Viroid purification and characterization

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J.S. Semancik
Department of Plant Pathology
University of California (Riverside)
United States of America

HOST PLANTS FOR PURIFICATION AND BIOASSAY

Indicator plants used for viroid purification and bioassay commonly respond with some form of stunting reaction which may be accompanied by leaf symptoms of rugosity, epinasty, mottling and chlorotic spotting, and vein browning. However, since viroid replication may occur in the absence of any discernible symptoms, all inoculated species should be extracted and analysed for viroid content. Plant species suspected of containing viroids can be analysed directly, provided that the extraction conditions necessary to obtain a quality nucleic acid preparation are determined.

In viroid transmission studies, seedlings which in most cases are viroid-free, or vegetatively propagated plant sources which have been made viroid-free by shoot-tip culturing, are preferred host plants. Woody plant species, such as citron and grapevine, can be inoculated by slashing the stem with a razor moistened with inoculum. With more succulent species, such as tomato, a needle or fine Pasteur pipette is used to puncture the hypocotyl of a very young plant at the point where a drop of inoculum has been applied.

Major indicator hosts

• Chrysanthemum (Chrysanthemum morifolium cv. Bonnie Jean) (see Brierly, 1953)
• Citron (Citrus medica cv. Etrog) (see Calavan et al.,1964)
• Cucumber (Cucumis sativus cv. Suyo) (see Sasaki and Shikata,1977; Van Dorst and Peters,1974)
• Gynura (Gynura aurantiaca) (see Weathers and Greer,1968)
• Petunia (Petunia hybrida) (see Weathers et al.,1967)
• Tomato (Lycopersicon esculentum cv. Rutgers) (see Raymer et al.,1964)

TISSUE EXTRACTION AND PURIFICATION

Ultimate success in detecting viroids as discrete bands on polyacrylamide gels is dependent upon the quality of the nucleic acid preparations obtained from infected tissues. High concentrations of phenolic and acidic compounds can seriously interfere with the recovery of all nucleic acid species. Therefore, the composition of the extraction medium must be custom-made to the particular tissue under investigation to assure the consistency of factors such as the maintenance of a pH of about 6.5 to 9.0, the presence of appropriate additives such as polyvinylpyrrolidone to neutralize the effects of polyphenols, and adequate concentration of antioxidants.

Two protocols commonly used for the extraction of a tissue will be presented. The procedures differ basically in the manner in which the aqueous phase from the initial phenolextraction step is treated. Concentration by ethanol precipitation is employed with tissue extracts from plants such as citron and tomato from which good nucleic acid preparations can routinely be recovered. "Trapping" of nucleic acids, including viroid RNA, on CF- 11 cellulose has been customized for use with direct extraction of grapevine tissue or others from which nucleic acids are difficult to recover.

Even though the primary focus of the procedures presented here is the analysis of citrus, the alternative approach as indicated for "grapevines" should be employed if nucleic acid preparations are either difficult to obtain or are of poor quality. The designation of the procedures for "grapevines" simply indicates the plant tissues for which the technique was developed and does not imply an exclusive application. To date, grapevine tissues have been the most challenging for recovery of nucleic acid preparations of high quality and adequate quantity for analysis of viroid content. Therefore, the information developed for this tissue may become valuable for the analysis of particular citrus species.

Unless one can demonstrate the recovery of a typical profile of host nucleic acids, it is difficult to evaluate the relative concentration or even the very presence of viroid molecules. Therefore, it is a good practice to inspect the presence and relative concentration of particularly the 4S and 5S RNA components of 2M LiCl soluble nucleic acids following electrophoresis in the native polyacrylamide gels.

Materials

• Infected tissue: fresh tip tissue which is actively growing and collected at least two to six weeks post-inoculation of herbaceous hosts and two to six months post-inoculation of woody species is preferred. If tissue is to be collected and stored for extraction at a later time, it should be powdered in liquid nitrogen and held at -20C.
• Extraction medium (EM- I) for citrus species and herbaceous plants:

Buffer (0.4 M Tris-HCl, pH 8.9)
SDS (sodium dodecyl sulfate), 1%
EDTA (ethylenedinitrilotetraacetate), 5 mM, pH 7.0
MCE (mercaptoethanol), 4%

• Extraction medium (EM-2) for grapevines and plants containing a high concentration of phenols and acidic compounds:

Buffer (0.5 M Na2SO3)
SDS (sodium dodecyl sulfate), 1%

• Resuspension medium (RM): TKM buffer:

Tris 10 mM
KCl 10 mM
MgCl2 0.1 mM
Adjust to pH 7.4 with HCl

• LiCl, 4 M
• PVP (polyvinylpyrrolidone) 20% (4X stock)
• Sodium acetate, 3 M, pH 5.5
• Phenol (water-saturated) adjusted to pH 7 with 1 N NaOH
• Ethanol, 95-100%
• CF- 11 fibrous cellulose powder (Whatman)
• STE buffer 108 stock(1.0 M NaCl, 0.50 M Tris-HCl, pH 7.2, 10 mM EDTA)
• Dialysis tubing
• Homogenizer: Virtis, high speed (50 000 rpm); "food" blender, low speed (15 000 rpm)

Note: Yield of viroid can be significantly affected by the method of homogenization.

• Centrifuge, low speed (5 000-10 000 rpm or 6 000-12 000 g), refrigerated
• Magnetic stirrer at 4C.

Extraction of citrus and most herbaceous species

1. Grind tissue in pre-cooled EM- 1 and phenol in a proportion equal to 1 g tissue: 1 ml EM- 1:3 ml phenol in an ice-bath if possible.

Note: Phenol can cause severe burns; therefore, protection for hands (disposable gloves) as well as a plastic barrier should be employed to guard against accidents during homogenization.

2. Transfer to centrifuge tubes or bottles and centrifuge for 20 min at 7 000-12 000 g.

3. A clear but pigmented aqueous layer is found over a solid interface of plant debris and a lower heavily pigmented phenol phase. The plant debris may also form a pellet below a liquid bilayer between the aqueous and phenol phases.

4. Remove aqueous layer and add a 1/10 volume of 3 M sodium acetate, pH 5.5 and a minimum of three volumes of 95-100 percent ethanol. Hold at -20C for 30 min or an indefinite period.

5. Centrifuge for 20 min at 7 000-12 000 g.

6. Discard supernatant and drain pellets containing nucleic acids until excess ethanol is removed.

7. Cover pellets with a minimum volume of RM (1-10 ml/5-100 g fresh weight tissue) and resuspend with agitation.

8. Transfer slurry to dialysis tubing.
Note: At this point the solution may appear quite turbid and particulate, but should clear considerably with dialysis.

9. Dialyse with rapid stirring on a magnetic stirrer at 4C overnight against 1 L of RM.

10. Remove sample from dialysis tubing to centrifuge tubes and add one volume of 4 M LiCl. Hold at 4C for 4 h or overnight.

11. Centrifuge for 20 min at 7 000-12 000 g.

12. Retain supernatant containing LiCl soluble nucleic acids (mainly DNA, 4S and SS RNA, dsRNAs, and viroids). Discard pellet of LiCl insoluble nucleic acids (mainly ribosomal RNA).

13. Add a minimum of three volumes of 95-100 percent ethanol and hold at -20C for 30 min or overnight.

14. Centrifuge for 20 min at 7 000-12 000 g.

15. Decant and drain ethanol from pellets and dry in vacuo.

16. Resuspend pellets in an appropriate volume of RM (100 ml/5g fresh weight of tissue).

17. Store at -20 to -80C.
Note: These preparations are sufficiently purified for routine viroid detection procedures and infectivity tests. However, the quality of the analysis will be markedly improved by further processing by cellulose chromatography.

Extraction of grapevines and tissues from which it is difficult to recover nucleic acids

1. Grind tissue as indicated above substituting EM-2.

2. Centrifuge as above.

3. Note as above.

4. Remove aqueous phase from above interface and lower phenol layer.

5. Make solution to 35 percent ethanol and 1X STE with stirring. Add dry CF-11 cellulose powder(1g/5 g fresh-weight tissue extract). Stir for 2 h or overnight at room temperature.

6. Collect cellulose by centrifuging at 7 000 g for 10 min.

7. Discard supernatant and wash cellulose pellet with a solution of 30 percent ethanol in 1X STE buffer with agitation.

8. Collect cellulose as in step 6.

9. Repeat washing procedure with 30 percent ethanol -STE solution two or three times until all traces of pigmented materials have been removed from the wash solution.

10. With the cellulose in 30 percent ethanol-STE, form a chromatography column and continue to wash cellulose with three to four void volumes of 30 percent ethanol-STE.

11. Elute bound nucleic acids by eluting with two to three void volumes of STE buffer, collecting the eluant in a serial manner and not as a single batch.

12. Add 10 percent volume of 3M sodium acetate, pH 5.5, and a minimum of three volumes of ethanol. Let solution stand at -20C for 30 min or longer as convenient.

13. Collect precipitated nucleic acids by centrifugation at 12 000 g for 20 min.

14. Discard supernatant and allow pellet to drain until reasonably dry.

15. Resuspend pellet in minimum amount of RM buffer.

16. Add one volume of 4M LiCl and let stand at 4C for 4 h or overnight.

17. Centrifuge at 12 000 g for 20 min. Retain supernatant of 2M LiCl soluble nucleic acids.

18. Add a minimum of three volumes of ethanol to the supernatant and let stand at -20C for 30 min or longer.

19. Collect precipitated nucleic acids by centrifugation at 12 000 g for 20 min.

20. Discard supernatant, drain liquid from pellet and dry in vacuo.

21. Resuspend pellet in a minimum volume of RM buffer usually in the range of 100 ml/5-10g fresh weight of tissue.

22. Store samples as aliquotes at -20 to -80C prior to analysis by polyacrylamide gel electrophoresis or infectivity.

(Duran-Vilaetal.,1986;Semanciketal.,1975;1987.)

CF-11 CELLULOSE CHROMATOGRAPHY

This technique can be utilized routinely as a preparative procedure for the removal of DNA and other pigmented components of viroid containing LiCl soluble nucleic acid preparations. The property of selective binding of viroid RNA at specific ethanol concentrations can also be exploited in recovering viroids from tissue extracts, as was demonstrated in the "trapping" procedure presented in the previous section.

More recently, an analytical approach to CF-11 cellulose chromatography has been introduced (Semancik,1986) to characterize different viroid RNAs by serial elusion with an ethanol gradient. This procedure can be utilized to remove contaminating host RNAs from viroid preparations as well as to separate individual viroids with selective elusion by different ethanol concentrations.

Materials

• CF- 11 cellulose powder, fibrous (Whatman)
• STEbuffer(0.1 MNaCl,1 mMEDTA,0.05 M Tris-HCl, pH 7.2)
• 95-100 percent ethanol
• syringe barrels (disposable) or chromatography columns
• GF/C glass microfibre filter discs (Whatman 2.4 cm)

Preparative chromatography

1. An aqueous sample containing nucleic acids is made to 35 percent ethanol in STE buffer.

2. Apply the solution to a CF-11 cellulose column which has been equilibrated with 35 percent ethanol-STE.
Notes: (a) The amount of cellulose used is dependent upon the amount of nucleic acid in the preparation. A proportion of 1 - 10 g cellulose/5100 g fresh-weight extraction is usually adequate.
(b) A "trapping" procedure can also be employed with dry cellulose added directly to the aqueous phase from a phenol extraction made to 35 percent ethanol-STE.

3. Wash the cellulose with sufficient 30 percent ethanol-STE to remove all traces of colour from the cellulose or with a volume equivalent to at least four to six column void volumes.

4. Elute the nucleic acids retained on the column with two to four void volume equivalents of STE (0 percent ethanol).

5. Precipitate nucleic acids with addition of 1 /10 volume of 3 M sodium acetate, pH 5.5 and at least three volumes of 95-100 percent ethanol and hold at -20C for 30 min or longer.

6. Centrifuge for 20 min at 12 000 g.

7. Dry pellet in vacuo and resuspend in TKM buffer (resuspension medium from extraction procedure).

Analytical chromatography

1. Nucleic acid sample from the extraction procedure or preferably after pre-treatment on a preparative CF- 11 column is made to 35 percent ethanol-STE.

2. Apply to a chromatography column containing an adequate amount of CF- 11 cellulose equilibrated with 35 percent ethanol-STE.

3. Wash column with 35 percent ethanol-STE (four to six column void volumes).

4. Elute with 25 percent ethanol-STE, collecting two to four column void volumes. Retain eluant. 5. Wash column with 25 percent ethanol-STE (four to six column void volumes).

6. Elute with 20 percent ethanol-STE, collecting two to four column void volumes. Retain eluant. 7. Continue alternating wash and elusion cycles with either a progressively reduced ethanol concentration, such as in 5 percent increments, or a decreasing linear ethanol gradient to a final elusion in STE buffer.

8. Precipitate nucleic acids with addition of 1 /10 volume of 3 M sodium acetate, pH 5.5, and at least three volumes of 95-100 percent ethanol and hold at -20C for 30 min or longer.

9. Centrifuge for 20 min at 12 000 g.

10. Dry pellet in vacuo and resuspend in RM buffer.

11. Analyse by sequential PAGE under native and denaturing conditions. (Barber,1986; Duran-Vila et al.,1986; Franklin,1966; Semancik,1986.)

POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE)

Optimum resolution of viroid RNA is obtained by a sequential gel electrophoresis procedure involving migration of the sample into a standard gel (5 percent PAGE), followed by excision of a piece of the gel which is then placed in contact with a second, denaturing gel (dPAGE) containing 8 M urea. This procedure exploits the unique properties of the single-stranded closed circular structure of the viroid for the separation of a distinct band. Placement of the excised gel piece in contact with the top (Semancik and Harper,1984) or the bottom (Schumacher et al.,1983) of the denaturing gel and migrating to the anode will produce similar results.

The discontinuous pH dPAGE (Rivera Bustamante et al.,1986) with the gel cast at pH 6.5 (TAE buffer), but migrated in a pH 8.3 running buffer (TBE buffer), enhances the separation between the circular and linear molecular forms of the viroid. In addition, the background of host nucleic acids is reduced, which aids in the recovery of pure viroid preparations for physical characterization and hybridization analysis.

Verification for the detection of a suspected viroid can be made by a PAGE analysis sequence involving:

• non-denaturing 5 percent PAGE: excise a strip of the gel in the "viroid zone" as defined by citrus exocortis viroid (CEV) and avocado sun blotch viroid (ASV, ASBV);
• dPAGE (pH 6.5): excise any slowly migrating band suspected of being a viroid circular form;
• dPAGE (pH 8.3): resolution of two distinct bands containing the viroid circular form and the linear molecular form, generated from circles during electrophoresis.

Staining with ethidium bromide to visualize nucleic acid bands is necessary when gels are to be subjected to a second electrophoresis and/or when biologically-active viroid is to be recovered. Increased sensitivity of detection can be achieved with silver nitrate staining. However, this procedure renders the viroid inactivated and immobilized in the gel.

Materials

• Electrophoresis chamber and casting apparatus: glass plates, spacers, clamps, sample-well comb. Variations in size are available through commercial sources or can easily be custom fabricated.
• Power supply(100mA, 1000 volt or greater capacity)
• Ultraviolet transilluminator
• Polaroid camera
• Stock solutions for 5 percent gels:

Stock A:

Acrylamide 30.00 g
Bisacrylamide 0.75 g
Dissolve in distilled water, bring to 100 ml and filter.

Stock B:
Tetramethylethylenediamine (TEMED) 2 ml to 100 ml with distilled water

Stock C:
Tris 120 mM
Sodium acetate.3H2O 60 mM
Sodium EDTA 3 mM
Dissolve in distilled water, adjust to pH 7.2 with glacial acetic acid.
Note: This solution is equal to 0.3X Stock D, therefore it can also be made by diluting 30 ml of Stock D to 100 ml with distilled water.

Stock D:
TAE buffer, pH 7.2(10X)
Tris 400 mM
Sodium acetate.3H2O 200 mM
Sodium EDTA 10 mM
Dissolve in distilled water, adjust to pH 7.2 with glacial acetic acid.
Note: Since Stock D is a 108 concentration, it should be diluted before use as a running buffer.

Stock E:
Ammonium persulfate (10 percent)
2.5 g in 25 ml H2O prepared fresh (weekly)

Stock F:
TAE buffer, pH 6.5
Tris 120 mM
Sodium acetate.3H2O 60 mM
Sodium EDTA 3 mM
Dissolve in distilled water, adjust to pH 6.5 with glacial acetic acid.
Denaturing gel buffer (TBE, pH 8.3 10X) stock

Tris 225 mM Boric acid 225 mM Sodium EDTA 5 mM Dissolve in distilled water, no adjustment of pH should be necessary. Dilute tenfold for use as a running buffer(1 X).

• Urea
• Glycerol (60%)
• Migration tracking dyes:
Bromophenol blue 0.3% in 60% glycerol Xylene cyanol 0.3% in 60% glycerol
• Ethidium bromide stock staining solution (5 mg ml) (30 ml/200 ml H2O for staining gels)
• Silver staining solutions:

Ethanol (50%) + acetic acid(10%) Ethanol (10%) + acetic acid (1%)
Silver nitrate(12 mM)
Potassium hydroxide (0.75 M) + formaldehyde (0.28%)
Sodium carbonate (0.07 M)
Native 5% PAGE

1. Assemble glass form to receive polymerization solution.
2. Mix contents of two beakers containing the following solutions in the indicated amounts or similar proportions:

Beaker 1

12.0 ml distilled water
10.0 ml Stock C
2.4 ml Stock B

Beaker 2
5.0 ml Stock A
0.48 ml ammonium persulfate

3. Fill form, place sample-well comb, and let stand for 30 min.

4. Withdraw sample-well comb and lower spacer. Attach to chamber and fill electrode reservoirs with 1/10 dilution of Stock D.

5. Mix samples with about 1/4 volume of glycerol and load into wells with fine-tip Pasteur pipettes. Load outermost wells with mixture of tracking dyes.

6. Apply constant current, 54 mA, at 4C for 2.53 h or until bromophenol blue dye has migrated about 8 cm and xylene cyanol has reached about 4 cm.

Note: The xylene cyanol is a useful marker for CEV since the migration of both molecules is very similar native conditions.

7. Remove gel from the chamber and form. Soak with gentle agitation in the ethidium bromide staining solution for 10 min.

8. View the gel directly over a UV transillumination source. Cut horizontal strip as defined by "viroid zone" (CEV-ASV) or smaller, depending upon viroid, and transfer to denaturing gel.

Denaturing PAGE (pH 6.5)

1. Assemble glass form for polymerization solution.
2. After the urea in beaker 1 is dissolved, mix rapidly the contents of two beakers containing:

Beaker 1

14.4 g urea
7.0 ml H2O
3.0 ml Stock F (TAE pH 6.5)
5.0 ml Stock A
dissolved on low heat

Beaker 2

2.5 ml Stock B
0.5 ml ammonium persulfate

3. Immediately fill form, leaving a flat surface with sufficient space for the excised native gel piece, and allow to stand for a minimum of 1 h.

4. Remove lower spacer and attach to chamber. Do not add buffer or any liquid to the gel surface until immediately prior to use.

5. After section has been removed from native gel, fill electrode reservoirs and cover top surface of gel with denaturing gel buffer (TBE, pH 8.3, IX).

6. Float excised section on to the top of the denaturing gel, making as close a contact as possible.

7. Add a few drops of xylene cyanol-glycerol mix next to the outer edges of gel strip.

8. Apply constant current, 15 mA, at 24C for about 4 h or until the xylene cyanol tracking has migrated to within 0.5 cm of the bottom of the gel.

9. Remove gel from form and stain with either:

• ethidium bromide:

a) for additional dPAGE (pH 8.3 gel) to confirm circular and linear forms;

b) for elusion of viroid bands for infectivity or for use as templates for cDNA probes; or:

• silver nitrate for maximum sensitivity of detection.

Denaturing PAGE (pH 8.3)

1. Follow the same set-up and running procedure as presented above.

2. Mix rapidly the contents of two beakers containing

Beaker 1

14.4 g urea
7.0 ml H2O
3.0 ml TBE buffer pH 8.3 10X
5.0 ml Stock A
dissolved on low heat

Beaker 2

2.5 ml Stock B 0.5 ml ammonium persulfate 3. Stain completed gel with either ethidium bromide as before or silver nitrate.

Silver staining

1. Gel can be stained with silver directly or following ethidium bromide staining without additional treatment.

2. Soak gel at room temperature in solution of 50% ethanol + 10% acetic acid for at least 1 h with gentle shaking. Overnight soaking can sometimes improve the background.

3. Soak gel at room temperature in solution of 10% ethanol + 1% acetic acid for 1 h with gentle shaking.

4. Soak in solution of 12 mMAgNO3 for 1 h with gentle shaking.

5. Rinse thoroughly (three times) with distilled H2O.

6. Rinse rapidly with small volume of developer solution (0.75 M KOH + 0.28% HCHO) and discard solution.

7. Add fresh developer solution (100-200 ml) and observe until bands appear, usually within 20 min.

8. Add excess distilled water and allow gel to expand. This process reduces the background and improves the quality of photographs.

9. Developing reaction can be stopped with 0.07 M Na2CO3.

10. Photograph gel over a light-box using Polaroid film. (Igloi,1983; Morris and Wright,1975; Rivera-Bustamente et al.,1986; Schumacher et al.,1983; Semancik and Harper, 1984.)

INFECTIVITY OF NUCLEIC ACID FRACTIONS AND VIROID MOLECULES

The infectivity of a viroid-containing sample can be influenced by the quality of the preparation. In many cases, viroid transmission by highly purified preparations can be more difficult than by a more complex, less purified preparation. This may result, in part, from the presence of host nucleic acids which may function to protect the viroid molecule from inactivation.

Therefore, a sample such as a 2 M LiCl soluble fraction may be valuable to demonstrate the transmission properties and host range of suspected viroid-containing preparations.

Nevertheless, an essential proof for the detection of a viroid is the transmissibility of the putative viroid-like molecule. This can be provided by the recovery of the unique, transmissible viroid structure, the single-stranded circular RNA molecule, in highly purified form followed by transmission to a host plant.

Electro-elution of the circular forms of viroids, as detected in denaturing PAGE by ethidium bromide staining, has proved to be a highly efficient procedure for the recovery of biologically-active, pure viroid.

Materials

• PAGE gel piece containing the viroid
• Electro-elution buffer (EB) (1/50 dilution of Stock D in PAGE procedure):

8.0 mM Tris
4.0 mM sodium acetate 0.2 mM EDTA
adjusted to pH 7.2 with acetic acid

• Electro-elution apparatus:

a) A chamber can be constructed to accommodate a piece of dialysis tubing filled with EB into which the gel piece has been introduced. When placed in an electrical field the viroid will migrate from the gel but be retained in the liquid phase inside the tubing.

b) A commercial apparatus (Unidirectional Electroelutor Model UEA) which does not require the dialysis tubing containment procedure is available from International Biotechnologies, Inc.

• Power supply (25 mA, 250 volt)
• 3 M sodium acetate, pH 5.5
• Ethanol

Electro-elution

1. Prepare gel piece to be eluted within dialysis tubing or according to IBI instructions.

2. Apply about 125 constant voltage for 30 min at room temperature.

3. Withdraw buffer sample containing eluted viroid, add 1110 volume of 3 M sodium acetate, pH 5.5, plus at least three volumes of ethanol and hold at -20C for 30 min or longer.

Note: The gel piece can be checked for incomplete elusion of viroid by restaining with ethidium bromide and viewing over a UV transilluminator. If viroid still remains in the gel piece, the elusion procedure can be repeated.

4. Centrifuge sample at 12 000 g for 20 min. Pellets may be extremely small or invisible. Nevertheless, sufficient viroid to be detected by PAGE and silver staining or infectivity can be recovered many times.

5. Dry decanted centrifuge tubes in vacuo and resuspend pellets in appropriate volume of TKM buffer (RM) or desired medium.

REFERENCES

Barber, R.1966. Biochim. Biophys. Acta, 114: 422-424.

Brierly, P.1953. Plant Dis. Rep., 37: 343-345. Calavan, E.C., Frolich, E.F., Carpenter, J.B., Roistacher, C.N. & Christiansen, D.W.1964. Phytopathol., 54: 1359- 1362.

Duran-Vila, N., Flores, R. & Semancik, J.S. 1986. Virol., 150: 75-84.

Franklin, R.1966. In Proc. Nat. Acad. Sci. USA, 55: 1504-1511.

Igloi, G.1983. Anal. Biochem., 134: 184- 188.

Morris, T. & Wright, N.1975. Amer. Potato J.,52: 57-63.

Raymer, W.B., O'Brien, M.J. & Merriam, D. 1964. Am. Potato J., 41: 311-314.

Rivera-Bustamante, R.F., Gin, R. & Semancik, J.S.1986. Anal. Biochem., 156: 91-95.

Sasaki, M. & Shikata, E.1977. In Proc. Jap.Acad., 55: 103- 108.

Schumacher, J., Randles, J.W. & Riesner, D. 1983. Anal. Biochem., 135: 288-295.

Semancik,J.S.1986. Virol., 155: 39-45.

Semancik, J.S. & Harper, K.L.1984. Proc. Nat. Acad. Sci. USA, 81: 4429-4433.

Semancik, J.S., Morris, T.J., Weathers, L.G., Rodorf, B.F. & Kearns, D.R.1975. Virol., 63: 160-167.

Semancik, J.S., Rivera-Bustamante, R. & Goheen,A.C.1987.Am.J.Enol.Vitic., 38:3540.

Van Dorst, H.J.M. & Peters, D.1974. Neth. J. Plant Pathol., 80: 85-96.

Weathers, L.G. & Greer, F.C. Jr.1968. Phytopathol., 58: 1071.

Weathers, L.G., Greer, F.C. Jr & Harjung, M.K.1967. Plant Dis. Rep., 51: 868-871.

FIGURE185 Diagram of the procedure for tissue extraction and purification

FIGURE 186 Diagram of the procedure for CF-11 cellulose chromatography

FIGURE 187 Diagram of the procedure for polyacrylamide gel electrophoresis (PAGE)

FIGURE 188 Diagram of the procedure for denaturing polyacrylamide gel electrophoresis (dPAGE)

FIGURE 189 Selection of citrus tissue for extraction

FIGURE 190 Selection of grapevine tissue for extraction

FIGURE 191 Components of extraction medium used for citrus

FIGURE 192 Components used in purification and concentration of nucleic acids

FIGURE 193 Components for resuspension of nucleic acid pellets

FIGURE 194 Components of extraction medium used for grapevines

FIGURE 195 Low speed refrigerated centrifuge

FIGURE 196 Virtis high-speed homogenizer

FIGURE 197 Apparatus used to agitate CF-11 cellulose for "trapping" of nucleic acids

FIGURE 198 Apparatus used for preparative or analytical cellulose chromatography

FIGURE 199 Apparatus used for polyacrylamide gel electrophoresis (PAGE) and denaturing PAGE (dPAGE)

FIGURE 200 Polaroid photography apparatus and transilluminator with ultraviolet light source for visualizing nucleic acid bands stained with ethidium bromide

FIGURE 201 Polaroid photography apparatus and visible light source for observing nucleic acid bands stained with silver

FIGURE 202 As for Figure 201 (with room lights off)

FIGURE 203 Silver-stained denaturing polyacrylamide gel as seen through the Polaroid viewer

Immunosorbent electron microscopy (ISEM) and antibody coating

Prof. G.P. Martelli
Dipartimento di Patologia Vegetale
Universit di Bari,
Italy

The principle of immunosorbent electron microscopy (ISEM) is the selective trapping of plant viruses on to electron microscope grids precoated with a specific antiserum. This technique has been described in a number of papers end review articles (Derrick,1973; Milne and Luisoni,1977;Garnseyetal.,1979;Roberts and Harrison,1979; Van Regenmortel,1982; Milne and Lesemann,1984) to which the reader is referred for comprehensive information.

ISEM may be combined with antibody coating (often referred to as "decoration"), a procedure whereby virus particles trapped on the microscope grid are exposed to the homologous antiserum, thus becoming visibly covered with antibody molecules.

The consensus is that ISEM is highly reliable (there are virtually no false positives), as sensitive as ELISA, fast (results can often be obtained within one or two hours), and operationally simple (it requires tools and reagents readily available in most laboratories).

Unfortunately, ISEM requires an electron microscope and is not suitable for large-scale routine testing. Specimens for ISEM, however, can readily be prepared in laboratories with no electron microscope facilities and then be shipped for observation (even over long distances) to properly equipped institutions.

BASIC TOOLS AND REAGENTS

The following are required (see Figures 204206):

•porcelain mortars 6 cm in diameter or smaller, or glass microscope slides and glass rods;
• carborundum powder(600-mesh), or quartz sand;
• bench centrifuge with relative glass or plastic conical tubes;
• fine straight-point tweezers;
• Petri dishes 9 cm in diameter;
• bars of dental wax, silicone-treated paper, or parafilm;
• Pasteur pipettes;
• electron microscope grids (400 mesh) covered with carbon film;
• protectants:

2.5 to 5% aqueous solution of nicotine
2% aqueous polyvinylpyrrolidone (PVP)
1% aqueous polyethylene glycol (PEG), MW 6000-7500;

• phosphate buffer 0.1 M, pH 7.0
• stock solution (IM):

(a) 136.09 g of KH2PO4 in distilled water to 1 litre
(b) 26X.077 g of Na2HPO4 in distilled water to 1 litre

Mix 3.86 ml of solution

(a) with 6.14 ml of solution
(b) and dilute tenfold.

• distilled water;
• staining solutions:

1-2% uranyl acetate in distilled water, pH not adjusted;
2% sodium or potassium phosphotungstate in distilled water, adjusted to pH 7 with NaOH or KOH;

• appropriate antiserum.

PREPARATION OF TISSUE EXTRACTS

Extracts may be prepared from tissues of different organs of field- or greenhouse-grown plants (leaves, roots, bark, dormant or breaking buds) or vectors (insects, nematodes). Plant tissues (usually 100-200 ma) are ground in a mortar in the presence of carborundum powder or quartz sand and 0.3-0.5 ml of phosphate buffer or, especially with grapevine and stone fruits, one of the above protectants (nicotine, PVP, PEG). When a smooth paste is obtained, 0.3-0.5 ml of buffer are added and the sample is ground again. The slurry is transferred to a centrifuge tube and centrifuged(I 500-2 000 g). The supernatant fluid is collected and used.

If a centrifuge is not available, tissue extracts can be further diluted with phosphate buffer to 1:15-1:20 with respect to tissue weight, and used as such.

Insect and nematode vectors are crushed with a glass rod on a glass slide in a droplet of buffer or protectant. A droplet of buffer is then added and the extract is used as such.

ANTISERUM DILUTIONS

The purpose of using an antiserum is twofold: coating EM grids for trapping virus particles and "decorating" virus particles by attachment of antibody molecules to the antigenic sites of the particles. Crude antisera are perfectly suitable for both uses, provided that they are properly diluted.

• Coating of grids: dilute antiserum near or above its end point (usually 1:1 000 to 1:5 000) with buffer.
• Decoration of virus: dilute antiserum to 1:10-1:100 with buffer.
(Use of freshly diluted antisera is advisable.)

PRECOATING OF EM GRIDS

In certain cases, precoating of EM grids with protein A, a bacterial wall protein that binds specifically to the basal part (Fc portion) of antibody molecules, can be advantageous. Protein A allows trapping of more virus particles owing to the richer antibody layer on the grid. It also allows the use of undiluted, low-titre(1:81 :16) antisera which would not be suitable after high dilution as required by ordinary ISEM.

Protein A is diluted in phosphate buffer at a final concentration of 10-100 g/ml, a drop is placed on the grid for 5 min at room temperature, and the excess is rinsed off before exposure to antiserum.

ANTISERUM COATING OF EM GRIDS

Drops of diluted antiserum(1: 1 000-1 :5 000) are placed on dental wax or other hydrophobic supports (parafilm strips, silicone-treated paper) in a plastic Petri dish containing moist filter paper (moist chamber). A freshly prepared carbon coated grid is gently placed, film-down, on top of each antiserum drop and floated for 510 min at room temperature. Grids are then removed with tweezers and rinsed.

RINSING THE EM GRIDS

Throughout the ISEM procedure, grids must be carefully rinsed to obtain clean preparations. Buffer rinse is used after protein A precoating, antiserum coating and incubation of the grid with tissue extract. Distilled water rinse is used after second antibody coating (decoration of virus particles), before negative staining, for uranyl acetate precipitates in presence of phosphate ions. or at neutral pH.

Two rinsing procedures can be utilized:

• grids are floated on drops of buffer or distilled water, as appropriate, for 5-10 mini
• grids are retained in tweezers, held vertically and rinsed with 25-30 drops of buffer or water from a Pasteur pipette held close to the grid.

NEGATIVE STAINING

Negative stain can be applied with either system used for rinsing, i.e. floating grids on small drops of the staining solution for 30 sec to 1 min, or applying the stain dropwise (five drops) with a Pasteur pipette.

SUMMARY OF THE PROCEDURE

• Prepare tissue extracts, place drops of extract in a moist chamber on a hydrophobic support (Figure 208).
• Float antiserum-coated grids film-down, one on each drop of extract (Figure 208) Incubate at room temperature or in the cold (4C) for 6-8 h.
• Rinse with 25-30 drops of phosphate buffer from a Pasteur pipette (Figure 210). Drain with filter paper.
• Place drops of antiserum diluted 1: 10- 1: 100 in the moist chamber on a hydrophobic support (Figure 211).
• Float grids on antiserum drops for 10-15 min at room temperature (Figure 211).
• Rinse with 25-30 drops of distilled water from a Pasteur pipette (Figure 212).
• Apply negative stain dropwise (five drops) from a Pasteur pipette (Figure 213). Remove excess with filter paper.
• Grids ready for observation (Figure 214).
• Observe with the electron microscope and read the results.

REFERENCES

Derrick, K.S.1973. Quantitative assay for plant viruses using serologically specific electron microscopy. Virol., 56: 652-653.

Garnsey, S.M., Christie, R.G., Derrick, K.S. & Bar-Joseph, M.1979. Detection of citrus tristeza virus. II. Light and electron microscopy of inclusions and virus particles. In Proc. 8th Conf. IOCV, p. 9-16. Riverside, CA, IOCV.

Milne, R.G. & Lesemann, D.-E.1984. Immunosorbent electron microscopy in plant virus studies. In Maramorosch, K. & Koprowski, H., eds. Methods Virol., 8: 85-101. New York, Academic Press.

Milne, R.G. & Luisoni, E.1977. Rapid immune electron microscopy of virus preparations. In Maramorosch, K. & Koprowski, H., eds. Methods Virol., 6: 265281. New York, Academic Press.

Roberts, I.M. & Harrison, B.D.1979. Detection of potato leafroll and potato mop-top viruses by immunosorbent electron microscopy. Ann. Appl. Biol., 93: 289-297.

Van Regenmortel, M.H.V.1982. Serology and immunochemistry of plant viruses. New York, Academic Press. 302 pp.

FIGURE 204 Basic tools for use with immune electron microscopy. From left to right: dental wax bars, Petri dish, carbon coated electron microscope grids, glass rods and slides, straight-point tweezers, Pasteur pipettes, porcelain mortar

FIGURE 205 Extraction and rinsing media: phosphate buffer (PO2), distilled water (H2O), and 2.5 percent aqueous nicotine

FIGURE 206 Uranyl acetate (1-2 percent solution in distilled water), tenfold and thousandfold dilutions of antiserum for "decorating" and "trapping" virus particles on the EM grid respectively

FIGURE 207 Plant organs commonly used for preparation of extracts: leaves, bark, roots, buds

FIGURE 208 Drops of plant extract, obtained by grinding tissues on a mortar, on which antibody-coated EM grids are being floated for particle "trapping" (upper wax bar). Lower wax bar suppports drops of phosphate buffer on which EM grids are being floated for rinsing

FIGURE 209 Petri dish with a dental wax bar on which EM grids are being floated on drops of a thousandfold dilution of antiserum for antibody coating

FIGURE 210 Rinsing EM grids with phosphate buffer applied dropwise

FIGURE 211 EM grids being floated on drops of tenfold diluted antiserum for particle "decoration"

FIGURE 212 Rinsing EM grids with distilled water applied dropwise

FIGURE 213 Staining EM grids with uranyl acetate applied dropwise

FIGURE 214 Petrl dish with EM grids ready for observation

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