purification of cucumber pale fruit viroid*

日植病報 50: 331-338 (1984)

Ann. Phytopath. Soc. Japan 50: 331-338 (1984)

Purification of Cucumber Pale Fruit Viroid*

Ichiro UYEDA**, Teruo SANO** and Eishiro SHIKATA**

上田一郎＊＊・佐野輝男＊＊・四方英四郎＊＊:Cucumber pale fruit viroidの純化＊

Abstract

Cucumber pale fruit viroid was purified from infected cucumber leaves and stems.

Nucleic acids were extracted from the frozen tissue by phenol-CHCl3-SDS, and precipitated

with ethanol. Polysaccharides were removed by ethylene glycol monomethyl ether treatment

and phenolic substrances by cetyltrimethyl ammonium bromide. After 2M LiCl soluble

fraction was treated with DNase, low molecular weight RNAs were obtained. The viroid

was further purified by CF-11 cellulose chromatography and 15% polyacrylamide gel electro-

phoresis. Yield of the purified viroid was about 3-6ƒÊg/200g of tissue. Five percent

polyacrylamide gel electrophoresis of the purified viroid in the presence of urea revealed

two bands both associated with infectivity. (Received December 16, 1983)

Key Words: cucumber pale fruit viroid, gel electrophoresis, purification.

Introduction

Sasaki and Shikata have shown that hop stunt disease is caused by a viroid15-19) and

Takahashi confirmed its viroidal etiology21). They15,17) also reported that hop stunt viroid

(HSV) is mechanically transmissible to several herbaceous plants including cucumber

and tomato. Descriptions of symptoms on cucumber and host range of cucumber pale

fruit viroid (CPFV) found in Europe22) were very similar to HSV. Sano et al.14) conducted

simultaneous comparison of HSV and CPFV, and showed that they were indistinguishable

by host range and symptomatology.

For further comparative studies of the two viroids by their physicochemical proper-

ties, it is essential to extract and purify them in a reasonably large quantity. In our

preliminary attempts, purification of CPFV and HSV by extraction methods of Morris

and Smith6) was unsuitable when a large amount of the infected tissue was handled,

because remained impurties prevented a sample to dissolve in a small volume of a buff-

er before 5% polyacrylamide gel electrophoresis (PAGE). Furthermore, 5% PAGE did

not separate the two viroids from 7S RNA of the host origin.

Although Sanger at al.13) and Muhlbach and Sanger7) reported studiess using purified

CPFV, detailed procedures have not yet been described. In this paper, we describe

*This research was supported in part by Research Grant No.56480037 and No.56360004 from the

Ministry of Education, Science and Culture of Japan.

*Plant quarantine permission number 54-Yokoshoku-1259 and 55-Yokoshoku-1035 .

**Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan北海道大学農学部

332 日本植物病理学会報第50巻第3号昭和59年7月

successful purification of CPFV by modification of the methods of Raymer and Diener11),

and Singh and Sanger20), and by the use of 15% PAGE.

Materials and Methods

Viroids. Cucumber pale fruit viroid was maintained and propagated on cucumber

plants (Cucumis sativus L. cv. Suyo) as previously described14).

Test sample were mechanically inoculated to 4 cucumber plants and infectivity was

quantitatively assayed according to Raymer and Diener11).

Purification of the low molecular weight RNAs. The methods of Raymer and

Diener11), and Singh and Sanger20) were combined and modified as follows.

Two hundreds g of frozen tissues were blended in 300ml of 1M K2HPO4, 4g of

sodium dodesyl sulfate (SDS), 0.4g of bentonite and 40ml of water-saturated phenol

for 5min at room temperature. After centrifugation at 6,500rpm for 20min, aqueous

supernatant was collected. Water-saturated phenol (200ml) and 200ml of CHCl3 were

added and stirred with a magnetic stirrer for 30min at room temperature. Aqueous

supernatant after centrifugation at 6,500rpm for 20min was collected and 2.5 volume

of 95% ethanol containing 0.2M sodium acetate were added. After storage at -30C

overnight, nucleic acids were recovered as a skin at the interface between the lower

phosphate rich aqueous phase and upper ethanol phase after the low speed centrifuga-

tion. Nucleic acids were dissolved in 100ml of distilled water, precipitated again by

2.5 volumes of 95% ethanol, and dissolved in 50ml TES buffer (0.1M Tris, 0.001M

ethylenediaminetetraacetate disodium salt (EDTA), 0.1M NaCl, pH7.5). Fifty ml

each of ethylene glycol monomethyl ether and 2.5M K2HPO4, pH8.3 was then added,

blended at 15,000rpm for 3min, and centrifuged at 3,000rpm for 20min. The up-

per aqueous phase is carefully recovered without disturbing the interphase and diluted

up to 625ml with distilled water to make 0.2M K2HPO4. While stirring with a mag-

netic stirrer, 10ml of 2% cetyltrimethyl ammonium bromide (CTAB) was slowly added.

And CTA-nucleic acids comlexes were precipitated by a low speed centrifugation after

incubation at 6C for 30min. The precipitate was washed twice with 95% ethanol to

remove CTA cations and stored in 70% ethanol at -30C overnight. After a low speed

centrifugation, the precipitate was disolved in TES buffer and an equal volume of 4M

LiCl solution was added. The solution was stood at 0C for 4hr and centrifuged at

3,000rpm for 30min. To the supernatant, which contained 2M LiCl soluble low

molecular weight RNAs and DNA, added 2.5 volumes of 95% ethanol and the nucleic

acid was precipitated by the low speed centrifugation. The precipitate was dissolved

in 10ml of DNase I (Worthington Biochemical) solution (25ƒÊg/ml)1), incubated at 37

C for 30min. The DNA digestion was stopped by phenol-CHCl3 treatment and the

RNAs were precipitated by ethanol. About 10mg of the low molecular weight RNAs

were recovered from 200g of tissue.

Column chromatography. DEAE cellulose (DE-32: Whatman) column chromato-

graphy was carried out at 6C. The column size was 10•~70mm and flow rate was

50ml/hr. The low molecular weight RNAs were fractionated by stepwise elution using

0.02M Tris-HCl, pH7.6, containing 0.3, 0.5, 0.6, and 0.7M NaCl.

Ann. Phytopath. Soc. Japan 50 (3). July, 1984 333

Sephadex G-100 gel filtration was carried out in a 16•~900mm column at 6C.

The flow rate was 10ml/hr. The low molecular weight RNAs were eluted in 0.05M

KCl containing 1% methanol.

CF-11 cellulose column chromatography was carried out at room temperature (25C)

according to the procedure of Franklin2). The low molecular weight RNAs were frac-

tionated by stepwise elution using 35% and 15% ethanol followed by ethanol free buffer.

Polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis under

nondenaturing condition was performed in 15% (acrylamide: bis=30:0.8) gel with 0.04

M Tris-0.02M sodium acetate-0.001M EDTA, pH7.2, buffer3) using 15cm•~19cm•~

1.5mm slab gel apparatus, at 15mA constant for 20hr at 6C. During electrophoresis,

the buffer was recirculated with a peristaltic pump.

Five percent PAGE under denaturing condition was performed in 7M urea, 0.075

M Tris, 0.045M boric acid, and 0.001M EDTA4). Ethanol-precipitated RNA sample

was suspended in 0.01M EDTA, pH7.0, 16% sucrose, 20% dimethyl sulfoxide, heated

at 50C for 10min, and electrophored, at 5mA constant for about 7hr at 40-45C.

After electrophorsis, gels were stained with 0.02% toluidine blue O and destained in

distilled water. The viroid band was cut out and homogenized in 0.5M ammonium ace-

tate, 0.01M magnesium acetate, and 0.1% SDS4). After stirring with a magnetic stirrer

overnight at room temperature, gel slurry was pelleted by a low speed centrifugation

and supernatant was collected. The viroid was precipitated with ethanol and dissolved

in 0.02M Tris-HCl containing 0.3M NaCl, pH7.6. The viroid was then applied onto a

DEAE-cellulose column equilibrated with 0.02M Tris-HCl containing 0.3M NaCl, pH

7.6, washed with the same buffer and eluted with 0.02M Tris-HCl containing 0.6M

NaCl.

Results

Detection of the viroids by polyacrylamide gel electrophoresis

In early experiments, the low molecular weight RNAs were directly eletrophoresed

on PAGE. As previously described, HSV- and CPFV- specific RNA were not detected

on 5% PAGE, because the migration rate of both HSV and CPFV was identical to that

of 7S RNA of host origin. Since it was known that electrophoretic mobility of viroids

in gels changes differently from host RNAs at different concentrations of acrylamide,

the low molecular weight RNAs extracted from CPFV-infected cucumber were electro-

phoresed on 5%, 7.5%, 15%, and 20% polyacrylamide gel in Tris-acetate-EDTA buffer.

These experiments revealeed that CPFV was clearly separated from host RNAs by 15%

20% PAGE. The result of 15% PAGE is shown in Fig. 1. An arrow indicates and

CPFV-specific RNA band that was not detected in extracts of healthy cucumber plants.

The gel was sectiond into 7 slices as indicated in Fig. 1 and assayed on cucumber plants.

Infectivity was mostly associated with the CPFV-specific RNA band.

The concentrated low molecular weight RNA preparation was usually colored and vis-

cous due to contaminated impurities and the bands were frequenly disturbed by them

and we sometimes failed to detect the viroid band after staining with toluidine blue O.

We therefore examined chromatographic procedures for further purification of CPFV.


Fig. 1. Fifteen percent PAGE of the low

molecular weight RNAs of healthy

(A) and CPFV-infected (B) plants

under non-denaturing condition and

its infectivity assay. An arrow in-

dicates the band specific to CPFV-

infected plant. Each well received

about 300ƒÊg of the RNA.

Fig. 4. Sephadex G-100 gel filtration of RNAs from CPFV-infected plant. The low molecular weight RNAs were

purified by DEAE cellulose chromatography and about 5mg of the RNAs were loaded onto the Sephadex column. Five ml/fraction was collected and absorba-nce at 260nm was monitored.

Fig. 2. DEAE cellulose chromatography of nucleic acids from CPFV-infected plant. About 17mg of the nucleic acids prior to DNase treat-ment were loaded onto the column and 5ml/fraction was collected. Absorbance at 260nm of fractions were monitored after 10 folds dilution.

Fig. 3. Fifteen percent PAGE analysis of the selected fractions of DEAE cel-lulose chromatography. One ml portion of the fractions 1 (A), 2 (B), 6 (C), 7 (D), 11 (E), 12 (F), and 16 (G) in Fig. 2 were treated with DN-ase, precipitated with ethanol and electropho-resed under non-dena-turing condition. An arrow indicates CPFV.

DEAE cellulose chromatography

Figs. 2 and 3 show the elution pattern of nucleic acid preparation prior to DNase

treatment and 15% PAGE analysis of the eluted fractions, respectively. The first peak

eluting with 0.3M NaCl contained mostly DNA and the colored impurities and degra-


Fig. 5. Fifteen percent PAGE analy -sis of the selected fractions of Sephadex G-100 chromato-

graphy. The RNAs in 1ml portion of the fractions 13 (A), 15 (B), 18 (C), 20 (D), and 24 (E) in Fig. 4 were precipitated with ethanol and electropho-resed under non-denaturing condition. An arrow indicates CPFV.

Fig. 7. Fifteen percent PAGE analy-sis of the fractions eluted with 35% (A), 15% (B), and 0% (C) ethanol of CF-11 cellulose chromatography.

Fig. 6. CF-11 cellulose chromatography of the low mo-

lecular weight RNAs from CPFV-infected plant.

About 10mg of the RNAs were loaded. Each

peak was assayed for infectivity on cucumber.

Fig. 8. Five percent PAGE under 7M urea-denaturing condition and infectivity assay of purified CPFV. Arrows indicate circular (C) and linear (L) molecules of CPFV.

ded nucleic acids. The second peak eluting with 0.5M NaCl contained almost all part

of the low molecular weight RNAs including the viroid. Thus most of the impurties

were excluded but a large portion of the host RNAs still remained in the eluted viroid

preparation.Sephadex C100 chromatography

Fig. 4 and 5 show the elution pattern and 15% PAGE analysis of the eluted fractions,

respectivery. The peak in exclusion volume contained viroid RNA, some cellular RNA

species of similar sizes to the viroid and highly viscous impurities that remained at

the top of the gel. Such impurities frequently disturbed the electrophoretic migration

of RNAs and made further purification of the viroid difficult.


CF11 cellulose chromatography

Fig. 6 and 7 show the elution pattern and 15% PAGE analysis of the eluted fraction,

respectively. The first peak eluted with 35% ethanol contained a large portion of

tRNA, degraded nucleic acids and impurities (Fig. 7). The second peak eluted with

15% ethanol contained viroid RNA, small amount of tRNA, 5S RNA, and some minor

species of cellular RNAs. The third peak eluted with the buffer alone scarcely

contained nucleic acids. The bioassay showed that almost all of viroid RNA was

eluted with 15% ethanol (Fig. 6).

Polyacrylamide gel electrophoresis of the purified viroid under denaturing

condition

After 15% PAGE and elution of the viroid from the gel, purity of the viroid prepa-

ration was assayed by 5% PAGE under denaturing condition. Two major bands (tolui-

dine blue O stain) were consistently detected after electrophoresis. The bioassay indi-

cated that both bands were highly associated with infectivities (Fig. 8).

It was assumed that the slower migrating and faster migrating bands were circular

and linear molecules of the same viroid, respectively, as has been reported for other

viroids5,9,10,13). However, in some preparation, several minor bands whose migration was

identical to RNAs of healthy tissue were detected indicating a trace of contamination

of cellular RNAs.

Discussion

Sanger and his co-workers7,13) purified CPFV from tomato cv. Rentina. However,

purification from the tomato plants had two disadvantages. First, the infected tomato

cv. Rentina did not produce distinct symptoms under greenhouse conditions. This

makes an additional procedure to assure that inoculated tomato plants are indeed

infected. Cucumber plant is an obvious choice of starting materials, because CPFV

produces distinct symptoms on this host. Second, low molecular weight RNAs prepar-

ations from tomato contained much more colored substances than from cucumber.

Although Sanger's attempts had been unsuccessful12), we were able to purify CPFV

from cucumber as shown in this paper.

It was found that CF-11 cellulose chromatography was the most effective for purifi-

cation among chromatographic procedures, because it could separate not only the impu-

rities but also a large portion of tRNA from the viroid. DEAE cellulose chromatography

could remove the impurities, particularly highly colored materials, but a large portion

of host RNAs eluted together with the viroid. Sephadex G-100 chromatography separa-

ted the viroid almost completely from both tRNA and 5S RNA but unable to remove

the viscous impurities which sometimes made further purification of the viroid by

PAGE impossible. Alternatively, two cycles of 15% PAGE effectively purify CPFV.

Ohno et al.8) also obtained satisfactory results using two cycles of 10% PAGE for

purfication of HSV. The combination of CF-11 cellulose chromatography and 15%

PAGE, or two cycles of 15% PAGE yielded 3-6ƒÊg/200g of tissue of purified CPFV.

The use of 15% PAGE was found to be effective to separate the viroid from the

cellular RNAs of the similar size. Hop stunt viroid migrated almost identically to CPFV


in 15% PAGE, and was purified by the method described here. Yoshikawa and Takaha-

shi23) used 7.5% PAGE for the purification of HSV in which HSV migrated between 7S

and 9S cellular RNAs. However, the RNAs banded sharper in 15% PAGE than in 7.5%

PAGE and were more effectively separated from each other. The migration pattern of

HSV in 10% PAGE8) was similar to that in our 15% PAGE. But we prefered 15%

PAGE because of the same reasons for 7.5% PAGE. Twenty percent PAGE also

effectively separated CPFV and HSV. However, it was difficult to make gels due to a

substantial shrinkage occured during gel formation in our electrophoresis apparatus.

Our purified viroid preparations still contain a trace of cellular RNAs as revealed

by 5% PAGE under denaturing condition. Further purification by 5% PAGE under de-

naturing condition will be necessary when viroids were used in the direct sequencing

or hybridization experiments where the highest purity is required.

Literature cited

1. Erikson, R.L. (1969). In Fundamental Techniques in Virology. Academic Press, New York and London. pp. 451-463.

2. Franklin, R.M. (1966). Proc. Natl. Acad. Sci. USA 55: 1504-1511.3. Loening, U.E. (1967). Biochem. J. 102: 251-257.4. Maniatis, T. and Efstradiadis, A. (1980). Methods in Enzymol. 65: 299-305.5. McClements, W.L. and Kaesberg, P. (1977). Virology 76: 477-484.6. Morris, T.J. and Smith, E.M. (1977). Phytopathology 67: 145-150.7. Muhlbach, H-P and Sanger, H-L (1977). J. gen. Virol. 35: 377-386.8. Ohno, T., Akiya, J., Higuchi, M., Okada, Y., Yoshikawa, N., Takahashi, T. and Hashimoto, J.

(1982). Virology 118: 54-63.9. Owens, R.A., Erbe, E., Hadidi, A., Steere, R.L. and Diener, T.O. (1977). Proc. Natl. Acad. Sci.

USA 74: 3859-3863.10. Palukaitis, P. and Symons, R.H. (1980). J. gen. Virol. 46: 477-489.11. Raymer, W.B. and Diener, T.O. (1969). Virology 37: 343-350.12. Sanger, L.H. (Personal communication).13. Sanger, L.H., Klotz, G., Riesner, D., Gross, H.J. and Kleinshmidt, A.K. (1976). Proc. Natl.

Acad. Sci. USA 73: 3852-3856.14. Sano T., Sasaki, M. and Shikata, E. (1981). Ann. Phytopath. Soc. Japan 47: 599-605.15. Sasaki, M. and Shikata, E. (1977). Proc. Japan Acad. 53B: 103-108.16. Sasaki, M. and Shikata, E. (1977). Ibid. 53B: 109-112.17. Sasaki, M. and Shikata, E. (1978). Ann. Phytopath. Soc. Japan 44: 465-477.18. Sasaki, M. and Shikata, E. (1978). Ibid. 44: 570-577.19. Sasaki, M. and Shikata, E. (1980). Rev. Pl. Protect. Res. 13: 97-113.20. Singh, A. and Sanger, H.L. (1976). Phytopath. Z. 87: 143-160.21. Takahashi, T. (1981). Ibid. 100: 193-202.22. Van Dorst, H.J.M. and Peters, D. (1974). Neth. J. Pl. Path. 80: 85-96.23. Yoshikawa, N. and Takahashi, T. (1982). Ann. Phytopath. Soc. Japan 48: 182-191.


和文摘要

Cucumber pale fruit viroidの純化

上田一郎・佐野輝男・四方英四郎

Cucumber pale fruit viroidを罹病キュウリ茎葉より純化した。核酸はフェノール・クロロホルム・SDS

法で抽出し,エタノールで沈澱させた。多糖類は,エチレングリコールモノメチルエーテル処理,またフェノール物質は,セチルトリメチルアンモニウムブロミド処理で除去した。更に2M LiCl可溶分画をDNase I

で処理して,低分子RNA分画を得た。ウイロイドはその分画よりCF-11セルロースクロマトグラフィー

と15%ポリアクリルアミドスラブゲル電気泳動を用いて純化した。ウイロイドの収量は,3～9μg/200g茎

葉であった。尿素を含む,5%ポリアクリルアミドスラブゲル電気泳動で,純化ウイロイドは2本のバンドに

分かれ,両者に感染性があった。

purification of cucumber pale fruit viroid*

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