Pinwheels and Crystalline Structures Induced by Atropa Mild Mosaic Virus, a Plant Virus with Particles 925 nm. Long

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1 J. gen. Virol. (I970, xo, Printed in Great BritMn 7I Pinwheels and Crystalline Structures Induced by Atropa Mild Mosaic Virus, a Plant Virus with Particles 925 nm. Long By B. D. HARRISON AND I. M. ROBERTS Scottish Horticultural Research Institute, Invergowrie, Dundee (Accepted I5 September 197o ) SUMMARY Atropa mild mosaic virus is transmissible from tobacco to tobacco, both by green peach aphids and by inoculation of sap. It causes mosaic symptoms and has elongated slightly flexuous particles with a modal length of about 925 nm. It induces the formation of pinwheels resembling those elicited by viruses of the potato Y group (potyviruses), and infected cells also contain crystalline structures resembling hexagonal arrays of tubules. INTRODUCTION There is increasing evidence that viruses with close affinities can have similar ultrastructural effects on infected cells. Thus the potato Y group of viruses (Brandes & Bercks, 1965), here referred to as 'potyviruses', characteristically induce the formation of 'pinwheels', which have otherwise been found only in plants infected with the mite-transmitted wheat streak mosaic virus (Shepard & Carroll, I967). These pinwheels were noted by Yamaguchi, Kikumoto & Matsui (I963), but not well understood and named until the work of Edwardson (I966), Purcifull & Edwardson (I967) and Edwardson, Purcifull & Christie (1968). They were found in cells infected with seven potyviruses and shown to consist of a series of plates radiating from a central axis and mostly curved in planes perpendicular to this axis. The plates can be digested by several proteolytic enzymes (Shepard, 1968; Weintraub & Ragetli, 1968) and therefore seem to contain protein which, moreover, is unrelated serologically to that in the virus particles (Shepard & Shalla, I969). In work started to study the cytological effects of viruses in other groups, we unexpectedly found pinwheels in cells infected with belladonna mottle virus, which has isometric particles and is allied to viruses of the turnip yellow mosaic group (Paul et al. I968; Gibbs, 1969). Further work showed that our virus culture also contained a second and apparently new virus, with elongated particles. This has been named Atropa mild mosaic virus (Bode, Brandes & Paul, 1969; Dr O. Bode, personal communication). We now show that Atropa mild mosaic virus has particles considerably longer than those of potyviruses (73o to 8oo nm.) but that, like them, it induces the formation of pinwheels. METHODS Infected tissue from young or well expanded leaves of tobacco plants (Nicotiana tabacum cv. Xanthi-nc) showing mosaic symptoms, was fixed, embedded, sectioned and stained by the 'modified method' of Harrison, Stefanac & Roberts (197o).

2 72 B. D. H A R R I S O N A N D I. M. R O B E R T S Fig. I. (a) Particles of Atropa mild mosaic varus m phosphotungstate-treated sap from inoculated tobacco leaves. (b) Plate, presumably derived from a pinwheel, seen in phosphotungstate-treated tobacco sap. (e) Portion of plate at high magnification.

3 Pinwheels and crystalline structures 73 RESULTS AND DISCUSSION In the first experiments, tissue was examined from leaves systemically infected with both belladonna mottle virus and Atropa mild mosaic virus. The cells contained abnormal chloroplasts, which had marginal vesicles of various sizes, similar to those found in cells infected with turnip yellow mosaic virus (Chalcroft & Matthews, I966). Many of the cells also contained large masses of cytoplasm, in which pinwheels and crystalline structures could be seen in addition to the isometric particles of belladonna mottle virus. When sap from such plants was mixed with 2 ~o sodium phosphotungstate and examined in the electron microscope, the isometric particles of belladonna mottle virus were found together with the slightly flexuous elongated particles of Atropa mild mosaic virus. l I I I 40 3O u Q. "6 E < > 90 Length (arbitrary units) Fig. 2, Particle length distribution of Atropa mild mosaic virus in sap from inoculated tobacco leaves 042 particles). One unit -- about I2.6 rim. Modal length = 925 nan. Atropa mild mosaic virus was freed from belladonna mottle virus by transmitting it to fresh Xanthi tobacco seedlings, using aphids (Myzus persicae Sulz.) fed for 5 min. on infected plants and then kept for 2 hr on healthy ones. When transmitted to further plants by inoculation of sap it produced mosaic diseases in Xanthi tobacco and Datura stramonium, but no symptoms in Chenopodium amaranticolor. The lengths of particles seen in phosphotungstate-treated leaf sap (Fig. I a) showed a sharply defined modal value of about 925 nm, (Fig. 2), considerably longer than that of potyviruses but close to the value of about 9oo nm. quoted by Bode et al. (1969). A similar value was obtained when the lengths of particles of Atropa mild mosaic and tobacco rattle viruses were compared by electron microscopy of mixed preparations. Preparations from plants infected with Atropa mild mosaic virus also

4 74 B. D. H A R R I S O N A N D I. M. R O B E R T S Fig. 3 to 5. Sections of tobacco leaves systemically infected with Atropa mild mosaic virus. Fig. 3. Section of infected palisade cell showing pinwheels (P) in transverse and longitudinal section, and crystalline material (C).

5 Pinwheels and crystalline structures 75 contained plates (Fig. ~b, c) showing obvious longitudinal lines about 5 nm. apart and less obvious transverse markings at a spacing of 3 nm. These plates therefore appear similar to those derived from the pinwheels induced by watermelon mosaic (Edwardson et al. ~968) and other potyviruses. Fig. 3 shows a large area of cytoplasm containing several pinwheels, some in cross-section and others in longitudinal section. They closely resemble the pinwheels induced by water- Fig. 4. (a) Pinwheel, showingcentral axis and radiating curved plates. (b) Mass of virus-likeparticles. (c) Longitudinal section of parts of pinwheels showing association of the plates with the endoplasmic reticulum (arrows).

6 76 B. D. HARRISON AND I. M. ROBERTS melon mosaic virus and, when examined in cross-section (Fig. 4a), dark spots of a similar diameter to that of particles of Atropa mild mosaic virus can be seen in contact with several of the plates. In longitudinal section, the ends of some of the pinwheel plates seem associated with the endoplasmic reticulum (Fig. 4c), a feature found also for two other potyviruses, pokeweed mosaic virus (Kim & Fulton, ~969) and maize dwarf mosaic virus (Krass & Ford, t969). In both systemically infected and inoculated leaves some cells contained small areas of vesiculate material not seen in virus-free plants; virus-like particles were visible in some regions of the cytoplasm, occurring either individually or in masses (Fig. 4b). Atropa mild mosaic virus had no obvious effect on the chloroplasts. Fig. 5. (a) Transverse section through crystalline structures seen in cytoplasm, showing honeycomb arrangement (arrow). (b) Longitudinal section through a similar crystalline structure.

7 Pinwheels and crystalline structures 77 Many of the infected cells contained crystalline structures (Fig. 3, 5a, b) apparently different from any previously reported in cells infected with plant viruses. They were often numerous in cells that contained them and were found only in the areas containing pinwheels. They mostly measured I5O to 3oo nm. in cross-section, and many had a central unstained, irregular spot; their lengths ranged up to 2 #m. When the crystalline material was examined at higher magnification it appeared in cross-section as a honeycomb-like arrangement of hexagonal units with darkly staining walls (Fig. 5 a). The centre-to-centre distance of these units was about I5 nm. In longitudinal section the crystalline material showed densely staining, parallel longitudinal bands at a spacing of about I2 nm. (Fig. 5b), depending on the angle of section. It seems unlikely that these patterns represent aggregates of virus filaments like those found in extracts of blue lupin infected with watermelon mosaic virus (Purcifull, Edwardson & Christie, 1968 ). They are interpreted more plausibly as hexagonally arrayed tubules of unknown composition. Although a possible interpretation of our results might be that our culture of Atropa mild mosaic virus was contaminated with potato virus Y or another potyvirus, we think this unlikely because our culture caused no lesions in Chenopodium amaranticolor and there was no peak at about 75 o nm. in the particle length distribution (Fig. 2). Also, pinwheels and crystalline structures were found in plants inoculated with preparations which had been diluted to IO -7 or heated to over 7o ; both treatments which should have resulted in loss of infectivity by most potyviruses. Like potyviruses, Atropa mild mosaic virus is transmissible by aphids in brief feeding periods, is transmissible by inoculation of sap, causes mosaic symptoms, has slightly flexuous elongated particles and induces pinwheel formation. It differs from potyviruses in reaching an apparently higher concentration in leaf sap and in the length of its particles. The description of the potyvirus group may need to be modified to include Atropa mild mosaic virus but more information is required about the properties and serological affinities of the new virus before a decision on this can be reached. At present it is not clear whether Atropa mild mosaic virus is best considered as a member of a previously undetected cluster of viruses distinct from the potyvirus group, or whether the group is larger than previously recognized, with Atropa mild mosaic virus at one extremity. We thank Miss A. Kinninmonth for technical assistance. REFERENCES BODE, O., BRANDES, J. & PAUL, n. L. (I969). Untersuchungen fiber ein neues, langgestrecktes Virus aus Atropa belladonna. Biologische Bunde~anstalt fiir Land- und Forstwirtschaft in Berlin und Braunschweig, Jahresbericht 1968, p. A6I. BRANDES, S. & BERCKS, R. (I965). Gross morphology and serology as a basis for classification of elongated plant viruses. Advances in Virus Research i1, I. CHALCROFT, S. *, MATTHEWS, R. E. F. (1966). Cytological changes induced by turnip yellow mosaic virus in Chinese cabbage leaves. Virology 28, 555. EDWARDSON, ~. R. (I966). Electron microscopy of cytoplasmic inclusions in cells infected with rod-shaped viruses. American Journal of Botany 53, 359. EDWARDSON, J. R., PURCIFULL, D. E. & CHRISTIE, R. G. 0968). Structure of cytoplasmic inclusions in plants infected with rod-shaped viruses. Virology 34, 25o. GIBBS, A. J. (I969). Plant virus classification. Advances in Virus Research 14, 263. HARRISON, B. D., STEFANAC, Z. & ROBERTS, I. m. (1970). Role of mitochondria in the formation of X-bodies in cells of Nicotiana clevelandii infected by tobacco rattle viruses. Journal of General Virology 6, ] 27. KIM, K. S. & FULTON, J. P. (I969). Electron microscopy of pokeweed leaf cells infected with pokeweed mosaic virus. Virology 37, 297. KRASS, C. J. & FORD, R. E. (1969). Ultrastructure of corn systemically infected with maize dwarf mosaic virus. Phytopathology 59, VrR IO

8 78 B.D. HARRISON AND I. M. ROBERTS PAUL, H. L., BODE, O., JANKULOWA, M. & BRANDES, J. (I968). Untersuchungen fiber ein neues isometrisches Virus aus Atropa belladonna L. I. Symptomatologie, Reinigung, Morphologie, physikalische und chemische Eigenschaften. Phytopathologische Zeitsehrift 6i, 342. PURCIFULL, D. E. & EDWARDSON, J. R. (I967). Watermelon mosaic virus: tubular inclusions in pumpkin leaves and aggregates in leaf extracts. Virology 32, 393- PURCIFULL, D. E., EDWARDSON, J. R. & CHRISTIE, S. R. (1968). Aggregated filaments in extracts from Lupinus infected with watermelon mosaic virus. Virology 35, 478. SHEPARD, J. F. (I968). Electron microscopy of subtilisin-treated tobacco etch virus nuclear and cytoplasmic inclusions. Virology 36, 2o. SHEPARD, J. F. & CARROLL, T. W. (I967). Electron microscopy of wheat streak mosaic virus particles in infected plar~t cells. Journal of Ultrastructure Research zt, t45. SHEPARD, J. V. & SHALLA, T. A. 0969). Tobacco etch virus cylindrical inclusions: antigenically unrelated to the causal virus. Virology 38, t85. WEINTr~AUB, M. ~ RA~ETLI, H. W. S. (I968). Intracellular characterization of bean yellow mosaic virus-induced inclusions by differential enzyme digestion. Journal of Cell Biology 38, 316. YAMAGUCHI, A., KIKUMOTO, T. & MATSUI, C. (1963). Electron microscopy of elongated particles associated with tulip mosaic. Virology 2o, I43. (Received 29 June 197o)