The Infection of Tobacco Protoplasts with a Variant of Brome Mosaic Virus

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1 J. gen. ViroL (1974), 25, Printed in Great Britain 31 The nfection of Tobacco Protoplasts with a Variant of Brome Mosaic Virus By F. MOTOYOSH,* J. B. BANCROFT t AND J. W. WATTS John nnes nstitute, Colney Lane, Norwich NOR 7oF, U.K. (Accepted 24 May 1974) SUMMARY Neither tobacco mesophyll protoplasts nor tobacco leaves could be infected with wild type brome mosaic virus (BMV wt) or its RNA, but both were infected when inoculated with 5o #g/ml or more of a variant of BMV (BMV VS). This is about OO x the amount required for efficient infection by cowpea chlorotic mottle virus (CCMV). Poly-L-ornithine stimulated the infection of protoplasts with BMV VS, but was not essential. BMV VS. RNA inoculum caused infection of protoplasts under conditions similar to those needed for their infection with CCMV-RNA. NTRODUCTON Tobacco mesophyll protoplasts can be infected with cowpea chlorotic mottle virus (CCMV) (Motoyoshi et al. 973a), which closely resembles brome mosaic virus (BMV) in its physical properties but which is much more electronegative than BMV. f tobacco protoplasts were also susceptible to BMV, the protoplast system would be especially useful for comparative studies on infection and on the genetic functions of both viruses, to complement those done with intact host plants (Bancroft, 1972; Bancroft & Lane, 1973). The present study describes the infection of tobacco mesophyll protoplasts with a variant of BMV that can also infect tobacco leaves. METHODS Two strains of BMV, the Norwich subcultures of wild type (BMV wt) and an electrophoretic variant (BMV V5) (Bancroft & Lane, 1973) which is slightly less electropositive than type BMV, were used for inoculating protoplasts obtained from leaf mesophyll of Nicotiana tabacurn cv. White Burley. Protoplasts were inoculated and cultured as described (Motoyoshi et al. 973a). Unless otherwise indicated, poly-l-ornithine ( #g/ml) was present during inoculation. Protoplasts were prepared from leaves of 4o-day-old tobacco plants as described (Motoyoshi et al. 973a). When poly-l-ornithine was used, virus (o to OO #g/ml) and polycation ( #g/m1) were pre-incubated at 25 C for O min before inoculation in o.o2 M-potassium citrate buffer, ph 5"2, containing 0"7 M-mannitol. Protoplasts ( to 2 lo 6) were centrifuged at 50 g for 2 min, the supernatant fluid was discarded and the pellet was immediately resuspended in O ml of the virus inoculum. This was further diluted with O ml 0"7 u-mannitol and maintained at 25 C for O min. The protoplasts were washed 3 times with 0"7 M-mannitol containing o -4 M-CaC12 and cultured as described (Motoyoshi et al. 973a). Contamination by microorganisms was controlled by * Present address: nstitute for Plant Virus Research, 959 Aobacho, Chiba, Japan. ] Present address: University of Western Ontario, London, Canada. 3-2

2 32 F. MOTOYOSH~ J. B. BANCROFT AND J. W. WATTS ~ ' ' ' ~ - ;' "-,, 'L, l % % ~/oo,,' "4 ~ 0"5 2 %1,, / \,, /d / / / ~, o L,,_,,, "-=--1o ~ 201 / N "", 0"61'2 3"6 7"2 1 ~ ~ i \ ", RNA,,ug/ml l / l/. \-,\ / "~ 10..s" 20 ~ g, 1.2" "2" ~ m ='-'-" 0"5 l Concentration of virus, pg/ml Fig.. The response of tobacco protoplasts to different amounts of wild type and variant V5 of brome mosaic virus, their RNAs and cowpea chlorotic mottle virus. noculation was at ph 5"2 in the presence of poly-l-ornithine ( #g/ml). Protoplasts were harvested at 44 h after inoculation, and virus yield (solid lines) and percentage of infected protoplasts (dashed lines) were measured. The two insets show the dose responses of protoplasts to BMV-R.NAs., BMV wt; [~, BMV VS; O, CCMV. antibiotics (Nystatin, 25 units/ml; carboxybenzyl penicillin OO/zg/ml). The percentage of infected protoplasts was assessed by fluorescent antibody staining using anti-bmv wt antibodies conjugated with fluorescein isothiocyanate as before (Motoyoshi et al. 973a). Virus yield was determined by sucrose-density gradient analysis of an extract of the protoplasts (Motoyoshi, Bancroft & Watts, 973b). Protoplasts were homogenized in ml o. M-sodium acetate buffer (ph 4"7) and centrifuged at 30o0 rev/min for 5 min. A sample (o- to 0"5 ml) of the supernatant fluid was layered on to 4"5 ml of a O to 4o% sucrose gradient in o- M-acetate buffer, ph 4"7, and centrifuged in a Beckman 5o"1 rotor for z'25 h at 36oo0 rev/min. The gradient was fractionated with an SCO density-gradient analyser using the o'z5 scale and a cm flow cell. When virus was present it appeared as a peak well separated from u.v. absorbing material at the top of the tube. Yield (in/zg) was calculated from the area of the peak using a calibration chart prepared with known amounts of virus. The lower limit of the method was about 0"5/zg virus. RESULTS The responses of protoplasts to BMV wt, BMV VS, their respective RNAs and CCMV are shown in Fig.. Protoplasts were not infected by BMV wt or its RNA at any inoculum concentration. The few protoplasts that stained with the fluorescent antibodies probably reflected the high inoculum level. The BMV V5 inoculum did infect protoplasts, but much higher concentrations of virus were required than with CCMV. BMV V5 infected fewer

3 nfection of protoplasts with BMV 33 15! 4 0 C~ ~2 o 10 5 /! /! ~ 40 - ~ 30 - o 20 - m / e / / ~ O- / i i "1....:1 / ~ 0 o-==~ i [] - ~. ; ~ " K / " ~ 1 Time (h) _ 2 o Y O u 0-5 e-~ -- -~/' / ~" ' " ;> 0-5 U u Time (h) 0-2 Fig. 2. Changes in the amount of brome mosaic virus associated with protoplasts during the period of culture after inoculation. Protoplasts were inoculated at ph 5"2 with BMV wt or BMV V5 at a concentration of 5o #g/ml, in the presence of poly-l-ornithine ([ ~g/ml). The inset shows percentage of infected protoplasts as scored by fluorescent antibody staining. Number of BMV V5 particles per infected protoplast was based on the final percentage of protoplasts that stained with fluorescent antibody (4 ~ at 24 h after inoculation), n--m, BMV wt; ~- - -rt, BMV VS. protoplasts even at 50 #g/ml or more than did CCMV at 0"5/~g/ml. Maximum infection with CCMV occurred when protoplasts were inoculated with the virus at TO #g/ml, fewer becoming infected at higher concentrations of virus. By contrast, the number of protoplasts infected with BMV 7/5 increased with increasing virus concentration. The number of protoplasts infected by BMV V5-RNA rose to a maximum with an inoculum containing x'2 #g RNA/ml and then declined with increasing RNA concentration; this pattern resembled that of CCMV-RNA which also had a maximum infectivity at -2 #g/ml (Motoyoshi et al. T973 a). Both CCMV and BMV Vs-RNAs therefore have about the same specific infectivity for protoplasts. CCMV has a higher specific infectivity than its free RNA (Motoyoshi et al 973a ). n contrast BMV V5 had a specific infectivity similar to that of its RNA (Fig. ). The difference in infectivity of intact CCMV and BMV V5 thus appears to be coat-related. The levels of BMV wt and BMV V5 detected in protoplasts with time of culture of inoculated protoplasts are shown in Fig. 2. Fluorescent antibody staining failed to detect any infection of protoplasts inoculated with BMV wt. The amounts of BMV wt and BMV /5, present at z h after inoculation were comparatively high in both the protoplast samples,

4 - 2"0 34 F. MOTOYOSH, J. B. BANCROFT AND J. W. WATTS Table. The effect of ph and poly-l-ornithine on the infection of tobacco protoplasts with a variant of brome mosaic virus, BMV V5 Virus particles Assay of virus yield ( i o -6) ncrease in Concentration Time < - - ~ ~ per (x o -e) of poly-l- after No. of Virus ~ protoplast per noculum ornithine infection protoplasts yield fluorescing that became infected ph (#g/ml) (h) ( x io -6) (#g) protoplasts* infectedt protoplasts 4'7 o '3.8 o '7 o 24 ~ 'o 35"4 45 9'8 7"7-4"7 t i i-i z6-4 o ' " "2 O 1.2 4'0 o 2.o -- 5'2 o 24 o.8 6"4 22 4"7 2'7 5"2 ' 'O 0 0"3 -- 5'2 24 0'9 9"9 38 3'8 3"5 * As scored by fluorescent antibody staining. t The values for the samples at h after inoculation were calculated from the percentages of infected protoplasts at 24 h. approximating to 3"7 x io ~ BMV wt and 1.3 x io 5 BMV V5 particles per protoplast assuming that the particles were evenly distributed amongst all the protoplasts. This represented the amount of the inoculum absorbed by the protoplasts. BMV wt particles associated with protoplasts steadily decreased to 6"5 x lo 4 per protoplast by 24 h, due either to virus degradation or release from the surface of protoplasts during the culture period. After 24 h, 4 fo of protoplasts that were inoculated with BMV V5 stained with fluorescent antibody. The virus appeared to have started multiplying by 7 h after inoculation because the yield at this time exceeded the amount measured at h after inoculation. The yield reached 3"5 x lo 6 particles per infected protoplast by 15 h after inoculation and thereafter remained at about the same level during the next 9 h. The fact that tobacco protoplasts were not infected by BMV wt or by its RNA suggests that failure to infect was due to the inability of BMV wt to multiply in tobacco protoplasts rather than to failure to enter them or to uncoat. ntact leaves of tobacco plants (Nicotiana tabacum cv. White Burley) could not be infected with BMV wt but could with BMV V5. Both poly-l-ornithine and the ph at inoculation affected the efficiency of infection of protoplasts with BMV ti5 (Table ). At ph 4"7, 45 ~o of protoplasts were infected without and 77 % with poly-l-ornithine. At ph 5"a, 2a % were infected without poly-l-ornithine and 38 ~o with it. These results suggest that (z) poly-l-ornithine stimulated infection of protoplasts with BMV VS, but as in the case of infection with pea enation mosaic virus (Motoyoshi & Hull, 1974) was not essential, in contrast to infection with CCMV, tobacco mosaic, cucumber mosaic and potato X viruses (Takebe & Otsuki, 1969; Motoyoshi et al. 973a; Otsuki & Takebe, 973; Otsuki et al. 1974). (2) ph 4"7 was more favourable to infection than ph 5"2. Protoplasts absorbed larger amounts of virus at ph 4"7 than at ph 5"a either with or without poly-l-ornithine, assuming that virus content at h after inoculation reflected amount of virus absorbed. Poly-L-ornithine appeared to promote virus absorption by protoplasts at ph 4"7, but to inhibit it at ph 5"2 despite its stimulation of infection. Thus, the amount of virus initially absorbed by protoplasts was not correlated with the number infected. Unlike CCMV (Motoyoshi et al a), BMV wt and BMV V5 did not precipitate with poly-l-ornithine over the range of concentration ratios examined (Table 2).

5 nfection of protoplasts with BMV 35 Table 2. The effect of poly-l-ornithine on brome mosaic virus Turbidity at 340 nm c Ratio poly-l-ornithine :BMV BMV wt BMV 1/5 o* o'74 o.26 o.2:~ 0'52 0'57 2 : 0"26 0"24 20 : 0"20 0"60 * The virus was at 2 mg/ml in o.o M-citrate buffer, ph 5"2, and was incubated at 25 C for to min. Results shown in Fig. 2 and Table indicate that individual protoplasts that became infected had absorbed io 5 to o 6 BMV V5 particles. This is almost certainly too high to be a measure of the actual number required to initiate an infection, because the Ds0 for CCMV is about 38o particles per protoplast (Motoyoshi et al. 973b). t is unlikely that this result was due entirely to a difference in the specific infectivities of CCMV and BMV VS. The RNA prepared from both viruses had similar specific infectivities (compare Fig. with Motoyoshi et al 973a). Further, the specific infectivity of the BMV V5 RNA was at least as high as that of the intact virus. t is probable, therefore, that the majority of the BMV particles failed to enter protoplasts. This might also explain the failure of protoplasts to stain with fluorescent antibody h after inoculation in spite of the presence of relatively large amounts of intact virus (Fig. 2). f these particles were on the surface of the protoplast they could readily be lost during the ethanol fixation and subsequent washing prior to staining. DSCUSSON The dosage response curves for BMV V5 and CCMV (Fig. ) suggest that electrostatic forces are of great importance in the process of absorption of virus by the protoplasts. With CCMV, which is negatively charged (isoionic point at ph 3"6, at #m -- o., Bancroft et al. i968), and forms aggregates with poly-l-ornithine (virus-poly-l-ornithine complex) in the inoculation buffer, ph 5"2 (Motoyoshi et al. 973a), the results suggest that there must be a balance between virus and poly-l-ornithine so that the net electrical charge of the complex is sufficiently positive to allow its ready approach to the negatively charged protoplast surface. There is an optimal ratio of CCMV to polycation; excess CCMV beyond this ratio increases the net negative charge of the virus-poly-l-ornithine complex and so reduces absorption. This seems also the case with the RNA of CCMV (Motoyoshi et al. 1973a) and BMV VS. BMV V5 has a positive charge at ph 5"2 (its isoionic point is not known but is lower than that of BMV wt which is at ph 7"9 at #m = o., Bockstahler & Kaesberg, 962) and does not visibly aggregate with poly-l-ornithine over a wide range of concentration ratios (Table 2). t therefore freely absorbs to the protoplast without the intervention of poly-l-ornithine. The reasons why much higher concentrations of BMV V5 were needed to infect the same proportion of protoplasts as were infected by CCMV are not clear. Differences in specific infectivities may account for part of the difference. Possibly also aggregation of CCMV with poly-l-ornithine gives infectious elements that contain many virus particles. Both BMV and CCMV are multicomponent systems (Lane & Kaesberg, 97i; Bancroft & Flack, 972), and the chance of infection when one of these CCMV aggregates absorbs to an entry site on the protoplast would be much higher than with BMV, which is either dispersed or only weakly aggregated in presence of poly-l-ornithine. The increase in infection when poly-l-ornithine was added to BMV V5 may be due to weak

6 36 F. MOTOYOSH, J. B. BANCROFT AND J. W. WATTS associations between virus and polycation. Additionally or alternatively, the poly-lornithine may interact with the surface of the protoplasts to produce more sites for entry of virus (Burgess, Motoyoshi & Fleming, 973). rrespective of the correct explanation, however, it is of considerable interest, that with two viruses with a net positive charge during inoculation, namely BMV 115 and pea enation mosaic virus (Motoyoshi & Hull, 974), poly-l-ornithine is not essential for infection. We wish to thank Mr. H. Flack and Miss D. M. Allen for technical assistance. REFERENCES BANCROFT, J. B. (1972). A virus made from parts of the genomes of brome mosaic and cowpea chlorotic mottle viruses. Journal of General Virology x4, BANCROFT, J. B. & FLACK,. H. (972). The behaviour of cowpea chlorotic mottle virus in CsC1. Journal of General Virology S, i. BANCROFT, J. B., HEBERT, E., REES, M. W. & MARKHAM, R. (968). Properties of cowpea chlorotic mottle virus, its protein and nucleic acid. Virology 34, BANCROFT, J. B. & LANE, L. C. (973). Genetic analysis of cowpea chlorotic mottle and brome mosaic viruses. Journal of General Virology x9, BOCKSTAHLER, L. E. & KAESBERG, P. (962). The molecular weight and other biophysical properties of bromegrass mosaic virus. Biophysical Journal 2, -9. BURGESS, J., MOTOYOSH, F. & FLEMNG, E. N. (1973). Effect of poly-l-ornithine on isolated tobacco mesophyll protoplasts: evidence against stimulated pinocytosis. Planta, Berlin xxx, 99-2o8. LANE, L. C. & KAESBERG, ~'. (1971). Multiple genetic components in bromegrass mosaic virus. Nature New Biology 232, 4o--43. MOTOYOSH, F., BANCROFT, J. B., WATTS, J. W. & BURGESS, J. (1973 a). The infection of tobacco protoplasts with cowpea chlorotic mottle virus and its RNA. Journal of General Virology 2o, MOTOYOSH, F., BANCROFT, J. B. & WATTS, J. W. (973b). A direct estimate of the number of cowpea chlorotic mottle virus particles absorbed by tobacco protoplasts that become infected. JournalofGeneral Virology 2x, 59-6L MOTOYOSH, F. & HULL, R. 0974)- nfection of tobacco protoplasts with pea enation mosaic virus. Journal of General Virology 24, OTSUK, V. & TAKEBE,. (1973). nfection of tobacco mesophyll protoplasts by cucumber mosaic virus. Virology 53, OTSUK, v., TAKEBE,., HONDA, Y., KAJTA, S. & MATSU, C. (t974). nfection of tobacco mesophyll protoplasts by potato virus X. Journal of General Virology z2, TAK~BE,. & OTSUK, V. (969). nfection of tobacco mesophyll protoplasts by tobacco mosaic virus. Proceedings of the National Academy of Sciences of the United States of America 64, (Received 8 March 1974)