Detection of virus-associated dsRNA from leafroll infected grapevines

Journal of Virological Methods, 31 (1991) 325-334

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© 1991ElsevierSciencePublishersB.V. (BiomedicalDivision) ADONIS 016885109100100G VIRMET 01127

Detection of virus-associated dsRNA from leafroll infected grapevines M.A. Rezaian, L.R. Krake, Q. Cunying and C.A. Hazzalin CSIRO, Division of Horticulture, Adelaide, Australia

(Accepted 15October1990)

Summary A simple procedure is described for reproducible detection of double stranded (ds) RNAs in leafroll infected grapevines. The procedure involves the extraction of tissues by a medium which preferentially yields dsRNA. The RNA is purified by CF 11 cellulose chromatography and gel electrophoresis. The dsRNAs varied in size in different vines. In the cases tested they did not cross hybridize and occurred at higher concentrations in stem cortex tissues than in leaves. They were not detectable in healthy vines, could be passaged with the disease to healthy plants by graft inoculation and removed by virus elimination procedures. These observations indicated that the dsRNAs are of viral origin and that a number of viruses are associated with the grapevine leafroll disease. dsRNA; Leafroll; Grapevine

Introduction Leafroll is a damaging disease of grapevines worldwide (Bovey et al., 1980). The disease is believed to be caused by phloem restricted viruses and a number of viruses with elongated particles (Bovey and Martelli, 1986; Gonsalves and Zee, 1986; Tanne et al., 1977) and isometric particles (Castellano et al., 1985) have been isolated from infected vines. However, the incidence of the disease has not been found to correlate completely with the presence of any single virus so far reported and even viroids have been suggested to be involved in the disease (Pena-Iglesias and Vecino, 1987). Correspondenceto: M,A.Rezaian,CSIRO,Divisionof Horticulture,GPO Box 350,Adelaide5001,South

Australia. 0168-8510/91/$03.50

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The study of leafroll associated viruses has been difficult because of the lack of satisfactory purification methods and assay procedures. Virus purification from grapevine is hampered by the presence of phenolic compounds which inhibit the extraction ofnucleoproteins (Loomis, 1974). Analysis of dsRNA has been used as a means of virus detection in various crops (Dodds et al., 1984) including grapevine (Mossop et al., 1985; Monette et al., 1989). However, dsRNA has not been consistently detected in all the leafroll infected vines (Monette et al., 1989a). We have examined the use of dsRNA isolation for detecting virus infections in leafroll infected grapevines and report a procedure for reproducible detection of dsRNAs and for purification of the RNAs in quantities req/iired for characterization purposes. The results indicate the involvement of a number of viruses in the etiology of grapevine leafroll disease.

Materials and Methods

Extraction of nucleic acidsfrom grapevines Cultivars of grapevine (Vitis vinifera L.) of known disease status, as determined by graft indexing, were grown in a glasshouse or obtained from the field. Samples of leaves or the green cortex of stems (normally 10 g) were pulverised in liquid nitrogen and mixed with one of the following extraction media (4 ml/g tissue). The first medium was that of Palukatis and Symons (1980) and was used up to the chromatography step as described (Rezaian et al., 1988). The second medium was based on NaC104-SDS (Rezaian and Krake 1987). The third medium, used in most of the experiments described here, was that of Laulhere and Rozier (1976) with some modifications. It contained 200 mM Tris pH 7.5,500 mM NaC1, 10 mM MgCIz, 3% SDS, 10% ethanol, 1% 2-mercaptoethanol. The homogenate was kept at 37°C for 10 min and extracted with 1/2 volume of chloroform by shaking at room temperature for 30 min followed by centrifugation at 10000 x g for 10 min.

Purification and analysis of dsRNA The clarified tissue extracts obtained after centrifugation were mixed with 2 g of Whatmann CF11 cellulose (Franklin, 1966) and adjusted to 17% with ethanol (disregarding the ethanol present in the extraction medium). The mixture was poured into a column and washed with 17% ethanol in STE (100 mM NaC1, 1 mM EDTA, 50 mM Tris-HC1, pH 7.0) until no further u.v. absorbing material was released. The dsRNA fraction was then eluted with STE (Jackson et al., 1971). The RNA in this fraction was concentrated by binding and eluting from a column of 0.2 g cellulose CF11. This step also resulted in further purification of dsRNAs. The dsRNA was precipitated with 2 volumes of ethanol and recovered by centrifugation at 200000 x g for 1 h. Samples of dsRNA corresponding to 0.5 to 2 g tissue were analysed in 6% polyacrylamide gels in TAE buffer system (Loening 1967) and RNA bands were visualised by staining with silver (Merril et al., 1981).

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Synthesis of cDNA cDNA copies of dsRNA were synthesized using avian myeloblastosis virus reverse transcriptase primed with calf thymus DNA fragments (Taylor et al., 1976). A mixture of dsRNA (derived from 0.5 g tissue) and the primer (7 l.tg) was dried and resuspended in 1 ktl of 90% DMSO and incubated at 65°C for 20 min (Asamizu et al., 1985). The reaction was carded out by diluting the mixture in 20 volumes of 50 mM TrisHC1, pH 8.3, 2 mM DTT, 10 mM MgCI2, 70 mM KC1, 1 mM each of dCTP, dGTP, dTTP, 2.5 I.tM dATP including 20 ktCi 32p-dATP (3000 Ci/mmol) and 4 units of reverse transcriptase. The reaction was incubated at 45°C, 30 min, then chased with cold dATP to a final concentration of 25 l,tM for 30 min at 45°C. The reaction was stopped by adding EDTA to 20 mM and the RNA template was removed by incubating with 0.3 M NaOH at 65°C for 2 h. After neutralising the mixture with acetic acid, the cDNA was purified by chromatography in Sephadex G-100.

Northern blot analysis Samples of dsRNA were electrophoresed through a 1% agarose gel containing formaldehyde (Maniatis et al., 1982). The northern transfer was performed as in Thomas (1980) except that the gel was soaked in 50 mM NaOH, 10 mM NaCI for 5 min and then neutralized in 1 M Tris-HC1, pH 7.5, for 5 min prior to transfer. Hybridization was as described by Thomas (1980) and the hybridization solution con. • 6 tamed probe at a concentrauon of-0.35x10 cpm/ml of buffer.

Results

Extraction of grapevine nucleic acids enriched in dsRNA It has been observed that conventional RNA extraction media containing phenol do not isolate RNA efficiently from grapevine tissues (Newbury and Possingham 1979; Rezaian and Krake 1987). We therefore examined three media for dsRNA extraction in the absence of phenol. Initially, an RNA extraction medium designed for the isolation of viroids from salt soluble nucleic acids fractions (Palukaitis and Symons, 1980) was used. The RNA was fractionated by chromatography in cellulose CF11 and analysed by gel electrophoresis. This procedure resulted in RNA preparations which contained a polydispersed population of molecules. Most of the RNA could be digested by RNase in the presence of 0.3 M NaC1 indicating that it was single-stranded. Although distinct RNase resistant bands were detectable, the procedure was unsatisfactory because of the high backgrounds in the stained gels (results not shown). Sodium perchlorate has been successfully used to isolate RNAs from grapevine tissues (Rezaian and Krake 1987). However, application of this procedure followed by CF11 chromatography also produced dsRNA fractions containing contaminating material. In a subsequent experiment, the extraction medium used by Laulhere and Rozier

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A 0

10

20

0

10

20

rRNAI

sRNA] Fig. 1. Extraction of total RNA and dsRNA from grapevines. (A) Total nucleic acids extracted with the medium A of Laulhere and Rozier (1976) as described in Materials and Methods. Ethanol concentration in the extraction medium was 0, 10 or 20%. RNA was analysed in 1% agarose gel and stained with ethidium bromide. (B) dsRNA in the same three extracts after purification by cellulose chromatography. The RNA was analysed in polyacrylamide gel and stained with silver.

(1976) containing Tris, NaC1, SDS, DEP and 20% ethanol was examined. The total RNA extracted was analysed by electrophoresis in agarose and visualised by ethidium bromide before CF11 chromatography (Fig 1A). The only RNA present in detectable amounts was soluble RNA. When the ethanol concentration in the extraction medium was reduced to 10% or omitted completely, significant amounts of RNA were extracted (Fig. 1A). The RNA obtained using the medium without ethanol appeared to be partly degraded (Fig. 1A). The RNAs extracted by the media containing 20%, 10% and no ethanol were purified by CF11 chromatography and the dsRNA fractions were analysed by electrophoresis and stained with silver. Figure 1B shows that significant amounts of nucleic acids were present as descrete bands in all the three extracts, despite the low yield of total RNA in the two extracts with the media containing ethanol. Since the presence of large quantities of single stranded RNA in extracts obtained by the medium not containing ethanol prolonged the purification of dsRNA by CF 11 chromatography and resulted in inferior levels of purity of dsRNA, in subsequent experiments an ethanol concentration of 10% was included routinely in the medium. DEP was also omitted from the medium without any significant effect on the yield or quality of dsRNA. The dsRNA preparations obtained by the above procedure were sufficiently pure and there was no need for including nuclease digestion steps in the purification method. The identity of dsRNA and its purity were confirmed by treatment with RNase in the presence and absence of 0.3 M NaCI (Fig. 2). Characteristically, the nucleic acids were degraded by RNase in the absence of NaC1 but were resistant to digestion in the presence of the salt. In each case a sample of single-stranded

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RdL RNase NaCI

+

D1H

+

+

--++

--

-I-

+--+

iii ~i iii

~i

C EV • Fig. 2. Effect of RNase on dsRNA fraction from grapevine in the presence and the absence of NaCI. dsRNA was extracted from each of two Cabernet franc sources previously inoculated with leafroll sources designated as RdL and DIH. The RNAs were analysed in polyacrylamide gel and stained with silver.

RNA (citrus exocortis viroid) included as an internal control was degraded by the enzyme (Fig. 2).

Leafroll infected grapevine samples suitablefor dsRNA Isolation It has been reported that a grapevine leafroll disease associated antigen occurs in higher concentration in the fully developed leaves compared to the young expanding leaves (Teliz et al., 1987). Therefore, initially we used fully expanded grapevine leaves for RNA extraction. Due to the low yields of dsRNA and since phloem limited viruses have been implicated in the etiology of leafroll disease (Castellano et al., 1985; Namba, 1986) we examined the occurrence of dsRNAs in the cortex of stems compared to leaf tissues. The results shown in Fig. 3 demonstrate that the highest concentration of dsRNA is present in the stem cortex of vines indicating they possibly occur in the phloem tissue. The level of dsRNA in mature leaves was found to be higher than that in the young developing leaves. This observation is consistent with the findings of a number of viruses in the thin sections of phloem tissues of leafroll infected grapevines (Namba et al., 1979; Castellano et al., 1985; Zee et al., 1987).

Are the dsRNAs isolatedfrom leafroll infected grapevines of viral origin? Due to the difficulties in purification of phloem limited viruses from grapevines, and the presence of many species of dsRNA in the vines tested (Fig. 4, unpublished results) direct comparison of the dsRNAs with the nucleic acids of putative viruses was not feasible. However, we have obtained evidence which indicates that the

330

CORTEX LEAVES 1

2

3

Fig. 3. The concentration of dsRNA in different tissues of leafroll infected grapevine (CF-RdL). Samples of young leaves (1) were compared with newly expanded full size leaves (2), old mature leaves (3) and cortex tissues of green stems, dsRNA derived from 1 g tissue was electrophoresed and stained with silver.

dsRNAs have a viral origin. The dsRNAs are widely variable in different grapevine cultivars, are absent in healthy vines and in grapevine seedlings (Fig. 4). These observations suggest that the RNAs are not a natural component of grapevine cells such as the dsRNAs found in healthy beans (Wakarchuk and Hamilton, 1985). The dsRNAs tested could be passaged by graft inoculation to vines free of dsRNA (Fig. 4, cf. tracks 1 and 2 and tracks 3 and 4). The same grafting method also resulted in the transmission of the leafroll disease to the indicator vines. The major dsRNA bands could be removed from the grapevines (Fig. 4, cf. tracks 5 and 6 and tracks 7 and 8,) by the 'virus elimination' procedure which freed the vines from leafroll and some other diseases (Barlass et al., 1982). These observations provide circumstantial evidence that the dsRNAs may be derived from viruses involved in leafroll disease. The patterns ofdsRNAs isolated from various grapevine sources infected with leaf-

331

1

2

3

4

5

6

7

8

9

Fig. 4. Diversity, transmission and elimination of dsRNAs in leafroll (LR) infected grapevines, dsRNAs were from: 1, LR infected DIH vine; 2, disease-free Cabernet franc (same as 9) but graft inoculated with 1; 3, LR infected RdL; 4, disease-free Cabernet franc inoculated with 3; 5, LR infected Sultana H23; 6, same as 5 but generated by fragmented shoot apex culture; 7, LR infected Sultana H5; 8, same as 7 but generated by fragmented shoot apex culture; 9, disease-free Cabemet franc, dsRNA from 1 g tissue was analysed in each track and stained with silver.

roll tissue (Fig. 4 and unpublished results) indicate that a number of viruses are present in these vines. Using the dsRNAs from flax rust as size markers, it was estimated that the sizes of leafroll associated dsRNAs range from lkbp to above 5kbp. It is possible that some of these RNAs are related species. However, we have compared the RNAs isolated from two of the leafroll sources which we selected for further study. A cDNA probe was synthesized for the dsRNAs isolated from a Cabernet franc (CF) vine previously graft inoculated with the leafroll infected Vitis rupestris du Lot (RdL). The probe was used in Northern blot analysis to compare dsRNAs isolated from CF-RdL with those obtained from another CF clone which was inoculated with leafroll infected Sultana clone DIH (DIH). Fig. 5 shows that no cross-hybridization was detectable and indicates that the dsRNAs present in these two leafroll infected vines are unrelated. This experiment shows that a cDNA probe can be made from small quantities of dsRNA and used for the detection of virus associated RNAs at higher levels of sensitivity compared to silver staining (cf. Figs. 4 and 5). However, in most cases examined, the dsRNAs were detectable by silver staining when RNA extracts of 0.5-2 g tissue were analysed and cDNA hybridization was not required.

332

1

2

3

4

I ID m

Fig. 5. Northern blot analysis of dsRNA from LR infected vines, dsRNA from the LR infected CF- RdL and CF-DIH were analysed and probed with a cDNA to CF-RdL dsRNA, Tracks 1, healthy vine; 2, virus-free seedling; 3, CF-DIH; 4, CF-RdL.

Discussion

We have described a procedure for detecting dsRNA in leafroll infected grapevines and have provided evidence that the dsRNAs are of viral origin. The combined sampling and extraction methods provide a reproducible dsRNA detection procedure and multiple samples can be processed. Although grapevine leafroll has characteristics of a viral disease, its etiology has not been determined. Plant viruses generally induce specific symptoms on a host from which (symptom and the host) the viral names are often derived. By analogy, viral agents for grapevine leafroll disease has been searched (Namba, 1986; Bovey and Martelli, 1986) and viral particles have been isolated. However, no satisfactory correlations have been established between the occurrence of the viruses isolated and the incidence of leafroll disease (Corbett, 1985; Gonsalves and Zee, 1986). These observations and the detection of several kinds of dsRNA patterns in different leafroll infected vines (Fig. 4, unpublished results) support the suggestion (Martelli, 1982) that more than a single virus may be involved in the disease and leafroll symptoms are probably a non-specific plant response to infection by a range of viruses which presumably invade the phloem tissues. Evidently, specific virus detection methods are not likely to be applicable to leafroll disease. The use of dsRNA for detecting viral infection has the limitation that the origins of the dsRNAs and the identity of the viru~s involved remain unclear. Nevertheless,

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given the difficulty in virus purification from grapevines, lack of assay systems for the viruses and the diversity of the viruses involved, the dsRNA detection procedure is a promising tool in the study of grapevine leafroll disease. This general detection method avoids the necessity to tailor the procedure to individual viral types. Our preliminary results (Fig. 4, unpublished results) indicate that the dsRNA detection method can differentiate the disease free vines obtained by fragmented shoot apex culture (Barlass et al., 1982) from the original leafroll infected vines. This method has therefore the potential to be used for assessing the success of eliminating leafroll by tissue culture techniques. While the dsRNA test can be done in 2-3 days, the graft indexing procedure requires 2 years to complete. The yields of dsRNAs obtained are sufficient to allow further characterization of these RNAs. Sequence information obtained from these RNA may reveal the identity of some of the viruses involved. The dsRNAs can also be used to produce detection probes (Fig. 5) which should enable assays of corresponding viruses in cellular fractions for developing purification procedures.

Acknowledgements We thank Martin Barlass for providing all the grapevine samples generated by fragmented shoot apex culture used in this work, Matt Dickinson for a sample of flax rust dsRNA and Nigel Scott for comments. The study was supported by a grant from the Grape and Wine Research Council.

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