By J. R. O. DAWSON, F. MOTOYOSHI,* J. W. WATTS AND J. B. BANCROFTt John Innes Institute, Colney Lane, Norwich NR 4 7UH, U.K. (Accepted t8 June ~975)

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1 J. gen. Virol. (1975), 29, 99-1o7 Printed in Great Britain 99 Production of RNA and Coat Protein of a Wild-type solate and a Temperature-sensitive Mutant of Cowpea Chlorotic Mottle Virus in Cowpea Leaves and Tobacco Protoplasts By J. R. O. DAWSON, F. MOTOYOSH,* J. W. WATTS AND J. B. BANCROFTt John nnes nstitute, Colney Lane, Norwich NR 4 7UH, U.K. (Accepted t8 June ~975) SUMMARY The multiplication of a temperature-sensitive (ts) mutant of cowpea chlorotic mottle virus (CCMV) in tobacco protoplasts has been examined. The results were similar to those obtained with intact cowpea plants. No intact virus was produced above 33"5 C, and no coat protein, either soluble or insoluble was detected in the absence of intact virus. Ribonucleic acid (RNA) of the ts mutant, however, was synthesized at 35 C. Virus particles made at 25 C were degraded in vivo when the infected protoplasts were subsequently cultured at 35 C. RNA-3 of ts CCMV was apparently encapsidated much more often than RNA-3 of wild-type (wt) CCMV in doubly infected protoplasts cultured at either 25 or 35 C. NTRODUCTON The properties of a temperature-sensitive (ts) mutant of CCMV, a spherical plant virus, have been described (Bancroft et al t972 ). The mutant multiplied wel! in cowpea (Vigna unguiculata L.) plants grown at 2i C. Unlike the wild-type (wt) form ts multiplied only slightly at 32 C although considerable amounts of uncoated infectious RNA accumulated in inoculated leaves. ts coat protein has two amino acid exchanges compared with wt. Temperature sensitivity and a low specific infectivity are thus associated with changed properties of the coat protein. The mutation appears to be located in RNA species 3, which codes for coat protein (Bancroft & Lane, 973). Tobacco leaf mesophyll protoplasts infected with CCMV provide a system for studying the synchronous development of infection under closely controlled environmental conditions. They have been used to analyse the behaviour of the ts mutant more precisely than was possible with intact cowpea plants. METHODS solates. Wt and ts CCMV were grown, assayed and purified as before (Bancroft et al. 1972). solation of coat protein. Cowpea primary leaf tissue ( 7 to 18 g) or tobacco protoplasts (~.6 to 4.2 x o 6) were ground in 2 ml and ml respectively of o.t M-tris-HC1 ph 8 buffer * Present address: nstitute for Plant Virus Research, 959 Aobacho, Chiba, Japan. t Present address: University of Western Ontario, London, Canada.

2 O0 J. R. O. DAWSON AND OTHERS ~: Z V f a b c d e f g Fig.. 0 % polyacrylamide gels of extracts of primary leaves of cowpea plants grown at 21 and 32 C. Comparison between healthy leaves and leaves inoculated with wild-type (wt) and temperature-sensitive (ts) mutant CCMV; gels stained with Coomassie brilliant blue. 2~ C: (a) Healthy, (b) ts mutant, (c) wt. 32 C : (e) Healthy, (f) ts mutant, (g) wt. (d) Standard purified wt virus (o.o2 mg virus protein). (van Loon & van Kammen, i968 ). To the extracts clarified by centrifuging at 9000 rev/min for 2o min, were added ml and o. ml respectively of Swank & Munkres' 097) buffer which included ~ ~ (w/v) sodium dodecyl sulphate (SDS) and 8 M-urea. Any precipitate formed after storage overnight at 4 C was removed by centrifuging at 9o00 rev/min for 2o min. The supernatant fluid was heated for 3 min at 2oo C and a precipitate was formed. This was pelleted at 9ooo rev/min for 20 rain and incubated at 37 C for 3 h in the Swank & Munkres' buffer (4 ml for leaf extracts and o.~ ml for protoplast extracts) before electrophoresis. 2oo # of this solution was added to a mixture containing 3/~1 of tracking dye (o'05 bromophenol blue in water), 5o/~1 of glycerol, 5 #1 of mercaptoethanol, and 5o/~1 of o.o2 M-sodium phosphate buffer, ph 7, containing o' ~ SDS and o. ~ mercaptoethanol (Weber & Osborn, 2969). oo#l of this was then placed on a o ~ acrylamide gel) (0. 5 x 7"0 cm) prepared according to Weber & Osborn (969), and the gels were subjected to electrophoresis for 3 h at 45 V and 8 ma/gel. Gels were stained for 25 min with Coomassie brilliant blue (~.25 g dissolved in 454 ml of 5 ~o methanol and 46 ml of glacial acetic acid), and destained over 48 h with two changes of methanol (2o ~) -acetic acid (7"5 ~) solution. The above procedures separated virus coat protein from most host proteins (Fig. ). For each experiment a standard stock of purified virus (equivalent to 2o/~g virus protein/gel) was included and used as a position and densitometry standard when assaying with a Joyce-Loebl 'Polyfrac' densitometer. To determine whether there was insoluble virus protein in the leaves and protoplasts,

3 ts mutant of CCMV in protoplasts samples from which soluble virus protein had been extracted with the tris-hc1 buffer were incubated with the Swank & Munkres' buffer. Any insoluble virus protein left in the tissue after tris-hc1 extraction would be dissolved by the incubation buffer and subsequently detected on the gels. Heat-denatured CCMV protein can be recovered quantitatively using this procedure. Tobacco protoplast and virus culture and identification. Protoplasts were prepared, inoculated, cultured and scored for infectivity using a fluorescent antibody staining procedure as described (Motoyoshi et al. 973a). Virus yields were determined physically (Motoyoshi, Bancroft & Watts, 1973b). The inoculum used to produce mixed infections contained 0"5 #g/ml wt and 0"5 #g/ml ts virus in the presence of #g/ml poly-l-ornithine, unless otherwise stated. When restrictive temperatures were used the protoplasts were first kept for h at 25 C. solates were characterized as wt and ts virus by a protein denaturation test (Bancroft et al. 1972). Radioactive labelling experiments. When proteins were labelled, 2o/zCi L-[G-a4C] leucine (specific radioactivity 3oo mci/mmol) were added to the lo ml culture medium containing about 2x io 6 protoplasts. The protein fraction was prepared and separated on ro polyacrylamide gels; the position of the bands was recorded by scanning the gels with the Joyce-Loebl 'Polyfrac' densitometer. Gels were sliced with a Mickel gel slicer. The slices were dissolved by the method of Goodman & Matzura (1970 using hydrogen peroxide and ammonia, and counted with a Beckman scintillation counter after dissolution in the usual PPO and POPOP mixture. When RNA was labelled, 300 lzci a2po 4 was added to o ml culture medium containing about 2 x io ~ protoplasts. RNA was extracted by the phenol method of Loening (967), and fractionated by electrophoresis on 2.6 ~ polyacrylamide gels (Loening, 967). Gel slices were placed in 5 ml water in a vial and radioactivity was counted by 12erenkov radiation (Bancroft et al. 975). O RESULTS Virus multiplication and degradation The wt virus multiplied well in protoplasts at both 25 and 35 C (Table ). The ts virus also multiplied at 25 C although not quite so well, but no ts virus could be detected in protoplasts cultured at 35 C. The mutant multiplied marginally at C (Table ). A more detailed examination of the effect of temperature showed that no intact ts virus could be detected at or above 33"5 C (Fig. 2). These results are in agreement with those obtained with intact cowpea plants (Bancroft et al. 972). Studies on the fate of the ts mutant in the primary leaves of cowpea indicated that mutant virus particles were denatured in vivo at 32 C whereas those of the wt were not, or only marginally so (Bancroft et al. 972). Fig. 3 shows that a similar process occurred with ts virus in tobacco protoplasts; mutant virus that had accumulated in protoplasts cultured for 40 h at 25 C was progressively lost when the protoplasts were subsequently cultured at 35 C. Survival of protoplasts at 35 C (about 5 lo5 protoplasts per sample) was satisfactory throughout the experiment. RNA multiplication Cowpea leaves infected with the ts mutant produce little virus at 32 C but substantial amounts of infective virus RNA (Bancroft et al. 1972). From this and from coat protein denaturation data it was concluded that restriction of virus synthesis was due to a

4 02 J.R.O. DAWSON AND OTHERS Table. The yields of wt and ts CCMV from protoplasts cultured at different temperatures Expt. nfection period (h) Virus yield ~ Virus particles per Temperature (#g) from infected infected protoplast noculum ( C) O 8 protoplasts protoplasts* (x io -8) wt wt 35 l o ts ts 35 ot 5 o wt wt "8 ts '9 ts 35 ot 6 o wt "3 wt ts o 14-o ts "5 * As scored by fluorescent antibody staining. t Not detectable.! X t~ G 30 o 8~ ~ i Temperature ( c) Fig. 2. The effect of temperature on yield of ts virus in protoplasts: 0--0, ~ fluorescing protoplasts; A--A, particlesfinfected protoplast x lo -8. maturation fault. This idea is consistent with a subsequent genetic analysis (Bancroft & Lane, 973). The present protoplast studies showed that virus RNA synthesized at 25 C in protoplasts infected with either wt or ts CCMV gave the characteristic labelling pattern described by Bancroft et al. (975; see Fig. 4). That is to say, all four species of virus RNA were present

5 ts mutant of CCMV in protoplasts o3-0 60q b ~ N "G t Time at 35 C (h) Fig. 3. The fate of ts virus in protoplasts transferred to 35 C after 4o h at 25 C: --0, ~ fluorescing protoplasts; --, particles/infected protoplast x h after infection. At 35 C both wt and ts RNA were synthesized, which is in agreement with the earlier work on intact cowpea plants (Bancroft et al. 972). The labelling patterns of wt and ts RNA were markedly changed at 35 C indicating that regulation of transcription was affected. Relative to the other RNA species there was an increase in RNA-2 at 35 C compared with 25 C; little or no RNA-~ could be detected at 35 C. Production of coat protein Table 2 gives evidence from 5 experiments with cowpea leaves showing that the amount of virus coat protein determined by polyacrylamide gel electrophoresis was similar to that coating intact virus of both strains at 2~ and 32 C. Neither ts coat protein nor virus were detected at 35 C. No heat-denatured coat protein was detected in leaves and protoplasts inoculated with these strains and grown at low or high temperatures. As the specific radioactivities of the coat proteins of both strains from protoplasts were similar at 25 and 32"5 C (Table 2) it is not possible to decide whether there has been preferential turnover of mutant coat protein at 32"5 C. Mixed inoculation of protoplasts Protoplasts were inoculated with a mixture of wt and ts CCMV and cultured at 35 C. After 24 h the protoplasts were homogenized in o-i M-acetate buffer (ph 5"o) and the clarified extract was used to inoculate Chenopodium hybridum L. leaves. Only the extracts from wt or wt + ts inoculated protoplasts produced lesions. Virus from single lesions was bulked on cowpea leaves and purified. The coat protein of these purified isolates was characterized by the heat-denaturation test. Table 3 shows that all isolates from protoplasts

6 ~o4 J. R. O. DAWSON AND OTHERS i wt 25 'C ts25 C C 25 :C 7 o X.E *6 15 wt 35 :C ~" ts 35 C C 35 ~C Fraction number Fig. 4. Polyacrylamide gel electrophoresis of phenol extracts of RNA from healthy and infected tobacco protoplasts cultured at 25 and 35 C in the presence of 3~P. Histograms of (~erenkov counts illustrating the effect of temperature on the species of RNA of wt and ts forms of CCMV. Migration is from right to left. C = control. Table 2. Yield of CCMV (wt and ts) coat protein from plants and protoplasts kept at different temperatures 2 C 3 2 C Plants* wt ts wt ts Virus coat protein (rag) per tz6_+ 8"7~: 34+9q m5+_ t8-6 o_+3"4 kg of fresh cowpea leaf materialt ' 41_' " "4 Protoplasts[ 25 C 32"5 C Virus coat protein (#g) per O 6 protoplastm: 87 (93) 73 (9o) 54 (82) 6 (4o) Specific activity of coat protein (ct/min/#g) based on gel data oi * o to 12 day infection period. t Average values and standard errors of five experiments. Calculated from yield of purified virus. Calculated from protein fractionated by gel electrophoresis. [[ 42 h infection period. Figures in parentheses are % infected protoplasts measured by fluorescent antibody staining.

7 ts mutant of CCMV in protoplasts O5 Expt. Table 3. Analysis of single lesion isolates from singly and mixedly-inoculated (ts and wt) tobacco protoplasts grown at 25 or 35 C nfection period (h) Number of isolates with either ls or wt coat* Temperature Lesions on c ---~" - -, lnoculum ( C) C. hybridum ts wt wt 35 Yes o 5 ts 35 No wt + ts 35 Yes 32 3 wt + ts 35 Yes 24 o wt 35 Yes o 5 ts 25 Yes 5 o ts 35 No wt + ts 35 Yes 32 2 wt 25 Yes o 5 wt 35 Yes o 5 ts 25 Yes 2 o ts 35 No w t + s 25 Yes 18 o wt + ts 35 Yes 15 o * 0"3 mg virus/ml heated at 43 C for 3 min in 1 ~-NaC, o.oz M-tris, ph 7"4 (Bancroft et al. 972 ). inoculated with wt were wt in character, but over 90 ~ of isolates from the mixed inoculations had a ts phenotype. The same result was obtained at 35 C with infection periods of 5, 24 and 4 h and with mixed inoculations after 24 h infection at 25 C. DSCUSSON The results obtained with tobacco protoplasts confirm those with cowpea leaves. For example, although both virus strains made comparable amounts of virus particles at low temperature, only wt made substantial amounts at 32 C and over. Both host systems showed that ts virus-rna was readily detectable at restrictive temperatures with small amounts only of soluble ts virus coat protein. The detailed analysis of the multiplication of CCMV ts in tobacco protoplasts gave more accurate values for the critical restriction temperature and the extent and rate of particle denaturation in vivo than could be obtained using intact plants. The recovery of infectious RNA in plant extracts at restrictive temperatures (32 C) and the polyacrylamide gel analyses of [32P]-labelled RNA from infected protoplasts at 35 C show that the temperature sensitivity of the mutant is not dt~e to a fault in RNA replication. The nature of the ts restriction is clearly concerned with maturation but the mechanism may differ from that found for TMV Ni 118 (Jockusch, 968) and ts 38 (Hariharasubramanian, Zaitlin & Siegel, 97o). The latter two mutants form a pool of insoluble coat protein at restrictive temperatures whereas this particular ts mutant of CCMV seems not to. The apparent lack of coat protein with CCMV at 35 C could be due to preferential coat protein turnover under restrictive conditions; denatured coat protein could be enzymically degraded into peptides not identifiable on gels as being derived from coat,protein. Alternatively, it is conceivable that coat protein of normal primary structure is not translated by the CCMV-ts mutant at high temperatures due to a thermal conditional nonsense insertion

8 O6 J.R.O. DAWSON AND OTHERS in the coat protein cistron. We have no way at present of distinguishing between the two possibilities. The high incidence of mixed infection of tobacco protoplasts by ts and wt strains of CCMV at 35 C with a high recovery rate of ts types was unexpected. Mixed infection of protoplasts at 35 C- at least 9o ~ of infections were mixed-was possibily due to poly-lornithine aggregating the virus. Aggregates causing infection would thus be likely to contain both strains. The incidence of genomic masking by TMV (Sarkar, 969; Atabekov et al. 97o; Kassanis & Bastow, 1970 was not so high, presumably because the possibility of doubly infecting cells of intact leaves was less likely. Also it is possible that CCMV strains induce mixed infections more readily than TMV strains; TMV has a singlecomponent genome and can infect with one virus particle, whereas CCMV has a multicomponent genome which requires three dissimilar nucleoprotein particles to initiate infection. Unlike TMV, therefore, part of the genome of a strain of CCMV may conceivably multiply in a cell containing the remaining parts of the virus genome, themselves derived from a different and related strain. The survival of ts types at 35 C suggests rescue of ts RNA-3 has occurred. t is not known, however, whether the high yield of ts types at low temperature (z5 C) represents the same phenomenon. n both cases ts RNA-3 appears to be preferentially encapsidated. This might be related to the ability of ts virus to uncoat more readily, at least in vitro, than wt. The level of mixed infection of tobacco protoplasts reported here suggests the technique may provide a potentially useful tool for the genetical study of plant viruses. We wish to thank Mrs D. B. Aldous and Miss D. M. Allen for technical assistance, and. H. Flack for help with RNA and virus determinations. REFERENCES ATABEKOV, J. G., SCHASKOLSKAYA, N. D., ATABEKOVA, T.. & SACHAROVSKAYA, G. A. (970)- Reproduction of temperature-sensitive strains of TMV under restrictive conditions in the presence of temperatureresistant helper strain. Virology 41, 397-4o7. BANCROFT, J. B. S, LANE, L. C. (973). Genetic analysis of cowpea chlorotic mottle and brome mosaic virus. Journal of General Virology x9, BANCROFT, J. B., MOTOYOSm, V., WATTS, J. W. & DAWSON, L R. O. (975). Cowpea chlorotic mottle and brome mosaic viruses in tobacco protoplasts. John nnes nstitute Symposium, 1974, PP, 349. BANCROFT, J. B., REES, M. W., DAWSON, J. R. O., McLEAN, G.D. & SHORT, M. N. (t972). Some properties of a temperature-sensitive mutant of cowpea chlorotic mottle virus. Journal of General Virology x6, GOODMAN, D. & MATZURA, H. 0971). An improved method of counting radioactive acrylamide gels. Analytical Biochemistry 42, HARHARASUBRAMANAN, V., ZATLN, M. & SEGEL, g. (197o). A temperature-sensitive mutant of TMV with unstable coat protein. Virology 4o, JOCKUSCH, n. (1968). Two mutants of tobacco mosaic virus temperature-sensitive in two different functions. Virology 35, 94-O. KASSANS, B. & BASTOW, C. (1971). Phenotypic mixing between strains of tobacco mosaic virus. Journal of General Virology t, LOENNG, U.E. (1967). The fractionation of high-molecular-weight ribonucleic acid by polyacrylamide-gel electrophoresis. Biochemical Journal o2, MOTOYOSH, F., BANCROFT, J. B., WATTS, J. W. & BURGESS, J. (1973a). The infection of tobacco protoplasts with cowpea chlorotic mottle virus and its RNA. Journal of General Virology 2o, MOTOYOSH, V., BANCROFT, J. B. & WATTS, J. W b). A direct estimate of the number of cowpea chlorotic mottle virus particles absorbed by tobacco protoplasts that become infected. Journal of General Virology 21, SARKAR, S. (1969). Evidence of phenotypic mixing between two strains of tobacco mosaic virus. Molecular and General Genetics lo5, 87-9o.

9 ts mutant of CCMV in protoplasts O 7 SWANK, R.T. & MUNKRES, K.D. (97t). Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulphate. Analytical Biochemistry 39, VAN LOON, L. e. & VAN KAMMEN, A. 0968)- Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana tabacum var, 'Samsun' and 'Samsun NN'.. Phytochemistry 7, 7Z WEBER, K. & OSBORN, M. (969). The reliability of molecular weight determinations by dodecyl sulphatepolyacrylamide gel electrophoresis. Journal of Biological Chemistry 244, 44o (Received ~ March 975)