J. gen. Virl. (I976), 33, 237-248 Printed in Great Britain 237 The Multiplicatin Cycle f Tbacc Rattle Virus in Tbacc Mesphyll Prtplasts By B. D. HARRISON, S. KUBO,* D. J. ROBINSON AND AILEEN M. HUTCHESON Scttish Hrticultural Research Institute, Invergwrie, Dundee, U.K. (Accepted t4 July I976) SUMMARY When prtplasts inculated with tbacc rattle virus (TRV) were sampled after successive intervals at 22 r 25 C in light, the fllwing sequence f events was detected. Infective TRV-RNA appeared at 7 h and apprached its maximum cncentratin by ~2 h. Antigen detected by staining with flurescent antibdy t TRV particles, infective TRV nucleprtein, and bth lng and shrt TRV particles were prduced by 9 h, and increased up t abut 4 h. Infective RNA synthesized at abut 9 h was apparently incrprated int nucleprtein particles abut 4 t 5 h later. Up t I6 h, lng virus particles utnumbered shrt, but their prprtin steadily decreased until 40 h, when it stabilized at abut 20 ~. Half the final yield f lng particles was prduced by 22 h, and half that f shrt particles by 30 h. Nearly all the lng virus particles were assciated with mitchndria at all times, but after the first few hurs the shrt nes were predminantly free in the cytplasm. Inculating shrt virus particles up t 8 h befre, r 4 h after lng particles instead f simultaneusly, did nt greatly affect the number f antigen-prducing infectins prduced, but smewhat decreased the prprtin f infective RNA incrprated int nucleprtein. These results prvide supprt fr the idea that separatin in time r place within the cell f the synthesis, translatin r assembly int nucleprtein f the different genme parts may be ne f the advantages f multipartite genmes as a mdus vivendi fr plant viruses. INTRODUCTION The great advantage f prtplast systems in plant virlgy is that the prcesses invlved in infectin and replicatin ccur relatively synchrnusly in a large majrity f the prtplasts in a ppulatin (Takebe, I975). In previus wrk with tbacc rattle virus (TRV) and tbacc mesphyll prtplasts we have studied the mechanism f infectin and established a time scale fr sme f the main features f virus multiplicatin (Kub et al. r975 b, I976). Fr example, we estimated that abut 3 lng particles and 85 shrt particles f TRV were taken up per prtplast at the IDs0 fr infectins in which virus cat prtein is prduced, and that abut I 6 prgeny virus particles per infected prtplast accumulated during incubatin fr 2 days at 22 C. In this paper we examine the virus multiplicatin cycle in mre detail. * Permanent address: Central Research Institute, Japan Tbacc and Salt Public Crpratin, Ykhama, Japan.
238 B. D. HARRISON AND OTHERS METHODS Experimental cnditins. The plants used as surces f prtplasts, preparatin f inculum, prcedures fr preparatin, inculatin by the indirect methd using phsphate buffer and culture f prtplasts, staining with flurescent antibdy t virus particles and titratin f antigen using antibdy-sensitized latex, were all essentially as described by Kub, Harrisn & Barker 0975a) and Kub et al. 0975b, ~976). In sme experiments, prtplasts were stred vernight at 4 C in 0"7 M-mannitl befre inculatin. They were incubated at 25 C after inculatin unless therwise stated. Infectivity assays. Samples cntaining virus nucleprtein r RNA were prepared and infectivity was assayed in Chenpdium amaranticlr as described by Kub et al. 0975b)- Each sample was assayed at three dilutins separated by factrs f five. The ttal numbers f lesins prduced by all samples at the three dilutins were cmpared. These numbers usually differed by a factr f 3"5 t 4"5 fr a fivefld dilutin, and the bserved factrs were used t cnvert the three lesin ttals fr ne sample t equivalent figures, which were averaged and multiplied by the actual dilutin t btain the relative infectivity f the sample. Prprtins f lng and shrt virus particles. T determine the rati f lng t shrt particles f prgeny virus, '5 t 2 millin infected prtplasts were sedimented in 2 min at 8 g, resuspended in I ml 'i7 M-phsphate buffer, ph 7"3, disrupted in a grund-glass tissue grinder and the extract stred at -2 C fr at least 3 days. Each sample was then thawed, centrifuged fr 3 rain at 9g, and the supernatant fluid layered n t '5 ml I5 ~ sucrse in phsphate buffer in a tube fr a Beckman SW 39 rtr. Each tube was then carefully filled with mre buffer, centrifuged fr x5 min at 36 rev/min, all but 2 drps f the supernatant fluid drawn ff and the sediment resuspended in this small vlume. Carbncated electrn micrscpe grids were first treated with a drp f.2 ~ bacitracin, which was drained ff and replaced by a drp f virus suspensin. This was drained ff after 15 t 30 s, and a drp f 2 ~ ammnium mlybdate, ph 6., placed n the grid fr 3 t 6 s, then drained ff and the grid dried. The specimens prduced in this way cntained, in additin t virus particles, cnsiderable amunts f phytferritin, but this did nt bscure the virus particles. At least tw grids f each sample were examined and the length classes f 5 t I particles/sample were recrded. Electrn micrscpy f ultrathin sectins. Prtplasts were fixed, embedded first in an agar blck and then in Araldite, stained and sectined essentially as described by Kub et al. (I976). At least tw blcks f each sample were sectined and examined. RESULTS Accumulatin f virus RNA, antigen and nucleprtein T study the accumulatin f virus prducts by a cmbinatin f methds, batches f prtplasts were harvested at intervals after inculatin and divided int three parts. The prtplasts in ne part were stained with flurescent antibdy t determine the prprtin cntaining TRV antigen. The secnd part was used t assay the amunt f TRV antigen using antibdy-sensitized latex and t assess the relative amunt f infective nucleprtein by lcal-lesin tests. RNA was extracted frm the third part and its infectivity assayed. The results indicate that virus antigen and nucleprtein infectivity increased in parallel, suggesting that there was n substantial accumulatin f virus cat prtein ther than in nucleprtein particles (Fig. I). Prgeny virus was detected ~ I h after inculatin by bth
Multiplicatin cycle f TR V in prtplasts 239 3-0 I! I 2-0,.-.1 1. It A I I I 20 40 60 Time after inculatin (h) Fig. i. Accumulatin f prducts f tbacc rattle virus replicatin in prtplasts. Q--O, prtplasts staining with flurescent antibdy t virus; A--A, virus antigen assayed using antibdysensitized latex; Ill--m, infective nucleprtein; O--O, infective RNA. In samples taken i h after inculatin, n infective RNA, virus antigen r staining with antibdy was detected and nly 0"3 units f infective nucleprtein. Hwever, I.O was added t each f these values, fr cnvenience f pltting. 80 infectivity and flurescent antibdy tests, but the amunt was less than 5 ~ f the final yield, t little t be detected using antibdy-sensitized latex. The intracellular distributin f antigen seemed t change smewhat during the curse f infectin; during the first day flurescent antibdy staining was seen as small bright granules in many prtplasts, whereas later it seemed t ccur mre generally thrughut the cytplasm, with the chlrplasts standing ut as dark unstained areas in a stained matrix. The results in Fig. I als indicate that althugh nly 3 70 f the ttal infective nucleprtein accumulated during the first 11 h after inculatin, at least half the infective RNA was prduced then. This phenmenn was cnfirmed by sampling the prtplasts every 2 h during the early stages f infectin (Table 0- Virus RNA was detected 7 h after inculatin, whereas nucleprtein particles were nt detected until 9 h. When the five- t twentyfld greater infectivity f a given amunt f TRV-RNA in nucleprtein particles as cmpared with prtein-free preparatins (Harrisn & Nixn, I959) is taken int accunt, it is estimated that althugh half r mre f the maximum cntent f virus RNA is achieved during 12 h incubatin at 22 r 25 C, nly abut 2 70 is then in the frm f nucleprtein particles. The prprtin f infective RNA in nucleprtein particles 3 h after inculatin was estimated by cmparing the infectivity f RNA extracted directly frm prtplasts with that f RNA extracted frm frzen and thawed samples f prtplasts. The results indicated that in different experiments 5 t I ~ f the infective RNA was in nucleprtein particles. When infectivity is assayed, the results relate t the larger RNA species r the lnger
240 B. D. HARRISON AND OTHERS Table I. Accumulatin f infective RNA and nucleprtein particles in the early stages f tbacc rattle virus replicatin Prtplasts stained with Hurs flurescent Infectivity f prtplast extractst after antibdy r ~ inculatin* (~) RNA Nucleprtein I O I II 5 O 0'5 7 7 O 25 6 9 2'8 282 IO I2 36 3150 7[6 30 90 2120 26000 * Prtplasts were incubated at 22 C. I" Arbitrary units; figures fr RNA and nucleprtein are directly~cmparable. 1 I I I "~ 50 0 ----.. I I I 20 40 60 Time after inculatin (h) Fig. 2. Prprtins f virus particles f different lengths at intervals after inculatin. O--O, prtplasts staining with flurescent antibdy t virus; A--A, lng particles; II--II, shrt particles; (3--(3, intermediate length particles. nucleprtein particles f TRV, and further experiments were therefre made t find whether synthesis f lng and shrt nucleprtein particles is synchrnus. Batches f prtplasts, incubated at 22 C after inculatin, were harvested at intervals and samples used t estimate the percentage f prtplasts staining with flurescent antibdy, the relative infectivity f buffer extracts and the prprtins f lng, shrt and intermediate length particles f prgeny virus. At 15 t I6 h after inculatin there were mre lng particles than shrt and abut half the prtplasts stained with flurescent antibdy. Hwever, during the next day the prprtins f lng and intermediate particles decreased t their final values and virtually all the remaining prtplasts became stainable by flurescent antibdy (Fig. 2). The speed f the change in prprtins f particles f different lengths varied smewhat frm experiment t experiment but the trend was always similar. It was difficult, by the technique used, t btain enugh virus t give accurate estimates f the prprtins f lng and shrt particles in extracts frm prtplasts infected fr less than ]5 h, but in ne experiment a sample taken at I2 h yielded lo3 particles, f which 6I
Multiplicatin cycle f TRV in prtplasts 24I 1000!!! 500 Y I! I 20 40 60 Time after inculatin (h) Fig. 3. Accumulatin f virus nucleprtein at intervals after inculatin. Q--O, lng particle nucleprtein; A--A, shrt particle nucleprtein. were lng, 25 ~ shrt and ~4 ~ intermediate, as cmpared t figures f 56 ~ lng, 36 shrt and 8 ~ intermediate fr samples taken after ~5 t I6 h. This indicates that sme shrt particles are prduced quite early in infectin, with their prprtin increasing frm the earliest time tested up t 40 h. The relative numbers f shrt particles in each sample can be calculated by multiplying the relative cncentratin f lng particles by the determined rati f numbers f shrt t lng particles. The relative nucleprtein cncentratins (w/v) were then btained by dividing the relative numbers f shrt particles by 3"8 (Fig. 3). The mean figures fr tw similar experiments indicated that half the final cncentratin f lng particles was attained after 2I'5 h at 22 C, with the main perid f accumulatin being I5 t 28"5 h. The cmparable figures fr shrt particles are 29-5 h and 23. 5 t 37 h. This is evidence f a switch frm prductin f lng TRV particles t that f shrt nes. The intermediate length particles recrded in Fig. 2 may represent either nascent lng particles, errrs in synthesis r breakdwn prducts, r sme cmbinatin f these pssibilities. Hwever, if they are predminantly breakdwn prducts their cncentratin relative t that f lng particles might be expected t increase with time after inculatin, and if they represent errrs in synthesis but are stable, their relative cncentratin shuld be steady. In fact their number, expressed relative t the number f lng particles, decreased steadily frm 22 ~ at I2 h after inculatin t 7 ~ at 64 h. Thus the pssibility cannt be ruled ut that at least sme f the intermediate particles are frms f nrmal lng particles in the curse f assembly. Events bserved by electrn micrscpy Several experiments were dne in which batches f prtplasts were fixed at intervals after inculatin, fr ultrathin sectining. In sme experiments prgeny particles were detected in a very few prtplast prfiles at 9 h after inculatin, and in ther experiments at x I h. The particles, therefre, were first detectable at almst the same time that virus antigen culd be detected by staining with flurescent antibdy and that virus nucleprtein infectivity began t increase (9 h in mst experiments; Table 0- In general, electrn micrscpy was smewhat less sensitive fr detecting virus particles than the ther tw methds.
242 B. D. H A R R I S O N AND OTHERS Fig. Fig. Fig. Fig, 4 t 6. Electrn micrgraphs f ultrathin sectins f prtplasts I I h after inculatin. 4. Tw small grups f lng TRV particles assciated with mitchndria. 5. Tw shrt TRV particles at the surface f a mitchndrin. 6. Lng TRY particle free in the cytplasm. T a b l e 2. Occurrence and lcatin f tbacc rattle virus in prtplasts at intervals after inculatin Hurs after inculatin ~ prtplasts cntaining virus antigen ~ prtplast prfiles with prgeny virus mitchndrial prfiles with virus in viruscntaining prtplasts I 6 O 50 96 33 67 45 54 68 79 3 5 7 9 II 3O I'6 8"5 6O O 3 (30* 64 (I4) ~ prtplast prfiles with mitchndria having prtuberances * Figures in parentheses are numbers f prtplast prfiles examined. B t h lng a n d shrt virus particles were f u n d in sectins f p r t p l a s t s 9 t I I h after i n c u l a t i n (Fig. 4 t 6). The lng particles p r e d m i n a t e d, a n d nearly all were a r r a n g e d with their ends in cntact with m i t c h n d r i a (Fig. 4, 6). T h e y were usually f u n d side-by-side in small grups assciated with a small p a r t f the surface f a m i t c h n d r i n, a n d careful searching revealed that, in the few virus-cntaining p r t p l a s t s fund, the particles were assciated with m a n y f the m i t c h n d r i a (Table 2). The few shrt particles f u n d 9 t ~ I h after i n c u l a t i n were als m s t l y assciated with m i t c h n d r i a (Fig. 5), b u t p e r h a p s a few were free in the cytplasm. Thse n m i t c h n d r i a ccurred singly, n t in grups. W i t h increasing time after inculatin, the n u m b e r f p r t p l a s t s c n t a i n i n g virus a n d the n u m b e r f particles per virus-cntaining p r t p l a s t increased. The vast m a j r i t y f the lng particles were n the m i t c h n d r i a where they were p a c k e d tgether a n d sme f the
Multiplicatin cycle f TRV in prtplasts 243 Fig. 7. Ultrathin sectin f a prtplast at 30 h after inculatin shwing a grup f mitchndria surrunded by lng TRV particles. Shrt TRV particles are visible free in the cytplasm (arrws). encrusted mitchndria assumed an abnrmal shape, pssibly as a result (Fig. 7). Where tw mitchndria were clse tgether, lng virus particles appeared t link them in sme instances, but mre usually each had its wn fringe f lng virus particles (Fig. 7), cnfirming bservatins n TRV in plant tissue (Harrisn & Rberts, ~968) and suggesting that ne end f the particles attached mre readily than the ther. In sme instances, the particles culd be seen t tuch, but nt t pass thrugh, the uter mitchndrial membrane (Fig. 8). Particles were nt fund n the membranes f the plasmalemma, tnplast, endplasmic reticulum, chlrplast r nucleus. The number f shrt particles increased during the perid frm 12 t 4 8 h after inculatin; by 24 h there were cnsiderable numbers free in the cytplasm and by 48 h even mre. In general, fewer shrt particles than lng were visible in the vicinity f mitchndria, despite the predminance f shrt particles in extracts made x t 3 days after inculatin. Hwever, shrt particles are less easily discerned than lng nes by electrn micrscpy f sectins, and many f them ccurred in cytplasm
244 B. D. H A R R I S O N AND OTHERS Fig. 8 t i. Ultrathin sectins f prtplasts. Fig. 8. Mitchndrin in regin pr in ribsmes, 3 h after inculatin. Nte TRV particle assciated with uter mitchndrial membrane. Fig. 9- Mitchndrin with a prtuberance, I2 h after inculatin with n.y.-inactivated TRV. Fig. i. Part f chlrplast cntaining crystalline bdy, 3 h after inculatin.
Multiplicatin cycle f TRV in prtplasts 245 away frm mitchndria, s that micrgraphs such as Fig. 7 d nt give a prper impressin f the relative numbers f particles f the tw types. Occasinal shrt particles were fund n the mitchndria at all times and there were mre f them n mitchndria after 48 t 7z h than earlier. A few prtplasts at 24 h after inculatin, and mre at 7z h, had cytplasm cntaining a smaller cncentratin f ribsmes than nrmal. The mitchndria in these prtplasts had virus particles f varius lengths, including sme shrt nes, scattered n their surface (Fig. 8). Als, the cytplasm cntained virus particles f varius lengths and sme f the chlrplasts seemed t be degenerating. We think these prtplasts are degenerating, thugh nt necessarily as the result f virus infectin. Several f the mitchndria had prtuberances, which became mre cmmn with increasing time after inculatin (Table 2). Typically these ccurred where cristae were clse t the periphery f mitchndria. Hwever, similar changes ccurred in prtplasts inculated with virus previusly inactivated by u.v. radiatin (Fig. 9) and the prtuberances are therefre nt a respnse t virus infectin. At all times after inculatin, crystalline bdies were bserved within and clse t the surface f the chlrplasts f virus-infected and cntrl uninfected prtplasts (Fig. I ). These bdies seem t be identical with the structures prduced when excised virus-free Nictiana glutinsa leaves are kept in light, and which are cnsidered t result frm degeneratin caused by starvatin (Ragetli, Weintraub & L, I97). Separate inculatin f lng and shrt virus particles Fr these experiments, lng and shrt particles were separated by tw cycles f density gradient centrifugatin. The gradients cntained EDTA, which was later fund t decrease infectivity (D. J. Rbinsn & J. H. Raschk6, unpublished results) s that the prprtin f prtplasts infected by remixed lng and shrt particles was smaller than with cmparable cncentratins f unfractinated virus. When inculated t separate batches f prtplasts, the samples f shrt particles were virtually nn-infective and thse f lng particles induced very few antigen-prducing infectins but much infective RNA (Table 3; Kub et al. I975b), In preliminary experiments nt described here in detail, we have fund that the yield f infective RNA frm prtplasts inculated with lng particles smetimes decreases at lnger times after inculatin, presumably because the RNA remains susceptible t degradatin, and this is mre extensive in sme batches f prtplasts than in thers. The result f this effect is seen in the lw yield f infective RNA given by prtplasts inculated with lng particles alne in Expt. 2, Table 3. When the shrt particles were inculated 4 t 8 h befre the lng nes, at least as many antigen-prducing infectins resulted as when mixtures f the tw were inculated (Table 3). In these inculatins the number f shrt particles per ml was abut 2 times that f lng particles; hwever, the prtplasts were nt heavily saturated with shrt particles because a fivefld decrease in cncentratin f shrt particles in the inculum resulted in a similar decrease in antigen-prducing infectins. When the shrt particles were inculated up t 4 h after the lng nes, the prprtin f antigen-prducing prtplasts was still mre than half that resulting frm inculatin f mixed lng and shrt particles. Althugh the percentage f prtplasts synthesizing TRV nucleprtein was small in these experiments, the results indicate clearly that shrt particles can enter prtplasts independently f lng nes, and that such independent entry is abut as efficient fr inducing antigen-prducing infectins as when the pprtunity exists fr entry f the tw kinds f particle at the same time and site.
246 B. D. HARRISON AND OTHERS Table 3. Effect f inculating shrt particles befre r after lng nes Expt. I Expt. 2 Relative time f inculatin f Prtplasts Infectivity/i ~ prtplasts'j" shrt particles* flurescing1",- (h) (~) RNA Nucleprtein NP/RNA O~ 0 0 0 -- x 0"4 605 I6 0"026 --4 6"7 835 455 O'55 --2 5"I 580 474 0"82 O 5'5 380 34t O'9O O I-~ -- I39 -- +2 4"4 515 341 0.66 +4 3"5 33 I47 '45 ~ 0'5 x 0.2 I7I 35 0.20 --24 0'7 56 28 0'50 --8 i t-7 700 53I 0-76 -- 4 8. -- 1340 -- 8.] 529 ]5 2'0 * Prtplasts inculated with either lng r shrt particles were incubated in light (3000 lux) at 22 C, in '7 M-mannitl cntaining antibitics, and were washed with mannitl immediately befre the secnd inculatin. Unless therwise indicated, lng particles were inculated at zer time; x = shrt particles were nt inculated. t Prtplasts were incubated at 22 C and were sampled 2 days after inculatin; figures fr RNA and nncleprtein infectivity are nt directly cmparable. ~: Lng particles were nt inculated. Shrt particles at '04#g/ml; therwise at.2 #g/ml. Lng particles were at '4 #g/ml thrughut. When shrt particles were inculated 24 h befre lng nes, few prtplasts prduced virus antigen, and yields f infective RNA and nucleprtein were lw (Table 3). The small number f antibdy-prducing infectins and small amunt f infective RNA are attributed t inefficient infectin by the lng particles. This was expected, because prtplasts becme much less susceptible t infectin when incubated fr 24 h in the cnditins f this experiment and nt previusly inculated with shrt particles. The results in Table 3 als suggest that the ttal yield f nucleprtein infectivity (lng particles) was greatest when shrt particles were inculated z t 4 h befre lng nes, whereas the ttal yield f RNA infectivity (larger RNA species) was greatest when the interval was 4 t 8 h. In these experiments, the assays f RNA and nucleprtein infectivity were made n different dates and used different batches f plants s that the figures fr the tw kinds f assay are nt directly cmparable. Hwever, trends in the rati f nucleprtein t RNA infectivity are meaningful. This rati, which gives an estimate f relative efficiency f cating the larger RNA species, was greatest when the lng and shrt particles were inculated tgether; efficiency f cating appeared t decrease gradually as the interval between inculatin f lng and shrt particles lengthened (Table 3). DISCUSSION The results described in this paper prvide a mre cmprehensive picture f the replicatin cycle f TRV than was available befre. The earliest change detected, at 7 h after inculatin, was the prductin f infective RNA (larger RNA species). N ultrastructural change was crrelated with this event, but the prductin f nucleprtein particles by 9 h was detected by electrn micrscpy as well as by infectivity assays and flurescent antibdy
Multiplicatin cycle f TRV in prtplasts 247 staining. The pattern that emerges is f infective RNA being synthesized, and incrprated int nucleprtein particles 4 t 5 h later. We suspect that n appreciable accumulatin f virus cat prtein ccurs befre nucleprtein particles are prduced, and that nucleprtein prductin may be limited by the rate f cat prtein synthesis. Infective RNA synthesis seems t be largely cmpleted by I2 h but a day r mre is needed fr its incrpratin int nucleprtein t catch up. Hwever, it is nt knwn whether synthesis f infective RNA ceases at later times, r is balanced by inactivatin. This pattern is remarkably similar t that fr tbacc msaic virus (Sakai & Takebe, 1974; Aki & Takebe, I975), the nly virus t have been studied previusly in prtplasts frm this pint f view. The mst nvel finding in ur wrk is the asynchrny f accumulatin f lng and shrt TRV particles. There are several pssible ways in which this effect might be prduced, ranging frm asynchrnus synthesis f the larger and smaller RNA species t preferential cating f the larger species, and further experiments are needed t differentiate between them. The reasn fr the cnsistent assciatin f the lnger virus particles with mitchndria whereas the shrter nes ccur mainly free in the cytplasm (Harrisn & Rberts, 1968) is nt clear. The predminance f synthesis f lng particles in the earlier stages f infectin may result in them ccupying mst f the sites suitable fr attachment n the mitchndrial surface befre shrt particles are prduced in large numbers. But even late in infectin many f the mitchndria are nt cmpletely surrunded by lng particles and if this explanatin is crrect it wuld be necessary als t pstulate that attachment sites are lcally distributed n the mitchndrial surface, r lse their affinity fr virus particles later in infectin. Anther factr may be a difference in affinity f mitchndria fr lng and shrt particles, which althugh cntaining the same cat prtein differ slightly in electrphretic mbility (Cper & May, 1972), suggesting they may als differ in surface charge density. A third pssibility is that lng and shrt particles have different sites f assembly within the cell. The asynchrny f accumulatin f lng and shrt particles, and their cntrasting distributins in the cell, may be pinters t an advantage f the multipartite genme as a mdus vivendi fr plant viruses, namely the pssibility fr separatin in time r space f the synthesis, translatin r assembly int nucleprtein f the different genme parts. That synthesis f the genme parts may be independently regulated is als suggested by wrk with cwpea chlrtic mttle virus, whse RNA-I accumulated in very much smaller amunts than did the ther three RNA species f this virus when tbacc prtplasts were incubated at 35 C fr a day after inculatin (Dawsn et al. 1975). Multipartite genmes bviusly lend themselves t kinds f regulatin f synthesis and expressin nt pen t unipartite genmes. Our attempts t alter the pattern f RNA and nucleprtein accumulatin by staggering the inculatins with lng and shrt particles did nt reveal any majr effects. In particular, inculatin f shrt particles befre lng nes did nt result in less accumulatin f the larger RNA species, and inculatin f shrt after lng particles did nt give increased accumulatin f the larger RNA, as might have been fund if the smaller RNA, r cat prtein, was instrumental in switching prtplasts frm synthesis f the larger t the smaller RNA species r frm assembly f lng nucleprtein particles t shrt. Indeed, smewhat the reverse effects were bserved. Our results therefre seem t raise mre questins than they answer, but in ding s they als direct attentin t several ptentially fruitful tpics fr further research. z7 vxg 33
248 B. D. HARRISON AND OTHERS We thank 1. M. Rberts and Margt A. Andersn fr the cunts f virus particles, and Agnes M. D. Dnald fr technical assistance. REFERENCES AOKI, S. & TAKEBE, I. (I975). Replicatin f tbacc msaic virus RNA in tbacc mesphyll prtplasts inculated in vitr. Virlgy 65, 343-354. COOPER, J. I. & MAYO, M. A. (I972). Sme prperties f the particles f three tbravirus islates. Jurnal f General Virlgy 16, 285-297. DAWSON, J. R. O., MOTOYOSHI, F., WATTS, J. W. & BANCROFT, S. B. (I975)- Prductin f RNA and cat prtein f a wild-type islate and a temperature-sensitive mutant f cwpea chlrtic mttle virus in cwpca leaves and tbacc prtplasts. Jurnal f General Virlgy 29, 99-I7. HARRISON, B. D. & NIXON, H. L. (I959). Sme prperties f infective preparatins made by disrupting tbacc rattle virus with phenl. Jurnal f General Micrbilgy 2x, 591-599. HARRISON, B. D. & ROBERTS, I. M. 0968). Assciatin f tbacc rattle virus with mitchndria. Jurnal f General Virlgy 3, I2I-I24. KUBO, S., HARRISON, B. D. & BARKER, H. (1975 a). Defined cnditins fr grwth f tbacc plants as surces f prtplasts fr virus infectin. Jurnal f General Virlgy 28, 255-257. KUBO, S., HARRISON, B. D., ROBINSON, D. J. & MAYO, M. A. (I975b). Tbacc rattle virus in tbacc mesphyll prtplasts: infectin and virus multiplicatin. Jurnal f General Virlgy 27, 293-304. KUBO, S., ROBINSON, D. J., HARRISON, B. D. & HUTCHESON, A. M. (1976). Uptake f tbacc rattle virus by tbacc prtplasts, and the effect f phsphate n infectin. Jurnal f General Virlgy 3, 287-298. RAGETLI, H. W. J., WEINTRAUB, M. & LO, E. (I970). Degeneratin f leaf cells resulting frm starvatin after excisin. I. Electrn micrscpic bservatins. Canadian Jurnal f Btany 48, I913-I922. SAKAI, F. & TAKEBE, I. (I974). Prtein synthesis in tbacc mesphyll prtplasts induced by tbacc msaic virus infectin. Virlgy 62, 426-433. TAKEBE, I. (I975). The use f prtplasts in plant virlgy. Annual Review f Phytpathlgy I3, IO5-125. (Received 29 April I976)