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1. WO1988003956 - PROCEDE POUR DETECTER UN VIRUS DANS UNE PLANTE

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[ EN ]

PROCESS FOR DETECTING PLANT VIRUS

The present invention relates to a process for detecting plant virus, more precisely by application of genetic engineering.
Every year- agriculture suffers heavy losses owing to virus attacks on the crops, above all on those which are vegetatively propagated. In Sweden, more than half of the seed potatoes are infected by the potato virus Y (PVY), but also by the potato viruses X, S, A and M. Barley yellow dwarf virus causes heavy losses, espec- ially on herbage, but also attacks cereals. Infected plants of meadow fescue grass, perennial rye grass and timothy grass can give up to 40% crop reduction (Lindsten 1974). The wintering of the barley yellow dwarf virus in grazing grounds is also of consequence to neighbouring fields of cereals, since the grazing grounds function as a source of infection. Since there is no equivalent to fungicides for fighting virus, extensive checks for virus must instead be carried out. The ability to diagnos virus is therefore of the utmost necessity so as to facilitate practicing of an efficient breeding for resistance. This requires a laboratory method which is

- sensitive
- specific
- reliable
- rapid with high capacity
- inexpensive.
By using recombinant DNA technology for detecting the virus genome, this ideal may be achieved, and virus diagnostics is the specific instance of plant breeding where it has been found that molecular-biological technology can be successfully used directly in practical breeding work.
Screening for virus resistance in a breeding prog-

E ramme usually involves measuring of the virus production after a test infection. This means that thousands of plants must be tested for the presence of virus in a relatively short time. Until some years ago, the most common method was simply to visually judge the plants for characteristic symptoms of a virus infection. The drawbacks of this form of selection are that an insignificant virus content provides no visible symptoms, and at the same time a trained eye is required to make a fairly safe judgement.
Since attacks of e.g. barley yellow dwarf virus in pasture grass provide no visible symptoms whatsoever, it is difficult to judge the presence of and the damage caused by this virus . Breeding for resistance against barley yellow dwarf virus in pasture grass must therefore be based on a laboratory method. One such method which has found widespread use -in plant virus diagnostics is the so-called ELISA method, a serological method which is based on the antigenicity in the virus protein envelope and implies that the virus particles are detected by the reaction of antibodies with virus protein. ELISA is the prevailing method for detecting plant virus and is routinely used for both seed control and breeding for resistance. The method has many advantages. It can be easily carried out, it is rapid and relatively sensitive, it may be used on a large scale and requires no expensive equipment. Unfortunately, also the ELISA test has its limitations by being unreliable at low virus frequencies, and when applied to e.g. barley yellow dwarf virus, it is difficult to obtain a pure virus preparation for the production of antibodies since the virus occurs merely in low frequencies.
The majority of plant viruses, about 75%, contains the nucleic acid RNA as the genetic material, enclosed by a protein envelope. The ELISA test uses the antigenicity of this protein, but the antigenicity represents only 10% of the genoitiic information obtainable from the virus.

*CT""_='J." "-! ;- -__*'_-- S__ SHEET By analysing instead the virus nucleic acid directl far more reliable results can be obtained in virus detection. This new technique of diagnosing virus is based on the fact that complementary nucleic acid strands hybridise with each other, which is a basic function in recombinant DNA technology. Such a method, nucleic acid hybridisation by means of "dot blot", has precisely the properties which make it desirable in breeding for resistance against virus. It is extremely sensitive and rapid, has high capacity and is, above all, reliable. In the British plant breeding institute PBI, Cambridge, such a molecular screening method has completely replaced the ELISA technique for detecting the potato virus X (Baulcombe et al 1984).
The object of the present invention is to provide a process for detecting plant virus. The process is characterised in that plant sap is treated with a protein decomposing enzyme for removal of endogenously bound biotin, that virus RNA from the plant sap is bound to a filter membrane, and that prehybridisation is carried out, whereupon virus RNA is hybridised by incubation of the membrane with biotin-labelled DNA which is homologous to RNA in the virus to be detected, whereby the presence of the specific virus RNA becomes visually detectable through the appearance of a colour after addition of a biotin-specific enzyme.
The process according to the present invention is a non-radioactive molecular hybridising technique for detecting plant virus. In the detailed description given below, potatoes have been used as model crop, and the process has been effected for detecting the potato virus Y (PVY) in both leaf and tuber juice. With certain modifications, the process can of course also be used for detecting other plant viruses, e.g. barley yellow dwarf virus.
The use of nucleic acid hybridisation for detecting PVY requires access to DNA complementary to the virus

SUBSTITUTE SHEET RNA genome. The nucleic acid hybridisation technique therefore comprises the following steps:
- isolation of virus
- isolation of virus RNA
- cDNA synthesis and molecular cloning
- spot test
Isolation of virus
PVY is produced in pure form from infected tobacco leaves according to Oxelfelt (pers. commun. ) . The cell walls of the leaves are decomposed by homogenisation in a buffer, which prevents aggregation of the virus particles. Both reducing and chelating substances are added to prevent/reduce the influence of polyphenol and enzyme activity, respectively. Organelles, ribo-somes etc. are separated by centrif.ugation, whereupon the virus is concentrated by precipitation with polyethylene glycol, followed by purification by means of ultracentrifugation. The purity of PVY is checked by measuring the absorption spectrum, while the infectivity is checked on tobacco, followed by an ELISA test.
Isolation of RNA
RNA is isolated from PVY produced in pure form, with common phenol extraction for nucleic acids according to a modification by Clemens (1985). The purity of the RNA preparation is checked on the one hand by measuring the absorption and, on the other hand, by agarose electro-phoresis.
Molecular cloning
DNA (cDNA) complementary to PVY-RNA is prepared by the RNase H method according to a modification by Gubler and Hoffman (1983). The advantage of this technique is that long, coherent DNA sequences are obtained, at the same time as the entire process takes place in a single test tube. The concentration and amount of synthesised cDNA are estimated by labelling with radio-active phosphorus ( 32P). cDNA is inserted into a plasmi which is linearised by treatment with a restriction

SUBSTITUTE SHEET 5
enzyme. The plasmid is transferred to and propagated in E. coli cells, whereupon colonies of plasmid + cDNA are identified on a selective medium. Bacteria and thus also plasmids from these colonies are propagated on a large scale, whereupon plasmid DNA is isolated by ultracentrifugation. The size of the plasmid and the inserted cDNA are determined by agarose electrophoresis .

In this way, several PVY clones with DNA complementary to virus RNA have been prepared, which can be used for detecting PVY by nucleic acid hybridisation. Spot test
The actual detection of the presence of virus is carried out with a so-called spot test, in which virus RNA from plant sap (leaf or tuber juice in the case of potatoes) is bound to a cellulose nitrate membrane and is recognised by and hybridises with complementary DNA as added which is made detectable by labelling.
Such labelling can be made in different ways, most commonly by means of radioactive isotopes. Use is fre- quently made of 32P which, however, suffers from certain
32
disadvantages. Since the half-life of P is only 14 day the labelling must be renewed constantly. For the safety of the laboratory staff, non-radioactive material is preferred.
According to the present invention, use is made of an optimal spot test which is based on non-radioactiv detecting by means of biotin. Since the process accordin to the invention is intended for screening, this is a great advantage. Thus, the process according to the in-vention comprises labelling by means of nick translation with biotin derivatives of deoxyribonucleotides instead of 32P.
An alternative method to nick translation for label ling DNA is to bond so-called photobiotin to DNA via a photochemical reaction. This labelling technique can be carried out far more easily, but unfortunately the detection limit is affected, which makes this technique less sensitive. If, in addition, use is made of the hybridisation and detection method recommended by the supplier (VECTOR) of the detection kit used, the result will deteriorate - the detection limit falls by 10 as compared to the method according to the present invention (see Fig. 1) .
The invention will be described in greater detail below in connection with the detecting of PVY in potato juice and with reference to the accompanying drawings in which
Fig. 1 shows the result of a comparison between two different hybridisation and detection techniques using a probe labelled with photobiotin, and
Fig. 2 shows the result of a comparison between biotin labelling and labelling with 32P for detection of PVY in potato tuber juice.
A small amount of potato juice from leaves or tubers is applied to a cellulose nitrate filter, after the juice has been treated with a protein-decomposing enzyme (protease). This is necessary since biotin endogenously occurring in potato leaves is bonded to protein (Nikolau et al 1985) which, like nucleic acids, also binds to cellulose nitrate. If the protein is not decomposed before application, the endogenous biotin will be detected as if there were a virus .
Pretreatment of plant sap with a protein-decomposing enzyme has previously been tested for the purpose of improving the virus detection degree in connection with

32
P detection. It was expected that the release of the virus nucleic acid from the protein envelope would lower the degree of detection, but treatment with a protein-decomposing enzyme had no effect whatsoever (Baulcombe et al 1984). When using biotin-labelled DNA it is, however, absolutely necessary that the protein in the plant sap which binds the endogenous biotin is decomposed, since otherwise falsely positive tests would be obtained. This has been reported by, among others, Forster et al (1985) who did not treat the plant sap with a protein- decomposing enzyme.
The cellulose nitrate filter is "baked" at a high temperature in vacuum for binding the nucleic acids, int. al. virus RNA, whereupon a prehybridisation is made. The prehybridising step is necessary so as to minimise the background disturbance in the filter owing to unspecific binding of■ macromolecules . This is avoided by treating the filter with a solution whose ingredients have precisely the function of reducing or preventing an unspecific background.
Subsequently, the actual nucleic acid hybridisation begins (Gatti et al 1984), when the filter is incubated with biotin-labelled DNA which is homologous to PVY-RNA. If a sample is infected with PVY, virus RNA bound in the cellulose nitrate filter will be hybridised with a homologous cDNA strand and detected by an enzymatic reaction with biotin, which results in a coloured product clearly visible to the eye. Blue-coloured spots on the filter thus indicate that a hybridisation has occurred between virus RNA and complementarily prepared DNA, and consequently indicates a virus infection. Where nothing is to be seen, no hybridisation has occurred, and this implies a virus-free sample.
In a comparative test, it was shown that biotin-labelled DNA homologous to virus RNA detects virus as adequately as 32P labelled-DNA. It appears from Fig. 2 that there is full agreement between the two detecting techniques when demonstrating the presence of virus in potato tuber juice. By means of the non-radioactive hybridising technique, amounts as low as 2 pg of virus

RNA are detected, which is the lower detecting limit
32
when using P. By changing the way of labelling with biotin, it is however possible to further lower the detecting limit (Forster et al 1985). For the detection of 2 pg of virus RNA by means of 32P, an exposure time of several days is required as compared to biotin label- ling, where there is a response after 1-4 hours only.
The agreement between nucleic acid hybridisation and ELISA is excellent in so far as samples containing virus according to the ELISA test also yield a positive result with nucleic acid hybridisation. However, a larger amount of virus-positive samples is detected in nucleic acid hybridisation.
This method thus is very sensitive and clearly superior to ELISA for detecting small concentrations of virus. The advantage of this test as compared to
ELISA is also that the virus isolation need be made just once. With the molecular test, the bacteria are propagated by cDNA if required, but with ELISA, continuous virus purifications must be made for preparing antibodies. The capacity of the hybridising technique is very high and comparable to ELISA, i.e. the screening of thousands of plants takes only a few days.
Since the sensitivity of the method according to the invention is much higher, the analysis can be carried out on potato tubers, which till now was not possible. This means that the presence of virus can be detected already in the growing crop, which is crucial to the grower, since he can judge whether the potatoes can be used as seed, or whether they are to be used as food-potatoes or industrial raw products. Except that the grower makes a direct economic profit by early planning, the method contributes to the national economy since perfect seed lots can always be selected.
The molecular hybridising test here described can be used for many other crops than potatoes. Thus, one may test, for example, the tolerance to barley yellow dwarf virus in pasture grass, which was previously not possible because laboratory methods were lacking.
The mode of action for performing the invention will be described in detail in the Example below which is not intended to restrict but merely to exemplify the invention. The results accounted for above have

SUBSTITUTE SHEET been obtained by the mode of action as described in the Example.
EXAMPLE

NUCLEIC AC^D_HYBRIDI ATION_FOR_DETECTING_PLANT_VI_RUS_
Preparation of samples
- Juice from potato tubers
- About 200 mg tuber tissue are homogenised in 150
μl sterile H-,0
- the extract is left on ice for 2 hours, whereupon
it is centrifuged for 3 min.
- 35 μl of the supernatant are transferred to new
tubes and are kept frozen until required
- Juice from potato leaves
- 2 drops of juice from a leaf pressed in a leaf press are added to 500 μl 0.1 M potassium phosphate buffer, pH 7.0
- The samples are kept frozen until required
- All samples are treated as follows:
- The samples are thawed
- to 35 μl extract are added:
6 μl proteinase K (50 mg/ml )
5 μl salmon sperm DNA (10 mg/ml)
- The samples are incubated at 37 C for 30 min.
- The samples are treated at 100°C for 10 min, followed by rapid cooling on ice so as to denature the nucleic acids
- The samples are centrifuged for 30 sec. prior to
application
Application of sample
- A cellulose nitrate membrane (8 x 12 cm) is soaked in 80 ml H20 for 10 min. and subsequently in 80 ml 20 x SSC for 10 min.
- The membrane is allowed to dry before being mounted in a vacuum manifold so as to facilitate the applica- tion of samples
- By means of a pipette, there are applied 35 μl of both samples and checks which consist of a buffer

SUBSTITUTE SHEET and plasmid DNA, respectively
- The membrane is treated at 80°C in vacuum for 2 hours, the nucleic acids being immobilised in the membrane
Prehybridisation
- The cellulose nitrate membrane is washed according to the instructions issued by the supplier of the detection kit used (BRL) , in 250 ml of
- 0.1 x SSC
- 0.5% SDS
at 65°C for 60 min. by shaking
- The membrane is transferred to a plastic pouch which is sealed after the prehybridising solution has been added which consists of the following components (modification by Gatti et al 1984):
- 4 ml 50 x Denhardt' s solution
- 4 ml 20 x SSC
- 1 ml 1 M NaHP04 (pH 6.5)
- 9 ml formamide
- 1 ml salmon sperm DNA (10 mg/ml)
- 200 μl 10% SDS
- The prehybridising solution is treated at 100°C for 10 min. followed by rapid cooling on ice before being added to the hybridising pouch.
- The cellulose nitrate membrane is incubated at 42 C in a shaking water bath overnight (at least 16 hours)

Hybridisation
- The prehybridising solution is replaced by a hybridising solution of the following composition (modification by Gatti et al 1984):
- 4 ml 20 x SSC
- 0.8 ml 50 x Denhardt' s solution
- 0.4 ml 1 M NaHP04 (pH 6.5)
- 9 ml formamide
- 200 μl salmon sperm DNA (10 mg/ml)
- 4 ml 50% dextran sulphate
- 1.4 ml H20
- 200 μl 10% SDS

SUBSTITUTE SHEET - The hybridising solution is treated at 100°C for 10 m followed by rapid cooling on ice before being added to the hybridising pouch
- 200 μl, probe, i.e. DNA complementary to virus RNA, are treated in the same way as above at 100 C for
10 min. followed by rapid cooling on ice before bei added to the hybridising solution in the plastic po

- The cellulose nitrate membrane is incubated at 42 C overnight in a shaking water bath (at least 20 hours) Posthybridisation washing
- This step is carried out according to the instruction issued by the supplier of the DNA detection kit used (BRL)
- Washing of the membrane in 250 ml 2 x SSC, 0.1% SDS for 3 min. at room temperature, repeated once
- Washing in 250 ml 0.2 x SSC, 0.1% SDS for 3 min.
at room temperature, repeated once
- Washing in 250 ml 0.16 x SSC, 0.1% SDS for 15 min. at 50 C, repeated once
- The membrane is quickly rinsed in 2 x SSC, 0.1% SDS at room temperature
Filter blocking
- This step is carried out according to the description issued by the supplier of the DNA detection kit used (BRL)
- The membrane is washed in 100 ml buffer (1) for 1 mi

- Incubation at 42°C in shaking water bath in 100 ml buffer (2) for at least 20 min.
- The membrane is dried between 2 Whatman 3MM papers in vacuum at 80°C for 10-20 min.
- Buffer (1):
- 0.1 M Tris,HCl pH 7.5
- 0.1 M NaCl
- 2 mM MgCl2
- 0.05% Triton X 100
- Buffer (2) :
- 3% BSA in buffer (1) Detection
- This step is carried out according to the description issued by the supplier of the DNA detection kit used (BRL)
- The membrane is soaked in buffer (2) for 10 min.
- Streptavidin is prepared in a polypropylene tube
- 6 μl stock solution (1 mg/ml) to 3 ml buffer (1)

- The membrane is incubated in the streptavidin solution for 10 min. during gentle shaking; the solution is pipetted over the membrane at intervals. The solution is decanted.
- The membrane is washed inJ buffer (1) in a volume which is 10-20 times the volume of the streptavidin solution, for 2 min; repeated twice.
- Poly(AP) solution is prepared as follows:
- 3 μl stock solution (1 mg/ml) to 3 ml buffer (1)

- The membrane is incubated in poly(AP) solution for

10 min. during gentle shaking; the solution is pipetted over the membrane at intervals. The solution is de- canted.
- The membrane is washed in buffer (1) in a volume which is 10-20 times the volume of the poly(AP) solution for 2 min; repeated once
- The membrane is washed twice in the same volume, buffer (3)
- Buffer ( 3 ) :
- 0.1 M Tris,HCl pH 9.5
- 0.1 M NaCl
- 50 mM MgCl2
Visualising
- This step is carried out according to the description issued by the supplier of the DNA detection kit used (BRL)
- 7.5 ml colour solution for the membrane of the
above-mentioned size is prepared
Mixed are:
33 μl NBT
SUBSTITUTE SHEET 7.5 ml buffer (3)
Added is:
25 μl BCIP
- The cellulose nitrate membrane is incubated in this colour solution in a sealed plastic pouch
- The colour is developed in darkness up to 4 hours.

- The membrane is washed in 20 mM Tris,HCL (pH 7.5), 5 mM EDTA so as to stop the development of colour.

SUBSTITUTE SHEET Bibliography:
Baulcombe, D., Flavell, R.B., Boulton, R.E. and Jellis, G.J. 1984. Plant Pathol. 33: 361-370.

Clemens, M.J. 1985. In Virology, A practical approach. (Ed. B.W.J. Mahy) . IRL Press: 211-230.

Forster, A.C., Mclnnes, J.L., Skingle, D.C. and Symons, R.H. 1985. Nucl. Acids Res. 13(3): 745-761.

Gatti, R.A., Concannon, P. and Salser, W. 1984. Bio-techniques 3: 148-155.

Gubler, U. and Hoffman, B.J. 1983. Gene 25: 263-269.

Lindsten, K. 1974. Nordisk Jordbruksforskning. 55(3). 354-356.

Nikolau, B.J., Wurtele, E.S. and Stumpf, P.K. 1985. Anal. Biochem. 149: 448-453.