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  • C12N2770/00011—Details
  • C12N2770/18011—Comoviridae

Definitions

• the present invention relates to a method for diagnosing blackcurrant reversion disease in plants, especially in blackcurrant ( R ⁇ be ⁇ ) plants.
• a reverse transcriptase-PCR protocol optionally in combi- nation with immunocapture, is used to detect a virus causing blackcurrant reversion disease, blackcurrant reversion virus, BRV.
• the invention also concerns a test kit for use in the method. In the work leading to the invention, the BRV RNA was partially characterized. This enabled primers to be constructed from the viral nucleo- tide sequence so determined for use in the method and the kit of the invention.
• blackcurrant reversion is unquestionably the most important for blackcurrant crops world-wide (1,2); it also affects redcurrant (3). It oc- curs in Ribes world-wide with the exception of the Americas. As its name suggests, the disease reflects the change in plant habit, mostly in the leaf appearance, that is suggestive of ' reversion ' to a primitive ancestral plant type (3). However, for diagnosis, the most reliable sy p- toms of reversion disease occur in flower buds as they open in early spring.
• E European form
• R A more severe form of the disease (R; 1, 2), found in Finland (4), Eastern
• the object of the present invention is, in a first aspect, a method for diagnosing blackcurrant reversion di- sease in a plant by detecting blackcurrant reversion virus (BRV) therein, the method comprising
• said method is combined with an immunocapture method comprising bringing the sample into contact with an antibody to BRV coated onto a substrate, in order to separate and concentrate the BRV containing particles from the sample, and disrupting the particles, e.g. by heating, to release the viral RNA.
• a further object of the invention is a diagnostic test kit for diagnosing blackcurrant reversion disease in a plant using a reverse transcriptase-polymerase chain reaction (RT-PCR) protocol for detecting blackcurrant reversion virus, BRV, in the plant, the kit comprising
• oligonucleotide primers for amplifying a cDNA fragment complementary to a frag- ment of BRV RNA.
• the invention is directed to the use of a pair of oligonucleotide primers flanking and capable of amplifying the 3' proximal 210 bp fragment of cDNA of the viral RNA made in the reverse transcription reaction, for use in the method and kit according to the invention.
• Figs la and lb show RT-PCR amplification of the 210 bp cDNA fragment of BRV RNA from nucleic acid extracts from ( lanes in parenthesis ) :
• Lane ' M' contains Pstl-digested ⁇ DNA mol. wt markers.
• Lane 1 contains a buffer control and lane 'M' a 1 kb ladder mol. wt marker ( Gibco BRL ) .
• Figs 2a to 2c IC-RT-PCR amplification of the 210 bp cDNA fragment of BRV RNA from nucleic acid extracts from plants or mites.
• lane 'M 1 contains a 100 bp DNA ladder (Gibco BRL) and lane ' C material following IC-RT-PCR of healthy blackcurrant. Extracts were from ( lanes in parenthesis ) :
• Figs 3a and 3b IC-RT-PCR amplification of the 210 bp cDNA fragment of BRV RNA from extracts of plant samples from Scotland.
• lanes ' C and 'M' contain the assay from healthy blackcurrant and a 100 bp DNA ladder (Gibco BRL) respectively.
• Samples are (lanes in parenthesis) extracts from :
• C. qu ⁇ noa were infected with strawberry latent ringspot virus (1), arabis mosaic virus (ArMV) - blackcurrant isolate (2) and - lilac isolate (5), raspberry ringspot virus - Scottish isolate (3) and uninfected healthy plant ( 4 ) .
• Blackcurrant cultivar samples are plants previously graft-inoculated with R ⁇ bes sources affected with:
• Fig. 4. IC-RT-PCR amplification of the 210 bp cDNA fragment of BRV RNA from extracts of the blackcurrant culti- var Silvergierters Schwarze from New Zealand. Lane 1, healthy plant; lanes 2 and 3, plants showing mild and severe reversion symptoms, respectively; lanes 4 and 5, control samples of healthy and reverted blackcurrant from Finland, respectively; lane M, a 50-1000 bp DNA marker ( FMC ) .
• the virus was isolated from a Scottish Crop Research Institute (SCRI) blackcurrant breeding line.
• the purified virus was used for the formation of an antiserum by immunizing a rabbit.
• the antiserum obtained was used for purifying and concentrating BRV particles in samples to be diagnosed.
• a virus was mechanically transmitted with difficulty to Chenopod ⁇ u qu ⁇ noa Willd. and, from this host, to other herbaceous test plants.
• the virus was purified, partially characterized, and an an- tiserum to it produced.
• Virus particles were isometric, c. 27 run in diameter and sedimented as two nucleoprotein components. They contained a protein species of M r 56,5 kD which was readily degraded into a 55 kD protein and two major RNA components of c. 6700 and 7700 nucleotides, each with poly-A tails. Most of these properties are shared by nepoviruses, but the virus was serologically unrelated to 14 nepoviruses, or putative confrontationviruses tested.
• RNA was isolated and analyzed on agarose gel and thereafter subjected to Northern blot analysis.
• cDNA was synthesized from the viral RNA, for the formation of recombinant clones. From selected recom- binant clones, DNA was isolated for preliminary restriction analysis and sequence analysis. This enabled an analysis of the sequence comprising 1260 3' terminal nucleo- tides and the poly-A tail of one of the RNA components (RNA2) of BRV. Said sequence is disclosed in SEQ.ID.N0:1.
• the sequence analysis enabled the design of primers for use in the method according to the invention. Specifically it enabled the design of primers for the amplifica- tion of a 3 ' proximal region comprising 210 bp of the DNA complementary to the BRV RNA directly upstream from the poly-A tail.
• the virus has been consistently associated with the reversion disease.
• the virus has also been inoculated back to healthy blackcurrant plants in which reversion symptoms occured and from which the BRV was again detected by IC-RT-PCR. This proves that this virus is the causal agent of the black- currant reversion disease. Consequently we call the virus "Blackcurrant Reversion Virus", BRV.
• an immunocapture reverse transcription PCR (IC-RT-PCR) protocol is used.
• a sample such as a plant extract can be used, which suitably is obtained from symptom bearing parts of the plant, such as from leaves, flowers or bark.
• BRV particles contained in such extracts are concentrated and purified by subjecting them to an immunocapture method, such as that described in (6).
• a substrate such as a PCR tube, microfuge tube or microtiter plate coated with antiserum can be used. After incubation, the virus particles trapped to the antiserum coated substrate are disrupted, for example by heating, for releasing the RNA.
• the RNA can be directly extracted from the plant samples .
• RNA so obtained is used for the first strand synthesis of cDNA in a reverse transcriptase reaction, using a reverse transcription mixture, for example a commercially available mixture, together with an oligonucleotide primer complementary to a viral nucleotide sequence e.g. in a position just upstream from the poly-A tail, and reverse transcriptase enzyme. After a suitable incubation time, the reverse transcriptase is inactivated.
• the cDNA so obtained is then amplified in a PCR reaction.
• a 'hot start' method is used (7) in the amplification.
• the DNA polymerase is activated only after the reaction has reached a higher temperature, thus minimi- zing non-target amplification and undesired side products.
• Hot start can be accomplished by adding an essential component to the reaction tube only after it has reached the elevated temperature.
• an oligonucleotide primer derived from the BRV RNA, being complementary to a first viral nucleotide sequence fragment is used in combination with a second oligonucleotide primer, corresponding to a second viral nucleotide sequence fragment, said first and second nucleotide sequence fragments flanking the target nucleotide sequence, cDNA, to be amplified.
• the first primer is complementary to the viral nucleotide sequence in positions 1-15 upstream from the poly-A tail and has the sequence 5' GAAAGGACATTTCAG 3 ' ( SEQ. ID. NO: 3 )
• the second primer corresponding to the nucleotide sequence in positions 199-210 upstream from the poly-A tail and has the sequence 5' CGCTGGTGTCTC 3 ' (SEQ. ID. NO: 4 )
• the primers can be synthesized according to known procedures, for example using the solid phase phosphoramidite method.
• the PCR amplification reaction can be carried out as an automated process in a thermal cycler, using for example 30 cycles of template denaturation at 95 °C for 1 min, primer annealing at 37 °C for 1 min and elongation at 72 °C for 1 min. After 30 cycles, a longer elongation period is used.
• PCR reaction products are elect- rophoresed in agarose gel, stained with ethidium bromide and viewed under UV light.
• the PCR reaction products can be electrophoresed in polyacrylamide gel and visualized by silver stain.
• the blackcurrant source material from which the virus was isolated was a Scottish Crop Research Institute ( SCRI ) breeding line, P9/5/1, growing in the experimental field of the Agricultural Research Centre, Institute of Horti- culture (ARC-IH), Piikki ⁇ , Finland. It was planted as virus-tested material but soon became infected with the agent of the R form of reversion disease, probably from the reverted bushes of Finnish blackcurrants growing near by. Soft wood cuttings were taken from this plant and the cuttings rooted in a peat-sand mixture in a glasshouse in the ARC, Institute of Plant Protection (IPP), Jokioinen. During rooting, pronounced chlorotic/yellow line patterns and ringspots developed on the new leaves of these cuttings.
• SCRI Crop Research Institute
• IPP Institute of Plant Protection
• Such leaves were ground in 2% nicotine solution, pH 9.5, the sap extract rubbed on carborundum-dusted leaves of herbaceous test plants, and the plants maintained in a glasshouse at c. 21 °C. Following one of these inoculations, symptoms were observed in the leaves of Cheno- podium qu ⁇ noa plants.
• the virus, BRV, found to be present in this plant was maintained thereafter by serial passages in C. guinoa and other herbaceous hosts. The virus was also preserved as infected C. qu ⁇ noa leaves at -20 °C and -80 °C.
• Virus pur ⁇ f ⁇ cat ⁇ on BRV was purified using a slight modification of the method described by Frison and Stace- Smith ( 8 ) for arabis mosaic nepovirus .
• Inoculated and/or systemically infected leaves of C. guinoa were harvested 15-28 days after inoculation and ground with 3 ml/g buffer (0.05M Na 2 HP0 4 , 0.02M ascorbic acid, 0.02M 2-mercapto- ethanol, pH 8.0). The homogenate was filtered through cheese cloth and clarified by centrifugation at 15 OOOg for 20 min.
• the supernatant fluid was adjusted to pH 5.0 with dilute HCl, kept over night at 4 °C, and then cen- trifuged at 15 OOOg for 20 min.
• To the supernatant fluid was added 1% NaCl and 8% polyethylene glycol (PEG 6000) and the mixture stirred at 4 °C for 1 hr before centri- fuging at 15 OOOg for 20 min.
• the pellet was resuspended in one tenth of the original volume of 0.05M Na-citrate buffer, pH 7 containing 1% NaCl and stirred for 1 hr before centrifuging again at 15 OOOg for 20 min.
• BRV was purified from N ⁇ cot ⁇ ana bentha- m ⁇ ana Domin. plants by clarifying the plant extracts with 50% (v/v) chloroform. The aqueous phase was further clarified and concentrated by one cycle of high and low speed centrifugation.
• Virus preparations were purified further by layering 100-500 ⁇ l of the final virus suspension on 10 to 40% sucrose density gradients in 0.05M citrate buffer, made up in Beckman S 41 or S 50.1 tubes.
• the sucrose density gradient tubes were centrifuged at 38 000 rp for 90 min (SW41) or 45 000 rpm for 50 min (SW50.1) and the gradients fractionated by upward displacement using an ISCO density gradient fractionator, monitoring absorption at 254nm.
• Virus-containing fractions were pooled, diluted with an equal volume of citrate buffer and centrifuged at 180 OOOg for 2 hr. The final pellets were resuspended in a small volume of citrate buffer.
• yields of virus were relatively small even when preparations were made from BRV-infected herbaceous plants at the optimum time of year for symptom expression. Maximum yields were about 4-5 mg/kg leaf.
• purified virus preparations usually formed two closely sedimenting light-scattering bands that were identified with the two main peaks of absorbance in the middle of the gradient. Fractions from these peaks were associated with maximum infectivity.
• virus preparations contained many isometric particles, some with angular ( icosahedral ) outlines and measured c. 27 nm in diameter. Many particles were penetrated by all four negative stains used but the proportion of particles penetrated by stain appeared greater in ammonium molybdate pH 7.0 than in PTA ( phospho tungstate acid), pH 7.0, methionine tungstate, pH 7.0, or uranyl acetate, pH 3.5. In uranyl acetate, those particles partially penetrated by the stain appeared to have either a central core, or a thicker protein shell that was not evident in particles in the other stains. Whilst these differences in particle appearance may be artifacts caused by the uranyl acetate stain, they have not been observed previously for particles of definitive nepoviruses in this stain.
• the size of the viral coat protein was determined by electro- phoresis in 10% SDS-PAGE gels, as described by Laemmli (9).
• Nucleic acid was isolated from purified virus preparations by using either the commercial Micro-FastTrack kit (Invitrogen) for isolation of poly-A tailed RNA, or by extraction with phenol, phenol-chloroform and precipitation with ethanol .
• the extracted nucleic acid was denatured with glyoxal and dimethyl sulfoxide, analyzed in 1% agarose gels and stained with ethidium bromide, as described by Sambrook et al . (10).
• RNAs were transferred from agarose gels to Hybond-N membrane (Amersham) and the membrane fixed by UV and de-glyoxylated by baking at 80 °C for 2 h.
• the blots were probed with digoxygenin-dUTP ( Boehringer Mannheim) labelled virus cDNA.
• Hybridization reactions were detected using the DIG Luminescent Detection Kit (Boehringer Mannheim).
• RNAs were estimated to be c. 6700 and 7700 nucleotides.
• First strand cDNA from the viral RNA was synthesized with oligo-dT primers using the First Strand cDNA Synthesis Kit (Pharmacia). The reaction mix was directly used for second strand synthesis according to Sa brook et al . (10). Notl-linkers were added to the cDNA, which was then ligated into the
• NotI-digested dephosphorylated Bluescript SK+ vector and transformed by electroporation into JM109 cells.
• DNA was isolated by alkaline lysis (10) either in minipreparation scale for preli- minary restriction analysis, or in large scale for sequence analysis.
• the sequence analysis was by automated sequencing, using the ALF Manager system, version 2.5.
• the sequence data was analyzed using various programmes in the Genetics Computer Group (GCG) sequence analysis programme package, version 8.0.
• GCG Genetics Computer Group
• Antiserum was prepared by immunising a rabbit subcuta- neously with c. 50-100 ⁇ g of purified virus in Freund's incomplete adjuvant. Two booster injections were adminis- tered intramuscularly with 14 days between injections.
• Serum was obtained from the animal 14 days after the last injection and stored at -20° C.
• Example 2 DIAGNOSING BLACKCURRANT REVERSION IN PLANTS
• oligonucleotide primers Based on the sequence for the 3' end of BRV RNA determined above, the following oligonucleotides were synthesized: 5' GAAAGGACATTTCAG 3' (primer 1), complementary to the viral nucleotide sequence in positions 1-15 upstream of the poly-A tail, and 5' CGCTGGTGTCTC 3 ' ( primer 2 ) , corresponding to the viral nucleotide sequence in positions 199-210 upstream of the poly-A tail. The 230 bp fraction of the viral nucleotide sequence, as converted to DNA, including the poly-A tail is disclosed in the appended SEQ.ID.N0:2. The primers were synthesized by the solid phase phosphoramidite method in 0.01 ⁇ mol scale and desalted before use.
• RNA extraction buffer 0.1 M-glycine, 0.1 M-Tris pH 8.6, 0.1 M-NaCl, 0.01 M-EDTA, 0.2 % SDS (sodium dode- cyl sulfate), 0.2 % sodium dodecyl sarcosine.
• RNA was extracted from this suspension by adding an equal volume of phenol/chloroform and the RNA precipitated from the aqueous phase with 1/10 volume of 3 M-NaOAc, pH 5.2 and 2.5 volumes of ethanol.
• the RNA-containing pellet was resuspended in 8 ⁇ l of water and used directly for first strand synthesis of cDNA.
• RNA extraction buffer 2 ml/g leaf
• the homogenate was extracted with an equal volume of phenol and phenol/chloroform and the nucleic acid was ethanol precipitated as described above.
• nucleic acid extraction from Rlbes leaves about 300 ⁇ g of young leaf material was powdered in liquid N in an Eppendorf tube with a glass rod. To the extract was added 500 ⁇ l of TE buffer (10 mM Tris, 1 mM EDTA), pH 7.4 with continuous grinding.
• the homogenate was frozen for 20 min at -20 °C, thawed at room temperature, and extracted with 0.25 volumes chloroform for 5 min.
• the emulsion was centrifuged at 2,000 rpm for 5 min, the aqueous phase removed and centrifuged at 12,000 rpm for lOmin.
• the pellet was resuspended in 500 ⁇ l RNA extraction buffer, extracted twice with phenol/-chloroform and the nucleic acid precipitated with ethanol at -70 °C.
• Plant samples for analysis Plant samples for analysis . Plant samples for PCR analysis were collected in late spring/early summer. In Finland, samples were from field plants showing flower and/or leaf symptoms of reversion disease from the IPP, Jokioinen, the germplas collection at the Agricultural Research Centre, Institute of Horticulture (ARC-IH), Piikki ⁇ , and from various private gardens in Southern Finland. Healthy plants (certified virus-tested plants) were originally from the Laukaa Research and Elite Plant Unit of the Agricultural Research Centre of Finland and subsequently maintained in a protected glasshouse at IPP, Jokioinen.
• RNA was either obtained directly from pu- rified BRV particles or extracted from BRV particles trapped from plant or mite extracts by immunocapture.
• the reaction mixture contained 8 ⁇ l RNA in water, 1 ⁇ l primer 1, 1 ⁇ l 200 mM-DTT and 5 ⁇ l reverse transcrip- tion (RT) mixture.
• the RT-mixture was either that of the First Strand Synthesis Kit from Pharmacia Biotech, or a mixture containing 135 mM-Tris-HCl, pH 8, 200 mM-KCl, 30 mM-MgCl 2 , 30 mM-DTT (dithiotreitol ) , 5.5 mM each of dNTP, 240 mg/ml RNase/DNase free BSA (Pharmacia Biotech), 2.7 U/ ⁇ l RNAguard (Pharmacia Biotech), and 3 U/ ⁇ l murine reverse transcriptase (Pharmacia Biotech). The reactions were incubated at 37 °C for 1 h and then at 90 °C for 5 min to inactivate the reverse transcriptase.
• I ⁇ x u ⁇ ocapture protocol Immunocapture of BRV particles from leaf and mite extracts essentially followed the method of Nolasco et al . (6) with minor modifications. Leaves (0.3g) were powdered in liquid N and extracted with a pestle and mortar in 2.1 ml of cold TE buffer, pH 8. About 0.5 ml of the extract was transferred to an Ep- pendorf tube, frozen at -20 °C for 20 min, thawed and centrifuged again for 5 min at 3,000 rpm at 4 °C.
• PCR tubes were emptied, washed twice with 100 ⁇ l PBS- Tween, and 8 ⁇ l of water was added and the tubes heated at 65 °C, for 10 min to disrupt the trapped virus particles. The tubes were transferred to ice and used directly for the first strand synthesis as described above .
• the "hot start” method (7) was used.
• the PCR reaction components were pipetted into the PCR tubes before the addition of the cDNA samples.
• the reaction mixture (50 ⁇ l ) contained 20-40 pmol of each primer and 400 ⁇ M of the dNTPs in lx DyNAzy eTM DNA polymerase buffer.
• the reaction mixture was covered with melted DyNAWaxTM ( Finnzymes ) , which quickly solidified on contact with the reaction mixture. On top of this wax layer was added 50 ⁇ l of a solution containing 2 U DyNAzymeTM DNA polymerase ( Finnzymes ) and 5 ⁇ l of the first strand cDNA reaction in 1 x DyNAzymeTM polymerase buffer.
• RT-PCR amplification was in a Hybaid thermal cycler ( Omnigene ) with 30 cycles of template denaturation at 95 °C for 1 min, primer annealing at 37 °C for 1 min, and elongation at 72 °C for 1 min. After 30 cycles the products were elongated at 72 °C for 10 min and cooled to 4 °C. Aliquots (30 ⁇ l ) of the
• PCR reactions were electrophoresed in 1.5 % or 2 % agarose gels in TBE buffer (0.045 M Tris-borate, 1 mM EDTA) and the gels stained with ethidium bromide and viewed under UV light.
• Figs 1 to 4 The results from the PCR reactions are shown in Figs 1 to 4. It can be seen that using the primers in question, reliable amplification of the product was achieved from the cloned cDNA when the 'hot start' RT-PCR method was used. The 'hot start' PCR method was therefore used throughout these studies and it successfully amplified the expected size product when used on RNA from purified BRV particles (Fig. la and lb, lanes 5), from BRV- infected C. guinoa (lanes 1 and 2) and N. occldentalls , (lane 3) (Fig. la).
• the 210 bp product was obtained from samples of reverted blackcurrant isolate, Piikkio (lane 2, Fig. lb) and Parikkala (lane 3, Fig. lb).
• the buffer controls are run in lanes 1 and samples of healthy plants in lanes 4.
• the 210 bp PCR product was detected more reliably and efficiently in Rlbes leaves throughout the season when IC-RT-PCR was used.
• the specific product was detected in leaves of 10 field-grown blackcurrant plants affected by the R form of reversion disease and obtained originally from ARC-IH, Piikkio and Parikkala (Fig. 2a, lanes 1-10) and also in groups of 50 mites ( Cecldophyopsls rlbls ) picked out of galled buds from these blackcurrant plants (Fig. 2a, lanes 11-12).
• Fig. 2a lanes 5-6 9 and 12
• Fig. 2a shows that only very little of the 210 bp PCR product was detected (Fig. 2a, lanes 5, 6, 9 and 12).
• IC-RT-PCR analysis was made on plants in an SCRI blackcurrant field trial at IPP, Jokioinen, established to assess the spread of the reversion agent and its gall mite vector.
• the tested plants were planted as mite- and virus-free cuttings amongst infector blackcurrant bushes infested with gall mites and showing leaf and flower symptoms of the R form of reversion disease. All plants tested were infested by gall mites and showed different extents of leaf and flower symptoms typical of the R form of reversion disease.
• Samples 1-5 were of cv Neosyp, and 6-10 of cv. Ben Tirran at the experimental field trial at Institute of Plant Protection (IPP), Jokioinen with the R form of reversion disease. Samples 11-38 were of unrecorded cultivars in locations where the E form of reversion disease was dominant .
• the 210 bp product was also detected in tests on three plants of Ben Nevis blackcurrant and in two plants each of the cvs Ben Lomond and Ben More that had been infested 4 years previously with gall mites from a blackcurrant bush showing symptoms of the E form of reversion disease and maintained subsequently in a heated glasshouse.
• BRV was transmitted by the mites to all 7 plants.
• the mites also transmitted the reversion agent, at least to the Ben Nevis plants.
• the absence of reversion symptoms in the other two cultivars may possibly be due to the known delay in expression of reversion symptoms after inoculation (3) and that this delay was accentuated by the low amount of inoculum (mites compared to graft-inoculation) used and by the warm conditions under which the plants were subsequently maintained.
• the 210 bp product was also detected by IC-RT-PCR in all the reverted samples of blackcurrant from New Zealand, whether they showed mild or severe symptoms (Fig. 4, lanes 2 and 3 ) , but it was not detected in healthy plants (Fig. 4, lanes 1 and 4). Lane 5 in Fig. 4 shows positive control of a sample of reversion infected blackcurrant from Finland.
• the two primer pairs amplify 210 nt and 481 nt DNA fragments, initiating at distance of 1 and 265 nucleotides upstream of the poly-A tail of RNA2 of the BRV genome, respectively.
• the primer pair (1,2) amplified the expected virus- specific fragments from all the tested virus isolates
• the primer pair (5,6) amplified the expected virus- specific fragments from nearly all the tested virus isolates, indicating that the viral sequences detected by these primer pairs are well conserved in all isolates, including the common (E) and strong (R) forms of the reversion disease.
• MOLECULE TYPE genomic RNA converted to DNA
• ORGANISM Blackcurrant reversion virus
• CTGCGTCCAC TCGGTTTGGT ATTTTACGAT TAAATACCCA
• GGTCTACTGC TTCCGAGCCT 600
• MOLECULE TYPE genomic RNA converted to DNA
• ORGANISM Blackcurrant reversion virus

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