JPH01281079A - Attenuated strain of plant virus and preparation thereof - Google Patents

Abstract

PURPOSE:To obtain a stable attenuated strain (will not cause reverse mutation and not be restored to nonattenuated strain) by transducing a mutation site to a noncode range of genome RNA. CONSTITUTION:An attenuated strain useful for preventing a virus by utilizing cross protection wherein a plant infected with a certain virus is protected from secondary infection with a relative virus is obtained by transducing a mutation site to a noncode range of genome RNA. The noncode range is preferably a noncode range positioning 3' end port of genome RNA. Especially the noncode range existing at 3' end part of genome RNA is preferably a noncode range between transfer RNA-like structure part at 3' end part of genome RNA and code range of coded protein.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an attenuated strain of a plant virus and a method for efficiently and artificially producing the same.

[Conventional technology]

The overwhelming majority of viruses that infect plants are RNA viruses. There are many types of plant RNA viruses, including uniparticulate ones whose genome consists of one RNA and multiparticulate ones whose genomes are segmented into two or more RNAs. Representative plant RNA viruses include tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), and alfalfa mosaic virus (
AIMV), Brome Mosaic Virus (BMV),
Cowpea mosaic virus (CPMV), potato virus X (PVX), potato virus Y (PVY)
) etc. can be given.

Although there are still only a few plant RNA viruses whose genetic structures have been elucidated, it is known that genomic RNA consists of a portion that encodes genes necessary for virus function and a portion that is not translated into proteins (non-coding region). The computational coding region exists at each end of the genomic RNA from several tens to several hundred bases, and is thought to play some role in the viral replication process. Non-coding regions may also exist between genes within the genome.

Diseases caused by viruses are causing severe damage to crops.
For example, in 1982, the amount of damage caused by the virus in Japan was estimated to be approximately 8 billion yen.

However, there is currently no effective treatment for the virus, and the main focus of its control is on preventing virus infection. One of the effective techniques for controlling such infections is a method that utilizes the interference between closely related viruses, and has been put into practical use for several viral diseases [Tochihara, Agricultural Technology 41.48H198
6)).

Interference is a phenomenon in which plants infected with one virus are protected from secondary infection by closely related viruses. Therefore, if a virus strain that is closely related to the virus that causes disease and causes no or very little disease damage, that is, a weakly virulent strain, can be used to infect crops with the weakened strain in advance to prevent disease. It can suppress the infection of viruses that cause

When trying to control viruses by using interference,
The most important point is whether an excellent attenuated strain that can withstand practical use can be obtained. Attenuated viruses currently in use include those isolated from nature (for example, attenuated strains of Citrus tris buzavirus (CT V )) and those artificially created (Tomato tobacco mosaic virus (TM)).
V), an attenuated strain of cucumber marginal locus mosaic virus (CGM)
Conventionally, the method used to artificially create attenuated strains is to induce attenuated mutant strains from highly virulent strains in the field through high temperature treatment, nitrous acid treatment, etc. [For example, Gogi 41, Hokkaido Agricultural Experiment Station Telegram 99.67 (1971);
Gonsalves, Phytopatholog
y 74+1086 (1984)], but its creation depends on chance and is not efficient. In recent years, attempts have been made to create attenuated strains of multiparticulate viruses by exchanging genome components and using satellite RNA [Hanada, BIOI
NDtlSTRY 4゜357 (1987)], but a practical attenuated strain has not yet been produced, and it cannot be applied to single-particle viruses or viruses without satellite RNA.

On the other hand, recently, it has become possible to generate infectious viral RNA from the full-length cDNA of several plant RNA viruses by in vitro transcription reaction [Ahlquist et al., Proc.

Natl, Acad, Sci, USA 81.7066
(1984); Meshi et al., Proc.
Natl, Acad, Sci, USA 83.504
3 (1986)], by modifying cDNA, it has become possible to intentionally introduce mutations into any location on the viral genome RNA.

(Problems to be Solved by the Invention) The present inventors have conducted extensive research to develop a stable attenuated plant virus strain that can be used to control crop diseases caused by viruses, and a method for systematically producing this strain, and have TMV-
As a result of introducing various mutations into the genomic RNA using the L strain and studying their effects, we found that, surprisingly, the genomic RNA
They discovered that an attenuated strain could be obtained by introducing mutations into the non-coding region of A, and completed the present invention.

Note that a "stable" attenuated strain here means that it does not undergo reversion and return to a highly virulent strain.

[Means for Solving the Problems] The present invention provides an attenuated strain of a plant RNA virus that has a mutation site in a non-coding region of the genomic RNA, and a non-coding region located at the 3' end of the genomic RNA. Genome R, an attenuated strain of plant RNA virus with a mutation site
An attenuated strain of plant RNA virus that has a mutation site in the non-coding region between the transfer RNA-like structure at the 3' end of NA and the coat protein coding region;
The present invention is a method for producing attenuated strains of plant RNA viruses in which each of the above-mentioned viruses belongs to Tobamovirus, attenuated strains of plant RNA viruses in which each of the above-mentioned mutations is a deletion mutation, and attenuated strains of these plant RNA viruses.

The plant RNA viruses of the present invention include tobacco mosaic virus (TMV), cucumber marginal locus mosaic virus (
CGMMV), Hibiscus yellow spot virus, Odontoglossum ringspot virus, Cucumber mosaic virus (CMV), Alfalfa mosaic virus (AIMV), Brome mosaic virus (BMV), Cowpea mosaic virus (CPMV), Potato virus x (PVX), Potato virus Y (PvY), etc.

The attenuated virus strain of the present invention can be created by introducing mutations into the non-coding region of its genome, and will cause no or only a slight amount of disease when it infects a systemically infected plant. be. Further, the attenuated virus strain of the present invention does not necessarily have the ability to infect the whole body. That is, the attenuated virus genome RN
A technology has been developed to create virus-resistant plants by introducing the cDNA of A into the plant chromosome (Japanese Patent Application No. 194038/1982). Using this technology, the ability of a weakened virus strain to infect the whole body can be reduced. do not need.

Next, the present invention will be specifically explained using the production of an attenuated strain of tobamovirus as an example.

Tobamovirus (Tobamovtrus) is a single-particulate rod-shaped virus, including TMV, which causes disease in tobacco, tomatoes, cowpeas, etc., CGMMV, which causes disease in cucumbers, melons, etc., Hibiscus macular virus, which causes macular spots on hibiscus, Cymbidium, and Cadrea. This includes Odontoglossosum lingspot virus, which causes variegation on the petals of flowers. TMV is a standard virus among tobamoviruses, and is one of the viruses that has been extensively studied as a plant virus. TMV has many stocks (s
Among these, one of the standard strains, Vulgare, a highly virulent strain of the l-mad strain and its attenuated strain L11A, has the full length of the genome, and that of the highly virulent strain of Cowpea, Cc, and CGMMV, approximately the 3' side of the genome. 1(
The primary structure of 100 bases is known [Goelet
et at, Proc, Natl, 8cad,
Sci, USA 79. 5818 (1982); O
hn.

et al., J.Biochem, 96.1915
(1984); NisN15hi old et al.
Nucleic Ac1ds Res, 13.558
5 (1985); Meshi et al., Mo1.
Gen, Genet, 184.20 (1981);
Shi et al., Virology127, 54
(1983)] *According to this, the genomic RNA of TMV is approximately 64 (10 bases long, 13 bases from the 5' side)
Four types of proteins are encoded: protein at 0/180, protein at 30, and coat protein.

Approximately 70 bases are present at the 5' end and approximately 2 (10 bases) at the 3' end.
There is a non-coding region of bases, but there is either no non-coding region or only a few bases between internal genes. The non-coding region at the 3' end can be divided into a transfer RNA-like structural part of about 1 (10 bases) from the 3' end and about 1 (10 bases) between this and the coat protein coding region, but the latter There is a pseudoknot (pseud) in the part.
It is known that there are three consecutive tertiary structures called okno t) structures [van Belkume
tal,, Nucleic Ac1ds Res, 1
3.7673 (1985)].

Among these structures, the transfer RNA-like structure is C
MV, BMV, turnip yellowing mosaic virus (TYMV)
This structure is also found in many other viruses.

The inventors focused on the region between this transfer RNA-like structure and the coat protein coding region, and created a preferred attenuated strain by introducing mutations there. The introduced mutations include substitution mutations, insertion mutations, and deletion mutations. Among these, deletion mutations are particularly preferred, but are not necessarily limited thereto. Deletion mutations are preferred because they are stable and reversion to a virulent strain, a problem that often arises when using attenuated strains, is unlikely to occur.

FIG. 1 shows the region between the transfer RNA-like structure' portion of tobamovirus and the coat protein coding region (hereinafter referred to as the pseudoknot region). As mentioned above, there are three pseudoknot structures in this region. Hereinafter, in this specification, in order to distinguish between these, the 5' side (
From the left side of Figure 1)! 5', center, 3' respectively
This is called a shade knot structure.

One form of the attenuated strain according to the invention has a mutation site in the central pseudoknot structure.

An example is one in which part or all of the central shade knot structure is missing.

Another form of the attenuated strain according to the invention is one that has a mutation site in the 3' shade knot structure. As an example, 3
′ An example is one in which a part of the shade knot structure is missing. However, according to the inventors' knowledge, 3′
The pseudoknot structure is important for virus replication, and introducing large mutations into this region is undesirable because it is highly likely that the virus will not be able to propagate.

Yet another form of the attenuated strain according to the present invention has a 5' pseudoknot structure and a mutation site in its 5' upstream region. However, introducing mutations in this part alone is not sufficient to weaken the virus;
It is necessary to combine the two types of mutations described above. An example of such a gene is one in which the entire region from just downstream of the coat protein coding region to the central pseudoknot structure is deleted. Such a large deletion mutation has the advantage of being more stable than the aforementioned deletion of only the central pseudoknot structure.

The method for creating an attenuated strain according to the present invention is to use viral genome RNA.
It consists of introducing mutations as described above.

During the introduction, in the course of experiments leading to the present invention, infectious viral RNA was extracted from viral cDNA by an in vitro transcription reaction, which is already known.
[8hlquist and J.
anda, Mo1. Ce11. Biol.

4.2876 (1984); Meshi et al.
, ,Proc, Natl., Acad.

Sci, USA 83.5043 (1986)],
This is because this experimental system has been judged to be the most efficient method at present for introducing planned mutations into viral genomic RNA. However, it goes without saying that the present invention does not necessarily require the use of this experimental system as long as the desired mutation can be introduced into the viral genome RNA.

When attempting to carry out the present invention using this experimental system, a planned mutation site is introduced into the cDNA of the desired portion by utilizing an appropriate restriction enzyme cleavage site on the viral cDNA, and then By performing an in vitro transcription reaction using this modified cDNA as a template, viral RNA having the planned mutation site can be obtained.

The restriction enzyme cleavage site to be used should be different depending on the base sequence of the virus of interest; however, if the base sequence is known, those skilled in the art can easily select the restriction enzyme to be used.

In the above case, if the mutation to be introduced is a deletion mutation, it is possible to use Bat 31 nuclease - by way of example.

Bat 31 nuclease is an enzyme that acts on double-stranded DNA and shortens the DNA from the ends at a nearly constant rate. Therefore, after cutting with an appropriate restriction enzyme, allowing Ba 131 to act for an appropriate time will shorten the length of the DNA. A desired deletion mutation can be introduced. The reaction conditions for Bal 31 nuclease are described in general laboratory books [for example, M
Aniatis et al., “Molecular
Clonjng”, Co1d Spring H
arbor Laboratory (19B2)]
.

The present invention will be described in more detail below with reference to Examples using TMV-L.

The present invention is not limited to this embodiment, but includes modifications and improvements commonly made in the art, and is applicable to other viruses.

Example-1 In this example, E. coli 88 was used for DNA cloning.
101 strain (manufactured by Zenshuzo ■, Cat, kg051) was used. In addition, steps in this example where reaction conditions etc. are not specified are described in general experimental books [e.g. Maniatis e
tal,, “Molecular Cloning”,
Co1d Spring Harbor Labora
Tory (1982)], it is a process that can be easily carried out by those skilled in the art.

In addition, in the following explanation, unless otherwise specified, TMV
Genomic RNA and its cDNA are numbered sequentially from the 5' end.

1) Creation of a transcription vector with a deletion mutation in the pseudoknot region The transcription vector plasmid pLFW 3, which incorporates the full-length cDNA of TMV-L, is already known and is available from Meshi.
et al, [Proc, Natl, Acad
, Sci, USA 83°5043 (1986)]
Created according to the method. In this vector, transcription into RNA starts from the 5' end of the viral cDNA, and the restriction enzyme (ul) (manufactured by Zenshuzo ■, Cat, No. 1071A) is added immediately downstream of the 3' end of the cDNA.
has a cutting site. Therefore, by performing an in vitro transcription reaction using this vector plasmid cut with old uI as a template, we were able to detect infectious TMV-L.
RNA can be obtained.

To facilitate the task of introducing a defect in the pseudoknot region, pLFga3 was added to ρnl (manufactured by Zenshuzo, Cat.

No. 1068A) and Bawtll (made by Zenshuzo ■, C
At, No, 101OA), the 3' side 2 kb of TMV cDNA containing the pseudoknot region was separated, and this was divided into pUclB (manufactured by Zenshuzo ■, Cat, No. 3218).
) to create the working plasmid pUc3L-1.
I got it. This allows restriction enzyme N5i to be used in the next step.
Since there is only one cleavage site for 1 on the plasmid, the following steps become easier.

TMV-L cDNA has an N5iI site at position 6183, which is located in the coat protein coding region (6182
3' immediately downstream of the Therefore, when introducing a deletion into the pseudoknot region, the 20N5il site was used as the starting point for the deletion.

After cutting p[1c3L-1 with N5il (NEBSff127) to linearize it, reaction solution 1 (107
71 (0,6M NaC1゜20+oM Tris-
HCI pH 8,0 (25°C), 12mM MgC
2.3U of Bal 3 in 1g, 12mM CaC1z)
1 nuclease (BRL Cat.

No. 80195A) was applied. The reaction was carried out at 30°C, and a portion of the reaction solution was removed every minute from 1 to 10 minutes and EGTA was added to stop the reaction.
I got an A. After mixing these reaction solutions with different reaction times and cleaving at the pUC-derived Bam1ll site, a mixture of fragments of various lengths containing the 3' end of the viral cDNA was separated and separated into BamHL of pucia and old n.
clT (manufactured by Toyobo ■, Code, No, 1lNC-2
01) to obtain the ptL3NC plasmid series.

The above steps are schematically shown in FIG.

Clones that had been deleted to an appropriate length by restriction enzyme mapping were selected, and the end point of the deletion was determined by DNA sequencing [Luckow et al., Nucl.
eic Ac1ds Res, 15.417 (1987
)] - Hereinafter, each clone obtained will be referred to as ptL3NG-X according to the endpoint X of the deletion. ptL3NC-)'/
Leeds Mlul and Pstl derived from pUc (Zen Shuzo ■
(Cat, No. 1073A) to obtain a fragment containing the 3' end of the viral cDNA, which was used to construct the following deletion mutant virus transcription vector. This fragment contains r derived from the polylinker of pUC.
Although GGTCJ was supposed to have been inserted, the inserted base was actually different for each clone. i.e. ptL3NC-6207, -6209, -62
33 has 'GJ, -6189, -6246, -625
3, -6263 has rGGJ, -6275 has rG
GT J, -6204, -6239 has r GGTCJ
were inserted in each. This is probably because the restriction enzyme 11incII used in this example was contaminated with exonuclease activity.

By replacing the N5il (6183)-Mlul fragment of pLFW 3 with the pLL3Nc-X/Mlul-Ps fragment, mutant virus transcripts with deletions of various lengths immediately 3' of the coat protein coding region were generated. A vector pLD3N-X series was created.

The Kono series contains a strain that lacks only a small part of the 5' upstream part of the 5' pseudoknot structure (pLD3N-6
189) to one lacking almost the entire pseudoknot region (pL03N-6275).

Next, ptL3NC-X was cut with Pstl and E. coli DN
A Polymerase ■Large Fragment (manufactured by Takara Shuzo ■,
After blunting the ends with 5naBI (Cat, No., 214 OA), the fragment containing the viral cDNA 3' end was obtained by cutting with old u■.
By replacing the NEB 1130) (No. 6231)-Mlul fragment, a mutant viral transcription vector pLD3S-X series having a deletion from the central shade knot structure to the 3' pseudoknot structure was created. In pLD3S-X, 5naB is added to the binding site.
The sequence rTACJ derived from pLD3S-6239, -6253, -6263 is actually rACJ, and -62'75 is rTAC.
Each J was lost. This is probably because the restriction enzyme 5naBI used in this example was contaminated with exonuclease activity.

Furthermore, a mutant virus transcription vector pLD3P-X series having only a deletion in the 3' pseudoknot structure was constructed as follows. In this case, since there is no suitable restriction enzyme site on TMV cDNA, first N5iI (
6183) and 6249.
long [Btoscience Reports
1,243 (1981)]. 5au3AI-EcoRI of pLFW3 (5934
-6354) fragment, 3mp9 (manufactured by Takara Shuzo ■, Cat, No. 3118) B of phage DNA
After cloning between the amHr and EcoRI cleavage sites, one 31 DNA containing the same sequence as TMV RNA was obtained from this phage. A 15-mer synthetic DNA having a sequence complementary to positions 6235 to 6249 of TMV RNA was annealed to this first-strand DNA, and this was used as a primer for first-strand synthesis using E. coli DNA polymerase I large fragment. Next, a second strand was synthesized using Ml3 reverse sea fencing primer, and the resulting double-stranded DNA was cut with N5iI to fractionate the target 6183-6249 fragment. This fragment was combined with the ptL3Nc-X/old uL Pstr (blunt-end) fragment, and these
The pLD3P-X series was created by replacing the N5iI-old ul fragment of LFW3.

The structure of the deletion site of the pL03N-X, pLD3s-X and pLD3P-X series explained above is shown in FIG.

2) Infectivity of defective mutant virus The defective mutant virus transcription vector prepared as described above was cut at the old ul site at the 3' end of the viral cDNA, and an in vitro transcription reaction using E. coli RNA polymerase was performed using this as a template.

The reaction is m'GpppG (RNA cap analog
gue + Pharmacia 27-4635-01) presence,
Known methods [Ahlquist et al., Pr.
oc, Natl, Acad, Sci, USA 81.7
066 (1984)]. RN used for reaction
A polymerase is Burgess anti-Jend.
risak [Biochemistry 14+4
634 (1975)] from E. coli strain A19. The RNA polymerase used in this reaction is RNA PoIymerase (Ho
loenzyme, E. coli) 11208 can also be used.

By incubating the obtained transcription RNA with the purified TMV-0M strain coat protein (4/m) in o, iM phosphate buffer at 20°C for 116 hours,
Reconstitution of virus particles was performed. The reconstituted particles were more than 10 times more infectious than naked RNA.

The reconstituted defective mutant virus RNA (hereinafter referred to as defective mutant virus for convenience) was inoculated onto tobacco leaves by rubbing it together with carbon random. First, local lesions are formed on tobacco (variety Xan).
The multiplication ability of the defective mutant virus was examined by inoculating it into tobacco plants (thin nc) and the presence or absence of local lesion formation, and then it was inoculated into systemically infected tobacco (variety Samsun) to determine whether the defective mutant virus would spread throughout the body and cause disease. Examined.

When inoculating tobacco plants with localized lesions, the virus concentration was adjusted to 4 n'g/μP in terms of transcription vector DNA, and 20-30μ2 of the virus was inoculated per single leaf. When inoculating systemically infected tobacco, use virus solution 20-60
μ! was inoculated.

The amount of virus in the virus solution was adjusted to form 50-1 (10 local lesions).

Defective mutant virus derived from pLD3N-X series (
(hereinafter referred to as N-X), N-6253 and the defective mutant virus with a smaller defective region produced local lesions, but N-6263 and N-6275 did not produce local lesions, and these two defects This showed that the mutant virus had lost its ability to proliferate. On the other hand, in systemically infected tobacco, N-
6209 virus and viruses with smaller defective parts caused typical mosaic symptoms throughout the tobacco plant, but N-6233, N-6239, N-6246 and N-6253 did not cause mosaic symptoms. Two weeks after inoculation, virus transfer to leaves above the inoculated leaves was examined for systemically infected tobacco plants inoculated with these four types of defective mutant viruses.

As a result, although N-6253 was not detected in the upper leaves, the virus was detected in the inoculated leaves, indicating that N-6253 was a weakened strain. On the other hand, N-6233
, N-6239, and N-6246, the virus was detected in the upper leaves at a concentration of about 115 to 1710 of the wild type virus. This is at least N-6233, N-6239
, N-6246 spreads throughout the body but does not cause disease, indicating that it is a weakened strain with the ability to infect the whole body. Note that virus detection was performed by local lesion assay and coat protein detection by gel electrophoresis.

To summarize the above, the deletion mutant viruses of the N-X series are attenuated strains from N-6233 to N-6253, and those with smaller deletions are almost the same as the wild strains, and are more virulent than these. Those with large defects had lost their ability to proliferate.

Defective mutant virus derived from pLD3S-X series (
(hereinafter referred to as S-X), S-6239, S-6246
and S-6253 produced local lesions, but S-626
3 and S-6275 did not produce local lesions, indicating that these two deletion mutant viruses were unable to proliferate.

In systemically infected tobacco, S-6239, S-264
6 and S-6253 did not cause any mosaic symptoms, but the virus was detected in the upper leaves of S-6246 and S-6253, indicating that these two defective mutant viruses are attenuated strains capable of systemic infection. I understand. Regarding S-6239, no virus was detected from the upper leaves, but virus was detected from the inoculated leaves, indicating that S-6239 was also an attenuated strain.

Defective mutant virus derived from pL03P-X series (
(hereinafter referred to as P-X), only P-6253 produced local lesions, and P-6263 and P-6275 had lost their proliferation ability.

P-6253 did not cause mosaic symptoms in systemically infected tobacco, but the virus was detected in the upper leaves, indicating that it was a weakened strain capable of systemically infecting.

The results of the infectivity test for the defective mutant virus described above are shown on the right side of FIG. L and S are the results of inoculation tests on locally infected tobacco and systemically infected tobacco, respectively, and indicate the presence or absence of lesion formation or disease symptoms.

To summarize the above results, among the deletion mutant viruses created in this example, the attenuated strain was N-62.
33, N-6239, N-6246, N-6253, S
-6239, S-6246, S-6253 and P-6
It was 253. In particular, it was confirmed that all of these strains, excluding N-6253 and N-6239, were attenuated strains capable of systemic infection. Viruses with a large deletion of the 3' pseudoknot structure in the pseudoknot region do not have the ability to proliferate, and conversely, deletion of only part of the 5' shade knot structure and its 5' upstream region does not show significant attenuation. There wasn't.

3) Interference effect of attenuated deletion mutant virus strains Among the attenuated deletion mutant virus strains explained in the previous section, N-6239 and N-62
The interference effect of 46 was investigated.

Virus particles recovered and purified from the upper leaves of systemically infected tobacco plants inoculated with reconstituted defective mutant virus RNA (in vitro transcription reaction product) were used in the test.

Systemically infected tobacco (variety Samsun) seedlings (true leaves 4
-6 sheets) and 0.1M phosphate buffer (pH 7,0).
:IAd N-6239 or N-6246 (5μg/
-) was inoculated.

As a control experiment, only the buffer solution was inoculated. 1 at 25°C
After 5 days, the newly developed upper leaves were infected with highly virulent strain T.
MV-L (1 μg/rnl) was inoculated and observed at 25°C.

One month after inoculation with the highly virulent strain, all of the cigarettes that were not inoculated with the attenuated strain showed mosaic symptoms, but cigarettes inoculated with N-6239 showed 1 out of 4 cigarettes, and cigarettes inoculated with N-6246 showed 1 out of 4 cigarettes. Three of them did not show a polisher. after that,
Observation was continued until 7 weeks after inoculation with the virulent strain, but no changes were observed. After 7 weeks, the uppermost leaves of each plant were collected and examined for the presence of viruses by local lesion assay, and it was found that even plants that did not exhibit abrasion, only plants that showed abrasion or only highly virulent strains. 1150 viruses were detected from 1/10 of the plants inoculated with . This result strongly suggests that the attenuated strain is proliferating even in plants that do not exhibit a grinder, and these plants are considered to be protected from disease by the interference of the attenuated strain. This result also clearly shows the attenuated toxicity of N-6239 and N-6246.

〔Effect of the invention〕

The present invention has made it possible to artificially create attenuated virus strains that are effective for controlling plant virus diseases much more efficiently and systematically in a shorter time than conventional methods. Furthermore, whereas attenuated strains created by conventional methods are base substitution mutant strains in most cases [for example, TMV-
Attenuated strain L11A of L; NishiN15hi et
al, Nucleic Ac1ds Res.

13.5585 (1985)], more stable mutations such as deletion mutations can be introduced into the attenuated virus strain produced by the present invention, and the highly virulent strain, which has often been a problem when using attenuated strains, can be introduced. The shortcomings of returning to can be solved.

[Brief explanation of the drawing]

The box in Figure 1 shows a model of the interaction in the pseudoknot region, and the box (■) shows the RNA sequences of various tobamoviruses. Figure 2 shows the preparation method of the ptL3NC plasmid series used to introduce a deletion mutation site into the pseudoknot SN region, and Figure 3 shows the method for preparing the ptL3NC plasmid series used to introduce a deletion mutation site into the pseudoknot SN region.
N-X, shows the structure of the deletion site of pLD3s-X and pLD3P-X series.

Claims (10)

[Claims] (1) An attenuated strain of plant RNA virus that has a mutation site in the non-coding region of its genomic RNA. (2) The attenuated strain according to claim 1, wherein the non-coding region is a non-coding region located at the 3' end of the genomic RNA. (3) The non-coding region at the 3' end of the genomic RNA is a non-coding region located between the transfer RNA-like structural part at the 3' end of the genomic RNA and the coat protein coding region. Attenuated strain. (4) The attenuated strain according to any one of claims 1 to 3, wherein the virus belongs to Tobamovirus. (5) The attenuated strain according to any one of claims 1 to 4, wherein the mutation is a deletion mutation. (6) A method for creating an attenuated strain of a plant RNA virus, which comprises introducing a mutation site into a non-coding region on genomic RNA. (7) The method for producing an attenuated strain according to claim 6, wherein the non-coding region is a non-coding region located at the 3' end of the genomic RNA. (8) A method for creating an attenuated strain in which the non-coding region at the 3' end of the genomic RNA is a non-coding region located between the transfer RNA-like structural part at the 3' end of the genomic RNA and the coat protein coding region. (9) The method for producing an attenuated strain according to any one of claims 6 to 8, wherein the virus is a virus belonging to Tobamovirus. (10) Claims 6 to 9 wherein the mutation is a deletion mutation.
A method for producing an attenuated strain as described in any of the above.