Plant Cell Reports

Plant Cell Reports (1986) 5:57-60

© Springer-Verlag 1986

Electroporation-mediated infection of tobacco leaf protoplasts with tobacco mosaic virus RNA and cucumber mosaic virus RNA M. Nishiguchi 1, 3, W. H. R. Langridge 2, A. A. Szalay 2, and M. Zaitlin 1 1 Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA 2 Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14853, USA Received November 4, 1985 / Revised version received December 31, 1985 - Communicated by J. M. Widholm

ABSTRACT Conditions were established for the introduction of both tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) R N A s into tobacco mesophyll protoplasts by electroporation. The proportion of infected protoplasts was quantified by staining with viral coat protein-specific antibodies conjugated to fluorescein isothiocyanate. Approximately 30-40 % of the protoplasts survived electroporation. Under optimal conditions, up to 75% of these were infected with T M V - R N A . Successful infection was demonstrated in 19 out of 20 experiments. Optimal infection was achieved with several direct current pulses of 90 psec at a field strength of 5 to 10 kV/cm. Changing the position of the protoplasts within the chamber between electric pulses was essential for achievement of high rates of infection. Optimal viral R N A concentration was about 10 ~g/ml in a solution of 0.5 M mannitol without buffer salts. INTRODUCTION There are m a n y reports concerning the introduction of foreign genes into plant protoplasts (reviewed by Steinbiss and Broughton, 1983), especially in the field of plant virology (M{ihlbach,1982; Takebe,1983; Sander and Mertes, 1984). The techniques employed include the use of polycations such as poly-L-ornithine (Aoki and Takebe,1969; Motoyoshi etal.,1973; Okuno and Furusawa, 1978), polyethyleneglycol (Dawson etal.,1978), liposomes (Fukunaga et ai.,1981) and direct microinjection (Steinbiss and Stabel~]-98-3). Electrical impulses induce permability changes in cell m e m b r a n e s ( N e u m a n n and Rosenbeck, 1972) (electroporation). Recently electroporation has been used to introduce foreign D N A into animal cells (Wong and Neumann, 1982; N e u m a n n etal.,1982; Potter et ai.,1984). Fromm etal. (1985) have reported that subsequent to electroporation they detected the transient expression of an artificially constructed chlora mphenicol acetyltransferase gene in carrot protoplasts. Langridge etal. (1985) obtained a transformation frequency between 1 and 2% in carrot protoplasts after electroporation with Ti-plasmid D N A . Little information is available concerning electroporation of plant protoplasts. In this paper w e report conditions for introducing foreign R N A into protoplasts by electroporation using tobacco leaf protoplasts and either T M V - R N A or C M V - R N A .

3 Present address: National Institute of Agrobiological Resources, Tsukuba Science City, Yatabe, Ibaraki 305, Japan Offprint requests to: A. A. Szalay

MATERIALS AND METHODS Isolation of protoplasts The seeds of tobacco plants, Nicotiana tabacum L. cv. Xanthi (originally obtained from the Institute of Agrobiological Resources, Tsukuba, Japan; these plants are very different in appearance from those grown from Xanthi seed in our collection) were sown every two weeks. After about 5 weeks, each seedling was transplanted into a pot (20 c m in diameter') filled with Cornell soil mixture (i part of Sphagnum peat moSs to 2 parts of composted greenhouse soil plus 3 kg of 5-I0-5 fertilizer per cubic meter). The plants were grown in a greenhouse; in winter the day-length was extended to 16 hr with fluorescent light. They were fertilized 5 times per week with a nutrient solution containing ammonium nitrate, 0.92 g/l, a m m o n i u m phosphate 0.46 g/l, and potassium chloride 0.46 g/l. For isolation of protoplasts, lower, fully-expanded leaves, usually the 3rd to the 5th leaf from the base, were taken from i0-12 week-old plants which had 24-26 leaves (greater than 2 cm). Protoplasts were isolated by a modification of the method devised by Takebe et al. (1968), as their two step method did not give good protoplasts from our tobacco plants. About 60 c m 2 of the lower epidermis was peeled from each leaf with the aid of forceps, or the lower' surface of the leaf was covered with 320 grit Carborundum and abraded with a plastic foam flask plug until it turned dark green. The leaf was floated in a 9 c m Petri dish, peeled or abraded surface down, on an e n z y m e solution containing 0.5 % M a c e r o z y m e R-10 (Yakult Honsha Co,, Tokyo), 0.5 % potassium dextran sulfate (Meito Sangyo Co. Ltd., Tokyo) and 2% Cellulase "Onozuka" R-10 (Yakult Honsha) in 0.5 M mannitol, p H 5.6-5.8, and incubated without shaking at 30-35°C for 2-3 hrs. (Several leaves were utilized in each experiment and were processed separately; only those dishes with a substantial proportion of intact, spherical protoplasts were utilized for the subsequent operations). During incubation, the dish was agitated gently several times. After protoplasts were released, the enzyme solution containing the protoplasts was filtered through two to four layers of cheesecloth into a round-bottom 40 ml centrifuge tube, and centrifuged at room temperature at I00 x g for 3 rain in an International table-top clinical centrifuge equipped with a swinging bucket rotor. After" aspirating the supernatant, the protoplast pellet plus a small volume (i.0ml) of the supernatant was resuspended in sterile 0.5 M mannitol at 4 ° and centrifuged again at low speed. Resu~pension and low speed centrifugation was repeated several times until the supernatant was clear'. The final protoplast pellet was resuspended in 1.0 ml of 0.5 M mannitol and held at 4 ° until use. Protoplasts were quantified by countIng in a 0.2 m m deep Fuchs-Rosenthal CountIng C h a m b e r (A. H. Thomas,

58 Philadelphia). Only viable protoplasts were counted; i.e., those which were round and were delimited by an intact plasmalemma. In viable cells, the chloroplasts were normally oriented along the inside of the membrane, although in s o m e instances they were clustered on one side of the cell. Preparation of viral R N A s Tobacco plants, N. tabacum L. cv. Samsun, were inoculated with the U I strain of T M V . The virus was purified (Otsuki et al., l~977) and R N A was extracted from virus particles using either SDS-lohenol (Zaitlin, 1979), or phenol-bentonite (Fraenkel-Conrat et al., 1961). The final RNA pellet was resuspended in sterile water at a concentration of 1.0-3.0 ~g/~ i, distributed into small polystyrene tubes and stored at -85 °. C M V , WL-strain without satellite R N A (Gonsalves et al., 1982) was purified from systemically-~nfected tobacco, N. tabacum L. cv. Xanthi nc, as described by Lot et al.~19-7"~. C M V - R N A was extracted from resuspended virus particles using the SDS-phenol-chloroform procedure (Peden and Symons, 1973; Gonsalves et al., 1982). The final R N A pellets were resuspended in sterile water and stored at -85 °. Electroporation Immediately before electroporation, the protoplasts were resuspended in a solution of 0.5 M mannitol, followed by low speed centrifugation. ATter removing the supernatant, sterile, 0.5 M ice cold mannitol solution was added gently to the peter to give a concentration of between 1.0 - 1.5 x 105/protoplasts/ml. The protoplast suspension was mixed with a viral R N A solution to give a final R N A concentration of 10~g/ml, unless otherwise stated. The protoplast-RNA mixture (0.4 ml) was transferred to a Z 1650 Helical Electrofusion C h a m b e r (200 l~m distance between electrodes; G C A Corporation, Chicago, Illinois) and subjected to electroporation using the square wave pulse generator of a G C A Model Z 1000 Electrofusion apparatus. The conditions normally used in our experiments consisted of 10kV/cm field strength, 9 x 90 ~sec pulses with 0.i sec between pulses. In each treatment, ].2 ml of protoplast suspension was used to get sufficient protoplasts for easy quantification of infection. The solution containing the protoplasts was held at 0°C for at least 10 rain after electroporation; protoplasts were collected by low speed centrifugation (50 x g) and resuspended in several ml of 0.5 M mannitol containing 10 m M CaCI 2. After centrifuging once or twice more, the protoplasts were resuspended in 1.0 ml of a culture m e d i u m devised by Otsuki et al. (1972), except that the mannitol concentration was 0.5 M instead of 0.7 M. The protoplasts were cultured in a 6 c m plastic Petri dish at 28°C under continuous fluorescent illumination of 500 lux. Detection of infected protoplasts After a 24 hr incubation, the percentage of infected protoplasts was determined by an indirect fluorescent antibody staining technique (Sulzinski and Zaitlin 1982) except that the protoplasts were prepared for staining by the method of Otsuki and Takebe (1969). One drop of protoplast suspension was placed on a glass slide coated with a very thin layer of M a y e ~ s albumin (Fisher Chemical), and dried with a hair dryer. The slide was i m m e r s e d in 95% ethanol for 15 min at room temperature to fix the protoplasts. After washing in phosphate buffered saline (PBS, 0.85% NaCl: 0.05M N a phosphate buffer, p H 7.0), for i0 rain, excess P B S around the edge of the drop of protoplasts was r e m o v e d with filter paper. O n e drop of l:10-diluted virus specific antibody solution was added to the protoplasts and incubated for 30 min. After immersing the slide in P B S for 10 rain, the protoplast-containing area was covered with a 20 ~i drop of ]:5 dilution of fluorescein isothiocyanate-conjugated goat anti-rabbit IgG ( Z y m e d Laboratories, Inc., South San Francisco, CA) in P B S for 30 min, followed by two i0 min washes in PBS. The proportion of infected cells was determined using a fluorescence microscope as described by Sulzinski and Zaitlin (1982). (That paper incorrectly denotes the Zeiss

barrier filter, which should be #50). RESULTS Electroporation parameters: field strength, pulse duration, n u m b e r of pulses, and time between pulses A mixture of protoplasts and T M V - R N A was subjected to electroporation at various field strengths; infection was first detected at a field strength of 0.5 kV/cm. As shown in Fig. I-A, 5 k V / c m was found to be sufficient to induce maximal levels of infection; higher field strengths did not increase that percentage. 80

A

~

6o

v

z

0

,o

40

n

_z 20

o o.5

I 2.5

,=,o.~-o

I 5

FIELD S T R E N G T H

I 7.5

I,,

I 10

ii

(kVlcm)

60

B

4C o

z

_o z 2C

PULSE DURATION (~sec)

Fig. 1 Effect of parameters of electroporation on the infection of tobacco protoplasts with T M V - R N A . A; Infection versus field strength. Electroporation: 8 pulses, 90 ~sec/pulse and 0.i sec between pulses. B; Infection versus pulse duration. Electroporation: 10 kV/cm, 9 pulses and 0.1 sec between pulses. Percentage of infection reached a plateau using a single 50 ~sec pulse (Fig. l-B); one pulse was sufficient to get maximal infection (data not shown). Thus, the time b e t w e e n pulses was not a critical factor in initiating infection. All the above-mentioned factors are interrelated in the poration of the protoplast m e m b r a n e ( Z i m m e r m a n and Vienken, 1982). However, even under the most severe electroporation conditions used, (ll kV/cm, 90 ~sec, 9 pulses and 0.5 sec between pulses) the protoplasts did not appear to be damaged, as no decrease in the proportion of infected protoplasts was detected. The effect of T M V - R N A and protoplast concentration on electroporation-mediated infection of tobacco protoplasts Fig. 2 shows the effect of T M V - R N A concentration on the percent of infected protoplasts. A few protoplasts were infected even at 0.I ~g/ml T M V - R N A . In one experiment (Fig. 2-A) 10 ~g/ml was sufficient for maximal infection. Concentrations of T M V - R N A greater than 50 pg/ml caused visible d a m a g e to protoplasts in treatments both with and without electroporation. In a second

59 experiment, concentrations of T M V - R N A from 2 ]jg/ml to 10 ~g/ml were used. The results (Fig. 2-B) show little difference in overall percentage of infected protoplasts in concentrations over that range. 60

using the latter method, the infection percentage was clearly improved, but w e found that removing the protoplasts from the chamber tended to d a m a g e them. As a compromise, w e adopted a procedure involving a first position change by cone removal and a second position change by protoplast removal. Table 1. Effects of position alteration on electroporationmediated infection of tobacco protoplasts with TMV-RNA a

4o v z

2c

Experiment number I 0.1

I

10

I

i

50

100

TMV-RNA (.ug/ml)

Fig. 2 Effect of the concentration of T M V - R N A on the infection of tobacco protoplasts mediated by electroporation. Protoplasts were electroporated, A; i0 kV/cm, 9 pulses, 90 usec/pulse and 0.5 sec between pulses. B; 10 kV/cm, 12 pulses, 90 usec/pulse and 0.i sec between pulses. The effect of protoplast concentration on percent of infection was tested, using the same preparation of protoplasts used in the experiment of Fig. 2-A at 10 ]Jg/ml of T M V - R N A . The percentages of infection at 1.0 X }06 , 1.3 x 105 and 3.2 x 104 protop]asts/ml were 28, 38 and 46%, respectively. Thus, the lower the concentration of protoplasts, the greater the proportion of protoplasts infected. Although with low concentrations of protoplasts, the proportion of infected cells is high, such low concentrations (3.2 x 104/ml) are not practical for further studies. Effects of osmotic concentration, buffer, calcium ion and looly-L-ornithine on infection by electroporation Concentrations of manhitol between 0.3 M and 0.7 M did not influence the percent of infection (da-ta not shown). These results contrast with those reported by Okuno and Furusawa (1978) using conventional infection procedures, which showed that the infection percentage peaked at 0.5 M mannitol. At all mannitol concentrations tested in our experiments, the presence of phosphate buffer (I to 50 mM__) reduced the infection percentage (not shown). W e found that poly-L-ornithine, normally used in protoplast infections (Takebe, 1983) was inhibitory to infection mediated by electroporation (data not shown). Calcium ions were found to have neither an inhibitory nor a stimulating effect on the percentage of infection of protoplasts with T M V - R N A at concentrations up to 8 m M . Effect of alteration of protoplast position in the discharge chamber on the percentage of infection In early experiments, the percentage of infection of protoplasts was never more than 60%, and was usually less. The discharge c h a m b e r contains some "dead space"; thus, some protoplasts are not exposed to the full electric field. To overcome this problem, experiments were performed in which the position of protoplasts in the chamber was changed between electroporation pulses. In Experiment l of Table l, this was effected by removing and replacing the electrical cone of the discharge chamber between sets of pulses. Clearly, this operation increased the percentage of infected cells. In this experiment, more than one position alteration did not generate a higher infection percentage. Thus, another method was used to change the protoplast position; i.e., to remove and return the protoplast suspension to the chamber between sets of pulses. As shown in Experiment 2 in Table i, a single position alteration by either method increased infection percentage equally. However, after the third alteration

Pulse number

Pulse number repeats b

Number of protoplast Infected position protoplasts changes (%)

8

1

0

4 4 2 1 1

2 2 4 8 8

0 Ic 3c 0 7c

36 32 58 60 23 47

0 4 2 2 2 1 1 1

0 1 2 2 2 4 4 4

0 0 0 1c Id 0 3e 3c

0 52 52 69 67 51 70 77

a) Electroporation conditions = i0 kV/cm, 90 ~sec pulse, 0.i sec between pulses. b) By pushing the power supply ON/OFF button, each set of pulses was repeated. c) The position of protoplasts in the solution was changed by removing and replacing the electrical cone of the discharge chamber between each set of pulses. d) The position of protoplasts in the solution was changed by removing and returning the protoplast suspension to the discharge chamber between each set of pulses, using a 1.0 ml Gilson Pipetman and a tip which had been cut off to enlarge the orifice. Infection of protoplasts with C M V - R N A CMV-RNA was introduced into tobacco protoplasts under the same electroporatinn conditions described above for T M V - R N A. Forty-six percent of the treated protoplasts b e c a m e infected with C M V - R N A . In the s a m e experiment with the same batch of protoplasts, 51% of the protoplasts b e c a m e infected with T M V - R N A . Under the same experimental conditions of electroporation, T M V and C M V virions used at either 10 ~g/ml or 100 ~g/ml gave no infection. Effect of electroporation on protoplast viability To determine the effect of electroporation on protoplast viability, the n u m b e r of apparently normal intact protoplasts was counted both before and after electroporation (including two position alterations), and after 24 hr further incubation in culture medium. A mock-~electroporation treatment (no viral R N A ) was also included. The numbers of living protoplasts after electroporation and incubation were reduced to 40% and 27 % of those counted before electroporation, respectively. In the mock-electroporation, the numbers of living protoplasts after electroporation and after incubation were reduced to 39 % and to 31% respectively. This suggests no effect of electroporation per se on protoplast viability,

60 although manipulation of the protoplasts during electroporation procedure appears to be deleterious.

the

D ISC USSIO N Our results indicate that electroporation introduced viral RNA into a high proportion of tobacco protoplasts. In comparison with existing methods of protoplast infection employing polycations and liposomes, electroporation is very simple and reproducible. In contrast, w e inoculated tobacco protoplasts with T M V - R N A in the conventional manner using poly-D-lysine under the conditions described by Otsuki (1982). In our most successful effort, approximately 20% of the protoplasts were infected and the percent of infection was highly dependent on the "quality" of the protoplasts. O n the other hand, the protoplast "quality" did not appear to be as important for infection via electroporation. Infection was demonstrated in 19 of 20 experiments; in the unsuccessful attempt, the protoplasts died. The eleetroporation chamber can a c c o m m o d a t e only a small volume of protoplast suspension because its volume is 0.4 ml (0.2 ml in the latest model). Thus, multiple aliquots of the same sample must be treated to obtain larger numbers of cells. A n additional problem with this method results from chamber design. W e found that position alteration of protoplasts between pulses, either by removing and replacing the cone of the chamber, or by removing and returning the suspension of protoplasts to the chamber, was essential to achieve a high percentage of infection. Unfortunately, both of these methods caused some physical d a m a g e to the protoplasts, so both must be done in as gentle a manner as possible. Upon electrofusinn it is accepted that high concentrations of electrolytes can cause heating which could result in disruption of the fusion process. Thus, protoplasts are normally subjected to electroporation in nonconductive solutions such as mannitol, sorbitol, glucose or sucrose with minimal concentrations of electrolytes ( Z i m m e r m a n n et al., 1984). W e were unable to demonstrate infection of protoplasts with virions of either T M V or C M V . This might be explainable by the observation that the pores in protoplast membranes are approximately 3-4 n m (Benz and Zimmermann, 1981). Thus it is reasonable to expect that neither virions of T M V (18 x 300 nm) nor C M V (30 nm) would be able to pass through. Electroporation is rapidly gaining acceptance as a means of introducing nucleic acids into plant cells (Ecker and Davies, 1985; F r o m m etal., 1985; Langridge etal., 1985; Shillito et al., 1985). In different laboratories, pulse chambers and pulse generators of different design have been utilized. The influence of the type of pulse wave pattern is of particular relevance in establishing parameters for electroporation. S o m e instruments employ a square wave pulse in which the voltage rise and decay is virtually instantaneous; we have used that system here. Others generate a pulse by discharging a capacitor, resulting in a high initial voltage which decays in milliseconds ( F r o m m etah, 1985, Shillito eta]., 1985). Unless one duplicates the equipment described in a given protocol, the exact parameters must be established. Recently, one of us (MZ) has used a m u c h less expensive pulse generator (Pulsar 4i, Frederick I-laer and Co., Brunswick, Maine) to successfully infect tobacco protoplasts with T M V - R N A , although the exact percentage of infected protoplasts has not yet been determined. The use of viral R N A for the optimization of conditions for effective electroporation of R N A presents several advantages over non-viral R N A s (such as m R N A s , or "anti-sense" RNAs). Principally, one can determine the proportion of the protoplasts which take up the R N A , rather than measuring an activity which is a reflection of the total cell population. Furthermore, because only those cells which support the replication of the R N A are scored as positive for uptake, only intact R N A molecules are counted. Electroporation should be valuable for the investigation

of the in vivo interaction between R N A s of different viruses and strains to resolve the phenomena of interference and cross-protection (Palukaitis and Zaitlin, 1984). In preliminary experiments, w e have found that two different R N A s can be sequentially introduced into protoplasts with this method, which would be an essential requirement for cross-protection studies. ACKNOWLEDGEMENTS N.M. is grateful to Drs. F. Motoyoshi, F. Sakai and Y. Otsuki, National Institute of Agrobiological Resources for providing valuable information about tobacco protoplasts. W e thank Dr. P. Palukaitis for providing the inoculum of C M V - W L and Dr. J. R. Aist for assistance with fluorescence microscopy. This research was supported in part by Grants P C M 84-09881 from the National Science Foundation and 83-0018l from the Competitive Grants Program of the United States Department of Agriculture to M.Z. and National Science Foundation Grant P C M 84-10753 to A.A.S. REFERENCES Aoki S, Takebe I (1969) Virology 39:439-448 Benz R, Z i m m e r m a n n U (1981) Biochim Biophys Acta 640:169-178 D a w s o n JRO, Dickerson PE, King JM, Sakai F, Trim A R H , Watts J W (1978) Z Naturforsch 33c:548-551 Ecker JR, Davies R W (1985) Proc First Int Congress Plant Mol Biol, Savannah, p 69 (Abstract) Fraenkel-Conrat I-I, Singer B, Tsugita A (1961) Virology 14:54-58 F r o m m M, Taylor LP, Walbot V (1985) Proc Nail Acad Sci U S A 82:5824-5828 Fukunaga Y, Nagata T, Takebe I (1981) Virology 113:752-760 Gonsalves D, Provvidenti R, Edwards M C (1982) Phytopathology 72:1533-1538 Langridge W H R , Li B J, Szalay A A (1985) Plant Cell Reports, in press. Lot H, Marrou J, Quiot JB, Esvan C (1972) A n n Phytopathol 4:25-38 Motoyoshi F, Bancroft JB, Watts JW, Burgess J (1973) J Gen Virol 20:i77-i 93 Mf]hlbach H P (1982) Current Topics in Microbiology and Immunology 99:81-129 N e u m a n n E, Rosenbeck K (i 972) J M e m b r a n e Biol 10:279-290 N e u m a n n E, Schaefer-Ridder M, W a n g Y, Hofschnelder P H (1982) E M B O J h841-845 Okuno T, Furusawa I (1978) J G e n Virol 4h63-75 Otsuki Y, Takebe I (1969) Virology 38:497-499 Otsuki Y, Shimomura T, Takebe, I (1972) Virology 50:45-50 Otsuki Y, Takebe I, Ohno T, Fukuda M, Okada Y (1977) Proc Natl Acad Sci U S A 74:1813-1817 Otsuki Y (i 982) A n n Phytopath Soc Japan 48:82 (Abstract) Pa]ukaitis P, Zaitlin M (1984) In: Kosuge T, Nester E W (eds) Plant microbe interactions: molecular and genetic perspectives, vol I, Macmillan, N e w York, pp 420-429 Peden K W C , Symons R H (1973) Virology 53:487-492 Potter H, Weir L, Leder P (1984) Proc Natl Acad Sci U S A 81:7161-7165 Sander E, Mertes G (1984) A d v Virus Res 29:215-262 Shillito RD, Saul M W , Paszkowski J, Mfiller M, Potrykus I (1985) Biotechnology 3:1099-i 103 Steinbiss HH, Stabel P (1983) Protoplasma I16:225-227 Steinbiss HH, Broughton W J (1983) Int R e v Cytology Supplement 16:191-208 Sulzinski M A, ZaitlIn M (1982)Virology 121:l 2-19 Takebe I, Otsuki Y, Aoki, S (1968) Plant Cell Physiol 9:115-124 Takebe I (i983) Int R e v Cytology Supplement 16:89-I 11 W o n g TK, N e u m a n n E (1982) Biochem Biophys Res C o m m u n 107:584-857 Zaitlin M (1979) In: Hall TC, Davies J W (eds) Nucleic Acids in Plants, vol II, C R C Press, Boca Raton, F L pp 3]-64 Z i m m e r m a n n U, Vienken J (1982) J M e m b r a n e Biol 67:165-182 Z i m m e r m a n n U, Vienken J, Pilwat G (1984) In: Chayen J, Bitensky L (eds) Investigative microtechniques in medicine and biology, vol l, Marcell Dekker, N e w York, Basel, pp 89-167