Archives of Virology 52, 307--313 (1976) © by Springer-Verlag 1976

Investigations on the Infection of Cucumber Mesophyll Protoplasts with Cucumber Mosaic Virus By I~. I-I. A. COUTTS and K. I£. WOOD Department of Microbiology, University of Birmingham, Birmingham, United Kingdom Accepted August 10, 1976

S uIllinalyy Isolated protoplasts from tile first leaf mesophyll of cucumber plants have been successfully infected in vitro with cucumber mosaic virus (CMV). Virus instability before, during and after inoculation of the protoplasts resulted in low infectivities when extracts were assayed on cowyea; however, viral t~NA extraction improved the bioassay technique. Attempts to optimize inoculation and incubation of protoplasts arc outlined, incorporating the improved assay.

Introduction

Protoplasts from tobacco leaf mesophyll, now used extensively for studies on virus infection and replication (17, 20) have Izeen infected with several spherical plant viruses, including the mu]ticomponent cucumber mosaic virus (12, 13). As a preliminary to using protoplasts from cultivars of cucumber (Cucumis ~ativus L.) which are either susceptible or resistant to CMV in studies on the biochemical basis of resistance, we have demonstrated that, such cells can be prepared in good yield, and are usable in a variety of cultural and metabolic studies (2, 4) including infection by tobacco mosaic virus (g). However, the presence of inhibitors in m a n y host, plants often leads to inefficient assay of CMV infectivity in crude plant extracts (e. g. 5, 6, 15, 18) and similar considerations m a y apply to protoplast studies. This and the relative instability of CMV compared to TMV, produce difficulties in the inoculation and Fostincubation stages concerning interactions of CMV with protoplasts. This communication outlines investigations to achieve a satisfactory measure of developing infectivity in protoplasts from a cucumber cultivar (Ashley) susceptible to CMV.

308

I4. H . A. COUTTS a n d K . R. W o o D :

Materials and Methods Virus and Plant tV!aterial Cucumis sativus L. cv. A s h l e y p l a n t s were g r o w n u n d e r c o n d i t i o n s o u t l i n e d prev i o u s t y (2). T h e P r i c e ' s No. 6 s t r a i n of CMV, p r o d u c i n g severe s y s t e m i c m o s a i c s y m p t o m s in i n t a c t p l a n t s (19), was p r o p a g a t e d e i t h e r i n Nicotania tabacum L. cv. X a n t h i n . c , , or cv. H a v a n a 425, a n d was p u r i f i e d b y t h e m e t h o d of ScoT~ (16), u s i n g t h r e e cycles of h i g h a n d low s p e e d c e n t r i f u g a t i o n . P u r i f i e d p r e p a r a t i o n s h a d d i l u t i o n e n d - p o i n t s of 10-5/t0 -~ w h e n a s s a y e d on p r i m a r y leaves of c o w p e a (Vigna sinensis L. cv. B l a c k e y e ) a n d were s t o r e d (2 m g / m l ) in 0.2 m l a l i q u o t s in 0.005 ~ b o r a t e b u f f e r ( p i t 9.0) a t - - 7 0 ° C.

Bullet and Poly-L-Ornithine ( P L O ) E//ects on Purl/led C M V P L O i n c i t r a t e - b u f f e r e d m a n n i t o l (CBM, 0.01 M, p H 5.0) a n d CMV were m i x e d in v a r i o u s p r o p o r t i o n s a n d t h e d e g r e e of f l o c e u l a t i o n of t h e P L O / C B M / C M V m i x t u r e assessed s p e c t r o p h o t o m e t r i c a l l y (11), e s t i m a t i n g u n c o m p l e x e d v i r u s i n t h e s u p e r n a t a n t b y a b s o r p t i o n a t 260 n m following r e m o v a l of P L O / C M V c o m p l e x b y e e n t r i f u g a t i o n (10,000 r p m , l 0 m i n u t e s ) . (See R e s u l t s . ) Similarly, P L O a n d CMV were m i x e d a t d i f f e r e n t p H v a l u e s a t 25 o C, in e i t h e r p h o s p h a t e - b u f f e r e d m a n n i t o l (PBM, 0.0125 lv~) (8), or CBM. I n f e c t i v i t i e s of t h e m i x t u r e s a f t e r v a r y i n g t i m e i n t e r v a l s were assessed b y local lesion a s s a y on c o w p e a leaves, u s i n g h a l f leaf inoculations, w i t h d i r e c t c o m p a r i s o n s t o o t h e r r a n d o m i s e d t r e a t m e n t s , on t h e o t h e r h a l f leaf; c a r b o r u n d u m w a s u s e d as a n a b r a s i v e t h r o u g h o u t .

Isolation el Protoplasts and In/ection with C M V P r o t o p l a s t s f r o m t h e first leaf m e s o p h y I I of c u c u m b e r were i s o l a t e d as d e s c r i b e d p r e v i o u s l y , u s i n g h i g h e r levels of e n z y m e s r o u t i n e l y , to o b t a i n m a x i m u m isolation efficiency t h r o u g h o u t t h e y e a r (3). I n f e c t i o n was a c h i e v e d u s i n g t h e r e p e a t e d direct m e t h o d (3) t h r e e t i m e s , s u s p e n d i n g CMV i n e i t h e r P B M or C B M a n d p r e - i n c u b a t i n g i n a s h a k i n g w a t e r b a t h a t 80 e x c u r s i o n s / m i n u t e , w i t h a r e c i p r o c a t i n g a c t i o n for 5 m i n u t e s , p r i o r to use for i n o c u l a t i o n . F r e s h l y w a s h e d p r o t o p l a s t s were also prei n c u b a t e d i n a similar w a t e r - b a t h i n e i t h e r P B M or C B M for 10 m i n u t e s p r i o r t o sedimentation (100×g, 6 minutes) and direct infection with the PLO/CMV/PBM o r / C B M m i x t u r e . T h e final c o n c e n t r a t i o n s of p r o t o p l a s t s , C1VIV a n d P L O u s e d d u r i n g t h e i n o c u l a t i o n were 1 to 5 × 10~/ml, 4 ~g/mI, a n d t ~ g / m l respectively. W h e r e C B M was u s e d as t h e i n f e c t i o n buffer, i t ' s final c o n c e n t r a t i o n was 0.01 M; w h e n P B M was u s e d i t ' s c o n c e n t r a t i o n was 0.0125 ~. T h e n o r m a l p H of i n o c u l a t i o n was 5.7, f u r t h e r v a r i a t i o n s in p H a n d b u f f e r m o l a r i t y b e i n g d i s c u s s e d in t h e t e x t ; all i n c u b a t i o n s were c a r r i e d o u t a t 25 ° C.

Culture el Inoculated Protoplasts I n o c u l a t e d p r o t o p l a s t s were c u l t u r e d i n t h e s i m p l e s a l t s m e d i u m of O~SUKI et el. (14) (l 0 m l aliquogs in 100 mI E r l e n m e y e r flasks) i n c l u d i n g m y c o s t a t i n a n d carbenicillin as a n t i b i o t i c s (3). S a m p l e s were c u l t u r e d in g r o w t h c a b i n e t s , a t 25 ° C, w i t h c o n t i n u o u s i l l u m i n a t i o n of 2000 l u x p r o v i d e d b y f l u o r e s c e n t w a r m w h i t e t u b e s . All i n c u b a t e d s a m p l e s were h a r v e s t e d 48 h o u r s a f t e r i n o c u l a t i o n .

In]ectivity Assays A f t e r collection b y c e n t r i f u g a t i o n (100 × g, 8 m i n u t e s ) a n d c o u n t i n g in a h a e m o c y t o m e t e r , i n c u b a t e d p r o t o p l a s t s were i m m e d i a t e l y e x t r a c t e d a t 4 ° C b y one of t w o m e t h o d s . P h o s p h a t e e x t r a c t i o n of cells (0.6 ml) w a s c a r r i e d o u t e i t h e r b y h a n d h o m o g e n i z a t i o n (all glass h o m o g e n i z e r , 5 m i n u t e s ) , or u s i n g a n o v e r h e a d s t i r r e r - d r i v e n h o m o g e n i z e r w i t h P T F E pestle a n d glass b o d y (1 m i n u t e ) , in 1 m l of 0.05 ~ s o d i u m

phosphate buffer (pH 7.5) containing 0.001 ~ EDTA; samples were then assayed immediately. Alternatively, infective viral ribon~icleic acid (RNA) was isolated from the inoculated cells by extracting total cell RNA using a phenol/sodium dodecy] sulphate (SDS) procedure outlined previously (4) using bentonite instead of diethylpyrocarbonate. RIgA present in the aqueous sodium phosphate buffer layer (i ml, 0.075 ~,

Virus Infection of Cucumber Pro~oplasts

309

pH 7.5), was freed from excess phenol by up to eight extractions with ether. InfecVivities of all the extracts were assessed on cowpea plants using half leaf inoculations as outlined previously with direct comparison to standard CMV suspensions (20 ~g/rnl) on the opposite half leaves. When the standard suspension produced less than 150± 75 lesions/half leaf, results were discounted. Results and Discussion

E//ects o/ Bu//er and Poly-L-Ornithine on Purl~led C M V

As with all systems studied previously, pre-incubation of virus with a net negative charge with PLO in a suitably osmotically stabilized buffer is an essential pre-requisite for successful infection of negatively charged plant protoplasts (7, 17). However, for C~V, the formation of any PLO/buffer/CMV complex results in a rapid loss of infectivity, which is not recoverable, a factor which applies to all CMV strains so far tested [e.g. CMV-W, CMV-No. 6; CMV-Y (12)]. These results contrast with those observed with TMV, but are similar to those found with potato virus X (PVX) in a similar system (1). As expected, CMV had lower infectivity at p H 5.0 than at either p H 5.7 or 6.0 following incubation in either buffer (Tables 1, 2). When PLO was introduced into either of the buffers (Tables 1, 2, 3) immediate loss of infectivity occurred in PBM (Table 1), whilst infectivity loss was progressive (though considerable) over a 15 minute period in CBM (Table 2), being rapid at all p H values. The degree of flocculation caused by a range of concentrations of PLO, assessed by treating CMV in CBM, sedimenting eomplexed virus, and determining uncomplexed virus in the supernatant speetrophotometrieally, is indicated in Table 3. I n the case of CBM (0.01 M, pH 5.0), optimal ratios of CMV/PLO for complex formation were between 1/1 and 2/t, while for PBM (0.0125 N, p H 5.7) a ratio of 4/1 was optimal (not shown). The degree of floceulation caused by PLO alone in CBM, was greater than in PBM, although the floeeulaTabte 1. The e/]ect o/poly-L-ornithine ( P L O ) on C M V ~ in/ectivity in 0.0125 ~ P B M b

pH

Sampling time (minutes)

Infectivity (lesion no./8 half leaves) PLO

Control

5.0

0 5 10 15

0 0 0 0

8 5 14 59

5.7

0 5 10 15

0 0 0 0

293 296 487 465

6.0

0 5 l0 t5

0 0 0 0

222 224 182 141

a CMV

concentration, l0 ~xg/ml; CMV:PLO ratio, 2 : 1 phosphate-buffered mannitol

b PBM

310

I4. H . A. COVTTS a n d K . R . WOOD:

t i o n i n C B M g r a d u a l l y i n c r e a s e d o v e r a 15 m i n u t e p e r i o d . S m a l l e r floecules w e r e p r o d u c e d i m m e d i a t e l y i n P B M . T h e s e f l o c c u l e s m a y well a s s i s t i n t h e f o r m a t i o n of the PLO/CMV/PBM complex, which has an infectivity too low to be detected by l o c a l l e s i o n a s s a y o n e o w p e a b u t is n e v e r t h e l e s s h i g h l y i n f e c t i o u s f o r p r o t o p l a s t s . P r o g r e s s i v e loss of i n f e c t i v i t y of C M V i n c o m b i n a t i o n w i t h P L O / C B M m a y b e c o - i n c i d e n t a l w i t h a s l o w e r i n c r e a s i n g f l o c c u l a t i o n o v e r t h e 15 m i n u t e t i m e p e r i o d m e a s u r e d . N o n e of t h e a g g r e g a t e s c o u l d b e d i s p e r s e d w i t h E D T A t o r e l e a s e infectious virus, and this stability possibly reflects buffer anion stability in binding t o P L O m o l e c u l e s , b i n d i n g of P L O t o v i r u s p a r t i c l e s , a n d P L O m o l e c u l e a l i g n m e n t . Previous work with tobacco rattle virus (TRV) in tobacco protoplasts has shown s i m i l a r r e s u l t s (9). T a b l e 2. The e]]ect o] poly.L-ornithine (PLO) on C M V a in/ectivity in 0.01 M C B M b I n f e c t i v i t y (lesion no./8 half leaves)

pH 5.0

5.7

6.0

Sampling time (minutes) 0 5 i0 15 20

E x p . 1°

Exp. 2c

E x p . 3¢

PLO

PLO

Control

PLO

Control

---

85 16 40 83

634 310 351

__a ----

170

.....

4 4 1 0 .

. 4 5

Control 53 100 54 68 . .

--

.

.

273 232

.

0 5 10 15

t22 29

---

1

121

0

--

0

100

1

--

0 5 10 I5

15 3 1 0

96 231 198 278

80 26 i2 1

-----

a CMV c o n c e n t r a t i o n , 10 ixg/ml; s t r a i n W u s e d i n e x p e r i m e n t 3 b CBM citrate-buffered mannitol c C M V : P L O r a t i o s were 2: 1, 1.6:1 a n d 10:1 i n e x p e r i m e n t s 1, 2 a n d 3 r e s p e c t i v e l y ~~ - - , n o t t e s t e d T a b l e 3. The e]]ect o/poly-L-ornithine (tJLO) on complexing C M V in 0.01 M CBM~

(pH 5.0) Ratio CMV/PLO

OptieM d e n s i t y b

% Virus eomplexed

0 0.2:1 t:1 2:1 I0:1 20:1

0.965 0.215 0.280 0.167 0.320 0.900

0 77.7 77.2 82.7 66.8 6.3

a CBM citrate-buffeled mannitol b CMV (200 ~g/ml) in 0,01 M C B M ( p H 5.0), was i n c u b a t e d a t 25 ° C for 10 m i n u t e s w i t h s h a k i n g before e e n t r i f u g a t i o n a t 10,000 r p m for 10 m i n u t e s . T h e optical d e n s i t y of t h e s u p e r n a t a n t was t h e n r e c o r d e d a t 260 n m

Virus Infection of Cucumber Protoplasts

311

Virus In/ection o/Isolated Protoplasts E i t h e r CBM or P B M could be used as buffer to inoculate c u c u m b e r p r o t o p l a s t s w i t h TMV a n d since P B M a p p e a r e d to be less toxic t h a n CBM a n d gave m o r e consistently successful results, it was used in preference to CBM (3). CBM could be used in infection of c u c u m b e r p r o t o p l a s t s w i t h CMV (results n o t shown) b u t it's t o x i c i t y to t h e cells led to its a b a n d o n m e n t , P B M being used instead. I n i t i a l l y the single direct infection was of little use in this s y s t e m b u t t h e m u l t i p l e direct infection p r o c e d u r e used p r e v i o u s l y (3) gave consistent results using three inoculations (Table 4). The o p t i m a l p H of P B M to be used in the inoculation was established using a range of values, assaying i n f e c t i v i t y of p h o s p h a t e e x t r a c t s of the i n c u b a t e d infected p r o t o p l a s t s on cowpea leaves. Good infections were achieved b e t w e e n p i t 5.7 a n d 6.0 in P B N (0.0125 M) a t 25 ° C (Table 5). However, infeetivities of p h o s p h a t e e x t r a c t s were lower t h a n those observed for i n o c u l a t e d tobacco p r o t o p l a s t s (12, 13), t h o u g h OTSUKI a n d TAKEBE a s s a y e d i n f e c t i v i t y after p a r t i a l p u r i f i c a t i o n which, t h o u g h r e m o v i n g inhibitors, is tedious for r o u t i n e use. Table 4. The e//ect o/ repeated inoculation on yield o/C2VIV /rein inoculated cucumber protoplasts Number of inoculations 1

2 3 3a

Period of incubation (hours)

Number of protoplasts/ml ( × 105)

Lesion no./ 8 half leaves

0

3.8

0

48 0 48 0 48

2.5 3.5 2.5 3.5 1.5

1 0 2 0 11

0

3.1

0

48

1.9

42

Samples subjected to B N A extraction (see text) procedures; all other samples were extracted in phosphate buffer. I n test groups using 3 inoculations duplicate samples from the same experiment were used Table 5. The e//ect o / p H o / P B M ~ on yields o/C2~1 V/rein inoculated cucumber protoplasts b pI-I (PBM)

Period of incubation (hours)

Number of protoplasts/ml ( × 105)

Lesion no./ 8 half leaves

5.0

0 48 0 48 0 48 0 48

2.2 1.4 2.7 1.5 4.2 1.2 2.3 0.8

0 20 0 60 0 84 0 2

5.7 6.0 6.7

a PBM phosphate-buffered mannitol b All samples were extracted in phosphate buffer (see text)

312

R. t t . A. COUT~S and K. R. WooD:

Since infectivities of phenol e x t r a c t s of p l a n t tissue c o n t a i n i n g v i r a l R N A r a t h e r t h a n n u c l e o p r o t e i n have often p r o v e d higher t h a n those of c o m p a r a b l e p h o s p h a t e e x t r a c t s (e.g. 10, 15, 18), v i r a l R N A was e x t r a c t e d from i n o c u l a t e d p r o t o p l a s t s w i t h p h e n o l / S D S a n d infectivities c o m p a r e d w i t h c o m p a r a b l e phosp h a t e e x t r a c t s (rather t h a n using a n u c l e o p r o t e i n p u r i f i c a t i o n procedure). As e x p e c t e d , t h e former h a d higher infectivities t h a n t h e l a t t e r , in all e x p e r i m e n t s (Tables 4, 6), a n d clearly g a v e a m o r e efficient m e a s u r e of infective I~NA synthesis. There was, of course, no i n d i c a t i o n of whe$her, in t h e i n t a c t w o t o p l a s t s , R N A h a d been in t h e form of n u c l e o w o t e i n , was u n c o a t e d , or a m i x t u r e of t h e two. I n feetivities were still, however, s o m e w h a t lower t h a n those o b t a i n e d from tobacco p r o t o p l a s t s , where 90 p e r cent of cells were infected (12). F l u o r e s c e n t a n t i b o d y studies n o w in progress, t o g e t h e r w i t h c o n t i n u a l l y i m p r o v i n g p r o t o p l a s t isolation a n d infection procedures, should enable e s t i m a t e s to be m a d e of the efficiency of infection of susceptible cells, and, t a k e n in conjunction w i t h i n f e c t i v i t y a s s a y s described herein, should allow p r e l i m i n a r y comparisons to be m a d e on t h e b e h a v i o u r of p r o t o p l a s t s from resistant, a n d susceptible cultivars t o w a r d s CMV inoculation. Table 6. Virus nucleoprotein and R N A yields /rom cucumber protoplasts inoculated with C M V, using P B M a as inoculation bu/]er

Infectivities (lesion no./8 half leaves)~) Nucleoprotein

~NA

Exp. No.

0 hours

48 hours

0 hours

48 hours

1 2 3

0 0 0

1t 17 12

0 0 0

42 163 67

PBM phosphate-buffered b I n experiments 1, 2, 3 at 3.1× 10 -5 protop]asts/ml, from 1, 2, 3 contained 1.75,

mannitol zero time (0 hours), samples contained 3.25, 4.55 and respectively. A t 48 hours after inoculation samples 3.25, and 2.45 × 10a protoplasts/ml, respectively

Aeknowledflments

Tile authors are grateful to the Royal Society for provision of environmental cabinets (Fisons 140--G2), to I.C.I. for a Research Fellowship (for RHAC), and to t h e Leverhulme Trust for financial assistance.

References

1. COUTTS, R. It. A. : The use of protoplasts as a model system for studying viral infection and replication. Ph.D. thesis, University of Nottingham (t973). 2. C o ~ s , 1R..I t . A., X¥ooD, K. 1~. : The isolation and culture of cucumber mesophyll protoplasts. Plant. Sci. Letters. 4, 189--193 (1975). 3. COUTTS, R. H. A., WOOD, K. R. : The infection of cucumber mesophyll protoplasts with tobacco mosaic virus. Arch. Virol. (in press). 4. Cou~Ts, R. I-I. A., BAR~E~T, A ~ . , WOOD, K. R. : Ribosomal R N A metabolism in cucumber leaf mesophyll protoplasts. Nuc. Acids Res. 2, 1111--1121 (1975).

Virus Infection of Cucumber Protoplasts

313

5. DIENEIt, T. O., S c o ~ , H. A., K ~ : ~ , J. M.: Highly infectious nucleic acid from crude and purified preparations of cucumber mosaic virus (Y strain). Virology 22, 131 141 (1964). 6. FI~&NcKI, 1%.I. B. : Inhibition of cucumber mosaic virus infectivity by leaf extracts. Virology 24, 193--199 (1964). 7. G~oc~, B. W. W., Cou~Ts, 1%. tI. A. : Additives for the enhancement of fusion and endocytosis in higher plant protoplasts: an electrophoretie study. Plant Sci. Letters 2, 397--403 (1974). 8. KuBo, S., H ~ I s o N , B. D., ROJ~I~SON, D. J. : Effect of phosphate on the infection of tobacco protoplasts by tobacco rattle virus. Intervirotogy 3, 3 8 ~ 3 8 7 (1975). 9. KUBO, S., ROBINSON, D. J., HAI/.RISON, B. D., HUTCHESON, A. M.: Uptake of tobacco rattle virus by tobacco protoplasts, and the effect of phosphate on infection. J. gen. ViroI. 30, 287--298 (1976). 10. MISAWA, T. : Histological and cytological observations in plants folIowing infection with cucumber mosaic virus. I%ev. Plant Protect. Res. Tokyo, Japan. 6, 59 74 (1973). 11. MOTOYOSHI, F., BANCROFW, J. B., WATTS, J. W., BURGESS, K. L.: The infection of tobacco protoplasts with eowpea chlorotic mottle virus and its RNA. J. gen. ViroI. 2@, 177 193 (1973). t2. OTSUKI, Y., TAK~BE, I.: Infection of tobacco mesopbyll protoplasts by cucumber mosaic virus. Virology 53, 433--438 (1973). 13. O~SUKI, Y., TAKEBE, I. : Double infection of isolated tobacco mesophyll protoplasts by unrelated plant viruses. J. gen. Virol. 3@, 309--317 (1976). 14. OTSUKI, Y., SmMOMU~A, T., TAKEBE, I.: Tobacco mosaic virus multiplication and expression of the N gene in necrotic responding tobacco varieties. Virology 50, 45--50 (1972). 15. StaLl, GEL, D. E. : Highly infectious phenol extracts from tobacco leaves infected with cucumber mosaic virus. Virology 11, 329--338 (1960). 16. SCOTT,It. A. : Purification of cucumber mosaic virus. Virology 20, 103--106 (1963). 17. TA~:E]3E, I. : The use of protoplasts in plant virology. Am]. ]~ev. Phytopathol. i3, 105 125 (1975). 18. To~Aau, K., UDAOAWA,A. :Infectivity of phenol extracts from leaf tissues infected with strains of cucumber mosaic virus and tomato aspermy virus. Ann. Phytopath. Soe. J a p a n 37, 73--76 (1971). 19. WooD, K. B., Cou:rTS, R. H. A.: Preliminary studies on the I~NA components of three strains of cucumber mosaic virus. Physiol. Plant. Pathol. 7, 139--145 (1975). 20. Z±I~LIN, M., BEAOHY, B. N. : The use of protoplasts and separated cells in plant virus research. Adv. Virus. I~es. 19, 1--35 (1974). Authors' address: Dr. K. /R. WooD, Department of Microbiology, University of Birmingham, P.O.Box 363, Birmingham B 15 2TT, England. I~eceived J u l y 20, 1976