JP3613090B2 - Production of attenuated viruses - Google Patents

Description

【００４４】
変異株ＲＭ２１のゲノムＲＮＡ の変異解析の結果、４６４６番目がウラシルからシトシンへ置換した ｓｉｌｅｎｔ ｍｕｔａｔｉｏｎ （アミノ酸はグリシンのまま） であった （図３）。
また、変異株ＲＭ２１を感染させた植物についてウエスタンブロットを行った結果、感染植物内におけるウイルス増殖量は、野生株とほとんど差がなく、病徴の軽減が単なるウイルス増殖量の低下によるものではなかった。また、植物体に感染後、遺伝子および表現型に新たな変異が起こることはなく、継代してもその病原性およびゲノムＲＮＡ の塩基配列は安定していた。さらに、ＲＭ２１の交叉防御試験では、病徴の発現が遅れ、野生株本来の病原性が低下、すなわち両者の間に干渉作用が働いた。
【００４５】
【発明の効果】
本発明によれば、植物のウイルス防除に効果的な弱毒ウイルスの新規な作出方法が提供される。この方法は、ウイルス遺伝子に部位非特異的に変異を導入することにより、安定した弱毒変異株を作製しうるものである。本発明で得られたＡＳＧＶの弱毒ウイルスは、病原性の強い野生株の感染を防ぐ干渉作用を有し、しかも使用中に病原性が回復することなく安全であり、かつ野生株と同等の増殖能力をもつ、効率のよい防御ウイルスである。従って、このウイルスを予め幼苗に接種しておけば簡便な方法で効率的にＡＳＧＶを防除することができる。
【図面の簡単な説明】
【図１】ＡＳＧＶゲノムＲＮＡ の遺伝子構造を示す図である。
【図２】ＡＳＧＶ弱毒変異株の作製方法を模式的に示す図である。
【図３】ＡＳＧＶ野生株と弱毒変異株ＲＭ２１の塩基配列を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing an attenuated virus (protective virus) effective in protecting against a viral disease of plants, an attenuated virus obtained thereby, and use thereof.
[0002]
[Prior art]
A phenomenon called interference that is resistant to a virus related to a plant when it is infected with a certain virus was discovered by McKinney about 70 years ago, and today it is used for a viral disease control method. Has been. In other words, a method has been put to practical use in which a plant is infected with a weakly pathogenic virus that does not cause abnormal growth to the plant even if it is infected, and a highly pathogenic virus that interferes with the virus is prevented from being infected. This is an effective method for biological control of viruses. The advantage of this control method is that it can be used in crops as soon as the attenuated virus is isolated or produced, and there is no concern about environmental pollution that impairs human health like pesticides. It can be said to be an easy biological control method.
[0003]
Such attenuated virus production methods include temperature treatment, selection from nature, nitrite treatment, and the use of satellite RNA that reduces pathogenicity ("Plant virus disease" Sadayoshi Fukushi, Naoji Suzuki, Eishiro Shikata, 1986, Yokendo, p. 514; “Attenuated Viruses and Plant Protection”, Masamichi Nishiguchi, 1993, Development of Vol.18, Hirokawa Shoten, p. 320-328; “Encyclopedia of Plant Pathology”, Kunihei Kishi and Hino (Tatsuhiko et al., 1995, Yokendo, 721-726). However, in these methods, the site at which the mutation is introduced is completely unknown, and it is completely unknown when and how much mutation is introduced. Further, it is a strain introduced by chance or under a certain stress environment due to temperature, chemical treatment, etc., and has a disadvantage that it is unlikely that it is a stable attenuated strain. In addition, some attenuated viruses that are currently in use are concerned about the possibility of recovery of pathogenicity during use.
[0004]
In addition, due to the development of molecular biology, methods to create viruses with reduced pathogenicity by introducing mutations in vitro are currently being studied (Agricultural microbes-utilization and prospects-, Umedani, Seiji, Kato, Kaoru (Coed., Yokendo, page 592). What has been generally considered as a method for producing an attenuated virus by introducing a mutation is a method for introducing a mutation in a site-specific manner using a gene involved in pathogenicity as a target. However, in this method of introducing a mutation into a known site by a known method, the stability is poor because the mutation is intentionally introduced without considering physicochemical and biological stability. In other words, there was a problem in practicality such as the possibility of recovery of pathogenicity. Moreover, since a mutation is introduced based on known knowledge, only a known level of effect is expected.
[0005]
[Problems to be solved by the invention]
As described above, it has been difficult to obtain a stable mutant strain that does not recover pathogenicity during use and effective for biological control of the pathogenic virus by conventional attenuated virus production methods. .
Therefore, an object of the present invention is to produce a safe attenuated virus having strong interference with wild type infection and stable pathogenicity.
[0006]
[Means for Solving the Problems]
The present inventors have conducted research based on a completely new idea that a site-specific (random) mutagenesis method can generate a stable mutant strain when introducing a mutation into a viral gene by a molecular biological technique. As a result, the present invention was completed. Such a method is a new method totally different from the conventional method of site-directed mutagenesis into a limited gene region known to be involved in pathogenicity.
[0007]
That is, the gist of the present invention is a method for producing an attenuated virus by introducing a mutation non-site-specifically into a virus genome that causes a viral disease in a plant and selecting an attenuated mutant. Is introduced by introducing a plasmid capable of synthesizing an infectious virus genome into a host having a deficiency in the DNA repair system, and culturing the host. This method is applicable to viruses in general, but the virus is an apple stem grooving virus, especially when the virus is of the genus Capirovirus and the plasmid is capable of transcribing infectious viral RNA. Or it is applied suitably when it is a citrus tatter leaf virus (citrus tatter leaf virus).
[0008]
The present invention also relates to an attenuated mutant strain of apple stem grooving virus or citrus tatter leaf virus of the genus Capirovirus, which is produced by the above method and attenuated by silent mutation generated in the viral gene. A preferred example of the attenuated mutant is an attenuated mutant RM21 of apple stem grooving virus, which is an attenuated mutant in which the 4646th position is mutated in the base sequence of genomic RNA compared to the wild strain. It is.
[0009]
Further, the present invention is characterized in that a seedling of a plant is inoculated with an attenuated mutant strain of Capirovirus genus virus produced by the method for producing an attenuated virus and attenuated by silent mutation generated in the viral gene. A method for controlling Pirovirus is also provided. In particular, the present invention relates to a method for controlling apple stem grooving virus or citrus tatter leaf virus, characterized in that seedling seedlings of apple stem grooving virus or citrus tatter leaf virus are inoculated into plant seedlings. Also provided are control agents for apple stem grooving virus or citrus tatter leaf virus containing these attenuated mutants as active ingredients.
[0010]
The present invention is described in detail below.
In the method for producing an attenuated virus of the present invention, a virus gene is site-specifically mutated to produce an attenuated mutant strain, and the mutation occurs randomly regardless of the function of the gene.
[0011]
A specific method for introducing mutations randomly into a viral gene is to introduce a mutation into a target gene by introducing a plasmid containing the target gene fragment into a strain having a deficiency in the DNA repair system and culturing the bacterium. is there. As a strain having a deficiency in the DNA repair system, for example, an E. coli strain having three deficiencies in the DNA repair system, Epicurian coli XL1-Red (Stratagene Co., Ltd.) may be used as compared to the wild strain of E. coli. it can. The three defective sites are mutS (mismatch repair gene), mutD (3′-5 ′ DNA polymerase III endonuclease gene), and mutT (gene involved in the hydrolysis of 8-oxoGTP). A method for introducing a mutation into an plasmid in vivo by introducing the plasmid into Epicorian coli XL1-Red and culturing the E. coli is described in Greener, A. et al. and Callahan, M .; , XL1-Red: A high efficient random mutation strain, STRATAGENE in molecular biology 7: 32-34. In the present invention, a stable attenuated mutant is obtained by utilizing this method for the production of an attenuated virus.
[0012]
In order to produce an attenuated virus by the method of the present invention, a plasmid capable of synthesizing an infectious viral genome (transcribed infectious genomic RNA in the case of an RNA virus) is prepared, and a random mutation is introduced into the plasmid by the above-described method. Select stocks.
[0013]
A method for producing an attenuated strain and confirmation of the obtained attenuated strain will be described in detail in the case of using an apple stem grooving virus (hereinafter referred to as “ASGV”) as a virus.
[0014]
ASGV is a genera of Capirovirus (The genus). Capillovirus ) Type species (representative virus), which occurs widely in regions around the world where apples, pears, pears (Japanese pears), apricots (Japanese apricots) are cultivated, such as graft graft abnormalities It is a virus that causes symptoms. In addition, there is citrus tatter leaf virus (hereinafter referred to as CTLV) as a virus very close to this virus and having almost the same base sequence of the gene. CTLV is a variety "Meyer lemon" introduced in 1908 from China to the United States. (Citrus meelii ) A latent infection in a tree was discovered (Wallace & Drake, 1962). This virus was named citrus tatter leaf virus because it showed tatter leaf (boro cloth-like deformed leaf) symptoms in Citrus excelsa. In addition, this virus is known to cause a budunion disease in Satsuma mandarin that uses Troyer citrange as a rootstock (Calavan, EC, Christiansen, D.W., and Roistacher). , C.N., 1963, Symptoms associated with tatter-leaf virus infection of Toroyer cigarette rootstocks.Plant Disease Reporter 47: 971-975). In Japan, it was reported that a virus that can be identified as CTLV was detected from an abnormal grafting tree of Citrus unshiu (Miyakawa, Kuni, 1975, citrus grafting abnormality and virus, plant protection 29, 371-376). Due to the failure of the grafted portion, a delamination is formed at the joint between the ear and the base, and the movement of nutrients between the ear and the rootstock is prevented. As a result, the vegetation is weakened and sometimes broken by strong winds. It is a virus important for citrus production, and it causes severe growth inhibition in lily of herbaceous plants by mixed infection with other viruses (Inoue Narunobu et al., 1979, citrus tatter leaf virus isolated from lily, Japanese Society of Plant Pathology) 45, 712-720).
[0015]
ASGV and CTLV are viruses belonging to the genus Capirovirus having a single-stranded plus-strand RNA having a total length of 6496 bases as a genome. Genomic RNA includes ORF1 that encodes a 241 kDa protein and ORF2 that encodes a 36 kDa protein in a different frame (Ohira, K., Namba, S., Rosanov, M., Kusumi, T. et al.). , And Tsuchizaki, T., (1995) Complete sequence of an infectious full-length cDNA clone of citrus tatter leaf capillous virus: 230, 76 The gene structure of ASGV and CTLV genomic RNA is shown in FIG.
[0016]
A method for producing an attenuated mutant of ASGV (or CTLV) will be described below. The outline of the method is as schematically shown in FIG.
(1) Construction of infectious transcription RNA synthetic plasmid
An infectious transcription RNA synthesis plasmid (for example, pITCL) is constructed using a plasmid vector such as pUC18 based on the ASGV cDNA. In the plasmid pITCL, a T7 promoter is inserted upstream of the insert, and polyA and Not I sites are inserted downstream.
[0017]
(2) Creation of mutant strains
(A) Introduction of random mutation
An infectious transcription RNA synthesis plasmid (for example, pITCL) is used to transform an E. coli strain having a defect in the DNA repair system, such as Epicurian coli XL1-Red. When this Escherichia coli is cultured and propagated, it is impossible to repair base pairing errors (mismatches) and DNA damage that occur during replication. 2000 bases). Next, colonies appearing on the selective medium containing antibiotics are cultured, and the plasmid is purified from the culture solution. Purification may be performed by PI-50 (Kurabo Co., Ltd.) or QIAGEN Tip-20 (Funakoshi Co., Ltd.).
[0018]
(B) Isolation of mutant plasmid
The purified plasmid is used to transform Escherichia coli lacking the repair gene, such as JM101 strain or HB101 strain. In the mutant plasmid prepared using the above-mentioned Epicolian coli XL1-Red, mutations are introduced continuously in the cells of Epicurian coli XL1-Red, so that they are introduced into Escherichia coli having no defect in the repair system, and the gene of the mutant strain is fixed. Is to do. Each mutant strain is cultured on an antibiotic-containing medium, and a single colony is isolated for each clone and cultured to purify the plasmid. Plasmid purification may be performed using PI-50, QIAGEN Tip-20 or the like.
[0019]
(3) Determination of the entire nucleotide sequence of the mutant strain
The entire base sequence of each mutant is determined using appropriate primers. For example, a -21M13 Dye primer (Perkin Elmer Japan Co., Ltd.) on pUC18, an M13 Reverse primer, and an ASGV-specific primer synthesized inside the virus sequence can be used. For the sequence, Dye Deoxy Terminator Cycle Sequencing Core Kit (Perkin Elmer Japan Co., Ltd.), Dye Primer Cycle Sequencing Core Kit (Perkin Elmer Japan Co., Ltd.) or the like may be used.
[0020]
(4) Transcription of infectious RNA
The purified plasmid pITCL is treated with the restriction enzyme Not I, cleaved immediately downstream of the ASGV cDNA, the enzyme is inactivated by protease treatment, and the protein is removed by phenol / chloroform extraction. DNA is recovered by ethanol precipitation and infectious RNA is transcribed using T7 RNA polymerase.
The infectious RNA transcript synthesized in this way is subjected to the following test and screened based on pathogenicity to obtain the target attenuated mutant.
[0021]
(5) Plant inoculation test with infectious transcription RNA
The infectious transcribed RNA obtained above is used as a test plant, for example, Kenopodium quinoa ( Cenopodium quinoa ) Inoculate the leaves. The establishment of the infection can be confirmed by observation of local lesions on the inoculated leaves, upper symptom symptoms, and virus detection by Western blotting, ELISA, immunocapture-RT-PCR or the like.
[0022]
(6) Passage inoculation test
In order to examine whether the infectious RNA transcripts of each mutant strain cause pathogenic mutations or genomic RNA base sequence mutations on the plant body, infecting the infected leaves with a buffer solution and crushing Inoculate test plants.
[0023]
(7) Cross defense (cross defense) test
Plants infected with a virus do not infect closely related strains of virus. This is called cross protection. Cross-protection is due to interference, and is used as a means of plant virus control.
Cross-protection test is a certified plant C. quinoa Next, infectious transcription RNA of an attenuated mutant of ASGV is infected, and after 1 to 2 weeks, a wild strain is inoculated, and expression of disease symptoms is observed.
[0024]
(8) Presence or absence of remutation of the base sequence of the mutant strain in the infected plant
Mutant strain test plant C. quinoa In order to examine whether the mutation introduced in the infected plant cell returns to the wild type or other mutations when inoculated into the plant, determine the total nucleotide sequence of the genomic RNA of each mutant. And confirm. That is, total RNA is extracted from an infected plant, cDNA is synthesized by RT-PCR, and its entire base sequence is determined using an ASGV-specific primer. The sequence reaction in the determination of the base sequence uses the same method as the determination of the base sequence described in (3) above.
[0025]
RM21, which is one of the attenuated mutants produced as described above, has a slower onset of symptom than the wild type and is almost asymptomatic. This mutant strain has a silent mutation in which the 4646th base of genomic RNA is substituted from uracil (thymine in cDNA) to cytosine. That is, even with such base substitution, the amino acid remains glycine. It is unexpected from the conventional knowledge that pathogenicity changes are caused by silent mutation without protein change.
[0026]
Moreover, when the mutant strain RM21 was infected, it was confirmed that the amount of virus propagation in the infected plant was almost the same as that of the wild strain. Therefore, it is clear that the reduction in disease symptoms is not simply due to a reduction in the amount of virus propagation. In addition, no new mutations occur in genes and phenotypes after infection of plants, and their pathogenicity and genomic RNA base sequence are stable even after passage. In addition, RM21 was pre-inoculated C. quinoa When inoculated with wild strains, it shows an interference effect that causes a protective reaction against infection of wild strains. Thus, RM21 is an efficient protective virus that is safe and has the same growth ability as the wild type.
[0027]
Although some of the protective viruses currently in practical use are worried about the possibility of recovery of pathogenicity during use, the mutants produced by the method of the present invention have stable pathogenicity. , Safe. In addition, the method using a bacterium having a defect in a DNA repair system, which is a method for producing a mutant virus of the present invention, is a very effective method. In other words, mutations introduced into the base sequence occur randomly regardless of the function of the gene, and only stable ones are selected in the process of establishing each mutant strain, which is a new method for creating a practical attenuated virus. .
[0028]
The attenuated mutant RM21 of the present invention is C. quinoa By inoculating seedlings of various fruit trees such as apples (non-symptoms) and citrus and plants such as lilies in advance, it is possible to effectively control capirovirus genus viruses such as ASGV and CTLV. Viruses cannot be cured once they are infected, and it is important to establish effective control methods in order to reduce the disease caused by viruses. The control method using the attenuated virus can be performed by a simple method such as inoculating the attenuated virus with a sprayer or the like in advance at the time of raising seedlings, and is also effective when handling a large amount of seedlings.
EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.
[0029]
【Example】
[0030]
[Example 1]
Production of ASGV attenuated virus
1. Construction of infectious transcription RNA synthesis plasmid pITCL
Based on the cDNA of ASGV genomic RNA, an infectious transcription RNA synthesis plasmid pITCLp was constructed using pUC18. In this plasmid, a T7 promoter is inserted upstream of the insert, and polyA and Not I sites are inserted downstream.
[0031]
2. Creation of mutant strains
(A) Introduction of random mutation
The plasmid pITCL was used to transform Escherichia coli Epicolian coli XL1-Red (Stratagene). The procedure is as follows.
[0032]
100 μl of Epicrian coli XL1-Red competent cells were thawed in ice and placed in a 1.5 ml Eppendorf tube. To this was added 1.7 μl of β-mercaptoethanol (1.42 M) to a final concentration of 25 mM. Keep on ice for 10 minutes with gentle mixing every 2 minutes. Then 300 ng of pITCL was gently mixed and allowed to stand in ice for 30 minutes, 42 ° C. for 45 seconds, then in ice for 30 minutes. Furthermore, the SOC medium (20.0 g of tryptone, 5.0 g of yeast extract, 0.5 g of NaCl dissolved in 1000 ml of distilled water was preliminarily kept at 42 ° C. and then autoclaved. Immediately before use, 1M MgCl ₂ 10 ml, 1M MgSO ₄ 900 μl of 2% 20% (w / v) glucose solution and then filter filtration sterilized) was added to the SOB medium prepared by adding 10 ml and filter sterilized by filtration, transferred to a falcon tube, and cultured at 37 ° C. for 60 minutes with shaking.
[0033]
100 μl of each culture solution was applied to a selective LB agar medium (tripton 10.0 g, yeast extract 5.0 g, NaCl 0.5 g / l, Agar 15 g / l) and allowed to stand at 37 ° C. for 24 to 30 hours. did. This selection medium contains 200 μg / ml of ampicillin. About 100 colonies that appeared on the selection medium were each cultured in 5 ml of liquid LB medium (containing ampicillin 200 μg / ml) for 16 hours. The culture was performed at 200 rpm using EYELA Multi Shaker MMS (Tokyo Science Machine Co., Ltd.). After the culture, the plasmid was purified with PI-50 (Kurabo Co., Ltd.).
[0034]
(B) Isolation of mutant plasmid
In order to immobilize the mutant plasmid, the purified plasmid obtained as described above was transformed into E. coli ( Escherichia coli ) It was introduced into JM101 strain (or JM109 strain). This was performed by the following procedure using a transformation system using Transformation Kit (Nippon Gene Co., Ltd.).
[0035]
E. coli 100 μl of JM101 strain (or JM109) competent cell was thawed in ice and placed in a 1.5 ml Eppendorf tube. To this was added 5 μl of the plasmid, and the mixture was allowed to stand in ice for 20 minutes, at 42 ° C. for 45 seconds, and in ice for 5 minutes. Furthermore, 400 μl of Hi-Competent Broth previously kept at 37 ° C. was added, transferred to a falcon tube, and cultured with shaking at 37 ° C. for 60 minutes. Each mutant strain was cultured on LB agar medium (containing ampicillin 100 μg / ml) for 12 hours, one single colony was isolated for each clone and cultured, and the plasmid was isolated by PI-50 (Kurabo Co., Ltd.). Purification was performed.
[0036]
3. Determination of the entire nucleotide sequence of the mutant strain
The nucleotide sequence of the mutant strain was determined by using the -21M13 Dye primer (Perkin Elmer Japan Co., Ltd.) on pUC18, the M13 Reverse primer, and 26 ASSGV specific primers synthesized inside the virus sequence, and using the Day Deoxy Terminator Cycler. It was carried out by a sequence reaction using Sequence Core Kit (Perkin Elmer Japan Co., Ltd.).
[0037]
4). Transcription of infectious RNA
Infectious RNA transcripts were synthesized as follows using the plasmid pITCL (pITCL-RM) into which the mutation was introduced.
[0038]
After culturing the E. coli strain (JM101 or JM109) carrying the mutant plasmid pITCL (pITCL-RM) in 200 ml of LB liquid medium (containing ampicillin 100 μg / ml) for 14 hours, QIAGEN-Tip500 (Funakoshi) was The plasmid was used for mass purification. 10 μg of the obtained plasmid pITCL-RM was treated with the restriction enzyme Not I, cleaved immediately downstream of the ASGV cDNA, and then treated with protease K (final concentration 100 μg / ml) at 37 ° C. for 30 minutes to inactivate the enzyme. Protein was removed by phenol / chloroform extraction. DNA was recovered by ethanol precipitation, dissolved in distilled water and adjusted to 1 μg / μl, and then an infectious RNA transcript was synthesized by mCAP (registered trademark) RNA capping kit (Stratagene). Transcription conditions were 5 × Transcription buffer 5 μl, plasmid DNA 1 μg / 10 μl, rNTP 1 μl, CAP analog 2.5 μl, 0.75M DTT 1 μl, RNase-free H ₂ O was added to a final volume of 24 μl, then T7 RNA polymerase was added to 10 u / μl, and incubated at 37 ° C. for 30 minutes. For the inoculation test, RNase-free H ₂ The concentration is adjusted by addition of O and used.
[0039]
5. Plant inoculation test with infectious transcription RNA
The infectious transcript RNA obtained as described above was inoculated into test plants. As a test plant, it was cultivated in a glass greenhouse at 25 ° C. under long-day conditions. C. quinoa Was used. After sprinkling carbon random (400-600 mesh) on the middle to second to fourth leaves, about 48 ng of transcription RNA was inoculated per inoculated leaf. Inoculated plants were managed in a greenhouse. Inoculated leaves and upper leaves were collected, and local lesions and upper leaf symptom of the inoculated leaves were observed. Subsequently, the amount of virus in the infected tissue was estimated by Western blot and ELISA. In Western blotting, an anti-ASGV polyclonal antibody was used as the primary antibody, and an anti-rabbit IgG-AP conjugate was used as the secondary antibody. In ELISA, an anti-ASGV polyclonal antibody was used as a primary antibody, and an anti-ASGV monoclonal antibody was used as a secondary antibody.
[0040]
6). Passaging test
About 10 times the weight of the infected diseased leaf, 0.1M phosphate buffer solution (containing 0.1% 1-thioglycerin) is added and ground. C. quinoa Inoculated. As a control, phosphate buffer was used as the inoculum.
7). Cross defense (cross defense) test
C. quinoa Infected with infectious transcription RNA of an attenuated mutant of ASGV, one week later, the wild strain was inoculated and observed.
[0041]
8). Presence or absence of remutation of the base sequence of the mutant strain in the infected plant
In order to examine the presence or absence of remutation in the infected plant cell of the produced mutant strain, the entire nucleotide sequence of the genomic RNA of the mutant strain was determined and confirmed. That is, the mutant strain C. quinoa And total RNA was extracted from the infected leaves, cDNA was synthesized by RT-PCR, and its entire base sequence was determined using ASGV-specific primers. For the sequencing reaction in the determination of the base sequence, the same method as the determination of the base sequence described in 3 above was used.
[0042]
〔result〕
As a result of screening the strains into which various mutations were introduced by the above-mentioned method 2 based on the pathogenicity, a mutant strain RM21 having a slower symptom expression than the wild strain and having almost no symptom was created. Wild strain and mutant strain RM21 were respectively infected. C. quinoa The following reaction is shown in Table 1.
[0043]
[Table 1]

[0044]
As a result of mutation analysis of genomic RNA of mutant strain RM21, the 4646th was a silent mutation (amino acid remained glycine) in which uracil was replaced with cytosine (FIG. 3).
In addition, as a result of Western blotting on the plant infected with the mutant strain RM21, the amount of virus growth in the infected plant was almost the same as that of the wild strain, and the reduction of disease symptoms was not simply due to a decrease in the amount of virus growth. It was. In addition, no new mutations occurred in genes and phenotypes after infection of plants, and the pathogenicity and genomic RNA base sequence were stable even after passage. Furthermore, in the cross-protection test of RM21, the onset of disease symptoms was delayed, and the original pathogenicity of the wild strain was reduced, that is, an interference action was exerted between the two.
[0045]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the novel production method of the attenuated virus effective in the virus control of a plant is provided. In this method, a stable attenuated mutant can be prepared by introducing a mutation into a viral gene in a site-nonspecific manner. The attenuated virus of ASGV obtained in the present invention has an interference action to prevent infection of wild pathogens with strong pathogenicity, and is safe without recovery of pathogenicity during use, and has the same growth as that of wild strains. It is a powerful and efficient defense virus. Therefore, ASGV can be efficiently controlled by a simple method if seedlings are pre-inoculated with this virus.
[Brief description of the drawings]
FIG. 1 shows the gene structure of ASGV genomic RNA.
FIG. 2 is a diagram schematically showing a method for producing an ASGV attenuated mutant.
FIG. 3 is a diagram showing the base sequences of an ASGV wild-type strain and an attenuated mutant RM21.

Claims (10)

It is a method of creating an attenuated virus by introducing a mutation non-site-specifically into a viral gene that causes viral disease in a plant, and selecting the attenuated mutant strain. The method, which is carried out by introducing a plasmid capable of synthesizing an infectious viral genome into a host having a defect in the system and culturing the host. The method according to claim 1, wherein the attenuated virus is attenuated by a silent mutation generated in the viral gene. The method according to claim 1 or 2 , wherein the virus is a virus belonging to the genus Capirovirus and the plasmid is capable of transcribing infectious viral RNA. The method according to claim 3 , wherein the virus is an apple stem grooving virus or a citrus tatter leaf virus. An attenuated mutant strain of apple stem grooving virus or citrus tatter leaf virus of the genus Capirovirus, which is produced by the method according to claim 1 and attenuated by silent mutation generated in the viral gene. Attenuated mutant RM21 of apple stem grooving virus. The attenuated mutant RM21 according to claim 6, wherein the 4646th position is mutated in the base sequence of the genomic RNA as compared with the wild type. Control of a capirovirus genus virus characterized in that a seedling of a capirovirus genus virus produced by the method according to claim 1 and attenuated by a silent mutation generated in a virus gene is inoculated into a seedling of the plant. Method. A method for controlling apple stem grooving virus or citrus tatter leaf virus, comprising inoculating a young seedling of the plant with the attenuated mutant according to any one of claims 5 to 7 . An apple stem grooving virus or citrus tatter leaf virus control agent comprising the attenuated mutant according to any one of claims 5 to 7 as an active ingredient.

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