria recorded in this paper was
observed by Weintraub and Ragetli (1970) in eowpea leaf cells infected with southern bean mosaic virus. The same type of abnormal mitochondria is evident in Fig. 3 of a paper by Harrison and Roberts (1971) although no mention is made of this. This suggests that this type of abnormal mitochondria may be more commonly associated with virus infection than has been noted. The filamentous inclusions found in this study differ from P-protein and X-tubules described by Esau and Cronshaw (1967) in morphology, size~ and cell type. P-proteins and X-tubules found in TMV infected tobacco leaves were 230 A_ and 280 A in diameter, respectively, while o the filaments in this study were 100-120 A. The individual filaments are not tubular as in P-protein and X-tubules. P-proteins occur only in sieve elements and phloem-related parenchyma cells while the BPMV-induced filamentous inclusion occurs in all types of infected cells. Filaments were not as electron dense as virus particles suggesting they are composed mainly of protein. Endoplasmic reticulum associated with filaments suggests that it may be involved in the synthesis of the inclusion. The material presented in this paper as well as other studies which have appeared recently (Kim and Fulton, 1971; Roberts and Harrison, 1970; van der Scheer and Groenewegen, 1971) dispels the impression that small, polyhedral plant viruses (25-30 nm) cause only limited cytological changes in infected cells (Matsui and Yamag~chi, 1966). In this study the striking changes were most evident at a very early stage of infection in a systemically infected host. Cells seemed to recover from these changes at later stages of infection even though the tissue showed good symptoms of disease. REFERENCES BA~CaOFrr, J. B. (1962). Purification and properties of bean pod mottle virus and associated centrifugal and eleetrophoretie components. Virology 16, 4!9-427. DE ZOETEN, G. A., and GAAItD, G., (1969). Possibilities for inter- and intracelhflar t r a n s l o e a t i o n of some icosahedral p l a n t viruses. J. Ceil Biol. 40,814-823.
BPMV-INFECTED PLANT CELL ULTRASTRUCTURE ESAU, K. and CRONSI-IAW,J. (1967). Tubular components in cells of healthy and tobacco mosaic virus-infected Nicotiana. Virology 33, 26-35.
FUJISAWA, I., RUBIO-HUERTOS,M., ~/IATSUI, C., and YAMAGUCHI,A. (1967). Intracellular appearance of cauliflower mosaic virus particle. Phytopathology 57, 1130-1132. GAYLORD, W. I-I.,and MELNICK, J. L. (1953). Intracellular forms of poxviruses as shown by the electron microscope (Vaccinia, ectromelia, molluscum contagiosum). J. Exp. Med. 98, 157-172. HARRISON, B. D., and ROBERTS, I. M. (1971). Pinwheels and crystalline structures induced by Atropa mild mosaic virus, a plant virus with particles 925 nm. long. J. Gen. Virol. 10, 71-78. KIM, K. S., and FULTON, J. P. (1971). Tubules with viruslike particles in leaf cells infected with bean pod mottle virus. Virology 43,329-337. MATSUI, C., and YAMAGUCHI, A. (1966). Some aspects of plant viruses in situ. Advan. Virus Res. 12, 127-174. PALIWAL, Y. C. (1970). Electron microscopy of
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bromegrass mosaic virus in infected leaves. J. Ultrastruct. Res. 39, 491-502. ROBERTS, I. M., and HARRISON, B. D. (1970). Inclusion bodies and tubular structures in Chenopodium amaranticolor plants infected with strawberry latent ringspot virus. J. Gen. Virol. 7, 47-54. VAN DER SCHEER, C., and GROENEWEOEN, J. (1971). Structures in cells of Vigna unguiculata infected with cowpea mosaic virus. Virology 46, 493-497. WEINTRAUB, ~V~., and I=~AGETLI,H. W. J. (1970). Electron microscopy of the bean and cowpea strains of southern bean mosaic virus within leaf cells. J. Ultrastruct. Res. 32,167-189. W~INTnAUB,M., and I~AGETLI,H. W. J. (1971). A mitochondrial disease of leaf cells infected with an apple virus. J. Ultraslruct. Res. 36,669-693. WEINTRAUB, M., RAGETLI, H. W. J., and I)WURAZNA,M. M. (1964). Studies on the metabolism of leaves with localized virus infections. Mitochondrial activity in TMV-infected Nicoliana glutinosa L. Can. Y. Bot. 42,541-545.