JPH07507457A - Viral resistance by plant transformation with the replicase portion of the plant viral genome - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Virus resistance by plant transformation with the rebricase portion of the plant virus genome This is our previously filed U.S. Patent Application No. 07/49 filed on March 12, 1990. This is a partial continuation of 1.473.

Transformed with the tobacco mosaic virus (TMV) coat protein gene and expressed it. 198 of P., Powell-Able et al. Since the 6th year literature [see Science 223: 738], various virus infections have been reported. Many of these concepts have undoubtedly important implications for the protection of many crop species. There are other examples. For example, to date, viral coat protein transmission resistance has been Fa mosaic virus, tobacco rattle virus, potato virus X1 cucumber Mosaic virus (CMV), potato virus and potato virus At least 15 taxa including plants transformed with Tetovirus Y coat protein Found in 25 viruses.

Plant viral sequences other than those encoding viral coat proteins can be transformed into Determining whether replanted plants can resist viral infection after transformation Tested for. Alfalfa containing almost full-length copies of RNA 1 and 2 Mosaic virus positive sense sequences can induce resistance in transformed plants. I couldn't [see Pyrology 163: 572 (1988)]. TMV and The antisense sequence of the potato coat protein gene confers low-level resistance in transformed tobacco. induced [Brondinges of the National Academy of Sciences] Nshizu USA86: 6949 (1989) and Hi EM B O') +-Nal ? + 1273 (1988)]. CMv genome (RNAI 5” sequence) A similar antisense RNA from one of the three parts tested showed low levels in one transformation system. Granted Bell resistance.

Using plant transformation with DNA prepared from satellite RNA of plant viruses Other forms of resistance have been reported. For example, the use of CMV satellites [Nichi 328 + 799 (1987)] and satellites with self-cutting capabilities. Concept of lipozyme based on sequence derived from human RNA [Nature 334, 585 ( 1988)].

The invention described herein provides an entirely new type of plant that can be transferred from one plant generation to another. Shows virus-induced resistance. The present invention is directed to the entire replicase portion of the viral genome. or a transgenic plant containing the coding sequence taken from , discloses that it is resistant to subsequent illness caused by the virus. The inoculated leaves are very Although there may be benign virus synthesis, the virus does not appear to be spreading and therefore causes disease. Qi does not develop. In the following description, the 54 kDa coding sequence from TMV is Use and RN encoding a component of the cucumber mosaic virus polymerase The modified cDNA of A-2 is described as two specific examples of the broad technique according to the invention. do. Thus, in its broadest embodiment, the invention provides a method for replicating pathogenic viral genomes. Infect plants transformed with DNA copies of fragments or sections taken from case parts. Identify the means to provide resistance to Furthermore, the present invention provides Transformation that retains the portion of the viral genome that encodes the portion of the precase genome Reveal plants and their seeds. According to the present invention, U. A transformed plant containing a portion of the virus rebricase gene is one in which that portion has been derived. resistant to subsequent viral diseases caused by viruses, and those plants also It is also resistant to subsequent illness caused by other (related) viruses.

In the description of the illustrative tobacco mosaic virus shown below, the presence of the 54 kDa sequence local chlorosis or necrosis development and symptoms associated with TMV infection or viral replication; It also prevents the development of

TM~' Genome organization is clearly well understood and accepted due to scientific commonality. It will be done. When decoding TMV RNA from the 5° to the 3° end, the open reading frame is 1 26 and 183 kDa proteins, 30 kDa motor protein and 17.5 kDa tegument protein. Code white. However, one aspect of the genomic strategy that has not been fully elucidated is An aspect is the precision of replicase enzymes involved in the synthesis of genomic and subgenomic RNA. It is a characteristic. The virus encodes four proteins, two of which are encoded by genomic RNA. and the other two are encoded by individual subgenomic RNAs. Although it is generally accepted that the virus Coding white is generally not acceptable.

N., D. Young, et al. [Journal of Cell Science Subs. 7:27 (1987)] consists of two co-initiating proteins, 126 kDa and 183 kDa. The 5° proximal portion of the genomic RNA encoding the a protein is a component of replicase. I reported that. The 183kDa protein is located at the UAG stop codon of the 126kDa protein. Produced by reading through. Two Pond proteins (with known functions), 3QkDa protein and coat protein each have separate subgenomic mRNAs to which each gene is 5° proximal. Synthesized from A.

However, what is generally not accepted is that we believe that distinct proteins exist. Therefore, there is an open reading frame in the reading part of the 183kDa gene. Knowing that it exists, we labeled the 54 kDa protein. Regarding the existence of this protein The proof-of-principle is that a third subgenome in TMV-infected plants, called l.RNA, NA is present, starting at nucleotide residues 3405 and 54 of the TMV genome. This comes from the discovery that it contains an open reading frame for kDa proteins [pyrology]. 145: 132 (1985)]. and subgenomic R as mRNA Support for its function as NA is that it is found in polyliposomes and that 1 There is a double-stranded RNA whose size corresponds to the double-stranded version of 1 subgenomic RNA. [Pyrology 113: 417 (+981) and and Pyrology 131:533 (1983)].

More specifically, the TMV genome containing the read-through portion of the 183kDa protein gene The following array for the part of the program is: 3405　34'72　3495　4919 [The complete genome of TMV is 6.39 5 nucleotides long, Golet et al. Le Academy of Sciences USA 79: 5818 (1982) [can be found]. l, RNA terminates at nucleotide 6395. During the main sequence, the main departure The 54 kDa open reading frame by Akira extends from nucleotide residues 3495 to 4919. The extended and underlined portions represent plant transformations that are more fully described in the Examples below. The sequence used is shown below.

For more details, see 1. The gene part for the 54 kDa protein within the RNA sequence is I To AU GAIIAAAG BA]Csu11＾GAA　1396α叉1tmltlAt+ AGAIJGGCLXl'LJWl425.

Unfortunately, the 54 kDa protein was not found in infected tissues. To Ethelina Cori β for 432 amino acids specific for the read-through of the expressed 126 kDa protein. - Once antibodies against galactondase fusion protein are prepared, protoplast extraction The 54kDa protein in the sample is immunoprecipitated, or the antibody detects the 183kDa protein there. It could not be detected by Western blotting under these conditions [T site etc. , Molecular and Noenetics 205: 82 (19 86)].

Similarly, the 54 kDa protein is sometimes fertilized in parts of the gel where such a protein is expected. Although intrabands are seen, Wester uses an antiserum that produces the entire protein. plot [G, J, Hills et al., Pyrology 158 + 488 (1987) reference]. However, antiserum that can be made up to the whole protein, In vitro analysis of either TMV RNA or T7 transcripts of the 54 kDa protein gene The 54 kDa protein produced from the translation product can be precipitated.

In an effort to fuse to the 54 kDa protein, we created a The coding sequence was transformed into tobacco. Unexpectedly, these transformations The replacement plants were then infected with uninoculated leaves of the U1 strain of TMV from which the 54 kDa sequence was derived. Completely resistant to replication. This resistance occurs when plants are exposed to high concentrations of viruses or viruses. It appeared when inoculated with either SRNA.

Furthermore, the resistance exhibited by transgenic plants (54 kDa) It differs from TMV coat protein transfer resistance in important respects. Resistance to TMV RNA and TM It was shown for both V-pilions. over a period of increasing inoculum concentration. , or did not seem to be lost along with it. Also, it is 54kDa white The gene has been induced and is effective against closely related mutant TMV strains. However, it was not effective against other TMV strains or other viruses.

Therefore, a novel aspect of the invention is that, in its "sense" orientation, Virus is introduced into plants that have already undergone transformation of their normal genome with part of the licase moiety. It is to convey the resistance to russ.

A more complete understanding of this and other aspects of the invention may be obtained by reference to the following drawings and examples. It can be obtained by

Figure 1 shows the CaMV35S promoter and tubalin synthase polyadenylation site. According to the invention, the TMV 54kDa coding sequence is inserted between A plant expression vector is shown.

Figure 2 shows the CaMV35S promoter and tubalin synthase polyadenylation site. A plant containing a modified Fny-CMV RNA-2 gene sequence inserted between Expression vectors are shown.

Figure 3 shows modified Fny-CMV RNA-2 chimera for integration into host plant genome. Showing the construction of the gene.

More specifically, FIG. 1 shows the Xho1 site of the polylinker part of pMON316 or Plasmids induced by insertion of TMV cDNA into either 5IIaI site. Indicates the mode. The numbers in these vectors refer to the nucleotides in the TMV genome. show.

The NPTn gene confers a selectable kanamayone resistance marker on transformed plants. Ru.

Figure 2 shows the CaMV35S promoter (35S) and tubalin synthase polyade modified Fny- according to the present invention inserted between the nylation site (NO5polyA) A plant expression vector containing the CMV RNA-2 gene sequence is shown. The following notes The present plasmid (pCMV N/B-23) is a binary The modified Fny according to the present invention is added to the BamH1 site of the sexual plant transformation vector pROK2. - Induced by inserting the CMV RNA-2 gene [Nature 321 :446 (1986) reference. This allows this plasmid to be linked to the RNA-2 cDNA sequence. By digesting pFny N/B-4 with 5 phl cut at the 5' site of achieved. The BamHl-3phl adapter is ligated into this 5phI site and then then digested with BaaHl to cut at the 3' position of the RNA-2 sequence, thereby cutting the entire RNA-2 sequence. Release the modified cDNA molecule.

This 3 kb fragment was subcloned into the BalHl site of pROK by standard techniques. , which produced pcMVN/B-23. Prior to plant transformation, this construct was transformed into Restock M~! Tripaternal mating transmitted by 294-pRK2013 [Merry's of Enzymoronoi 118: 627 (1986)] It was transferred to Kukankomu Tsume 772172 strain LBA-4404. trans conjugate Kanamainone and streptomycin at 5o and 125 ag/ml respectively Selected based on resistance to The numbers in the figure are in the Fny-CMV RNA-2 sequence. Refers to the nucleotide of

The neomynon phosphotransferase II (NPTn) gene is transformed into Provide plants with a selectable kanamayone resistance marker. LB and RB = Agroba T. tumefaciens transferred into the plant genome during plant transformation DNA left and right borders. ○ri=Reproduction origin.

Figure 3 shows the construction of pCMV N/B-23, which was done in two steps. first Is the 94 base pair part a full-length cDNA clone of Fny-CMV RNA-2? It was deleted. Second, subtract this deletion derivative into a plant transformation vector, pROK. I cloned it.

Fny-CMVR in plBl-76, as more fully described by Example Store Plasmid pFny206 containing the full-length cDNA clone of NA-2 is Digested with restriction enzymes Ncol and BstEI. This DNA is then subjected to DNA modification. treated with the Flenow fragment of E. coli DNA polymerase, which acts as an enzyme, and A terminal-terminated molecule was obtained. The plant terminal DNA is then ligated and ligated using standard methods. E. coli was transferred into JMI01. The plasmid obtained from this is labeled pFnyN/ It was B-4. This plasmid was digested with various restriction enzymes and DNA sequenced. was analyzed and shown to have a 94 base pair deletion. Deleting the book also results in opening the book. As a result, this gene produces a truncated protein of approximately 75 kDa. Coded. The cloning results also indicate the potential for an additional 29 amino acids at the amino terminus of the protein. Latent translation ininator-87 upstream of AUG in the RNA-2 gene that undergoes sexual translation As a result, AUG was retained.

The modified Fn contained in pN/B-4, as further described in Examples and Xl. The y-CMV RNA-2 gene was subcloned into the plant transformation vector pROK. It turned on. To facilitate this subcloning, the BamH1 site was ° Added to the end. pN/B-4 was digested with 5 phl and BamHl-Sphl ada Place the protein at the 5' end of the gene! It bound to the 5phl site. Following the main binding reaction pB/N-4 contains Ba5H1 complementary ends at both the 5' and 3' ends. An approximately 2960 base pair fragment of the site was digested with liberated BamHl. It is pRO This fragment is ligated to the BaaHl site in K, and the resulting plasmid is pCM It was named VN/B-23.

This plasmid was applied to tobacco for the following tests as initially described in Example H. Decorative Fny-CMV was used to transfer the RNA-2 gene.

In particular, with regard to Figure 3, which structures of Fny-CMV RNA-2 are present in plants? what culture was used for the specific transformation, and whether the specific transformation They are named with the code indicating the producing plant. rN/BJ is Fny-CMV RNA -2 constructs were transformed into specific plants. In the case of N/B1-8, The construct in this plant is derived from pCMV N/B-23 (see Figure 2). Number " 1 or 2 indicates which of the two culture tubes was used to transform tobacco with the N/B construct. This indicates that it was used to The second number seen in the nomenclature simply indicates the specific plant number. vinegar.

For example, the number "8" in the above example refers to the eighth product obtained in this transformation experiment. It simply indicates that it is a plant.

Example I Cultivation and maintenance of plant and virus strains TMV strain U, A, Aceline et al. [Pyrology 91: (1978) Reference Co. Purified from infected N. tabacum Cv. tabacum plants as described by Ta. Viral RNA was isolated by phenol extraction and ethanol precipitation. N, Tabacum CV.

xanthine nn as a TMV-susceptible systemic host, Sanchi nc was used as a local lesion host. Plants have a light cycle of 14 hours per 24 hours. Maintained in a greenhouse or growth room at 24°C.

Example■ Coding of the 54kDa gene The TMV 54kDa gene sequence is located at base pair 4906 of the TMV RNA sequence. A 22-salt consisting of a BamH1 site attached to the 5′ end of a sequence complementary to Ishi 4923. obtained by using base oligonucleotide primers. The second strand DNA is M- Synthesized by MIV reverse transcriptase, Freno by reverse transcriptase and loopback synthesis double-stranded by continuous treatment with C. [T. Maniatis et al. Ningnia Laporatory Manual (Cold Spring Harbor-9N) Y) (1982)]. Double-stranded cDNA was digested with BamHl and M13mp It bound to 18 BamH1 sites. The clones tested were given by primers. The BamH1 site was locked. As a result, deletion of the 54 kDa stop codon , and an extension of the 54 kDa protein by five amino acids at its C-terminus.

The 54 kDa insert was removed by digestion with Haell and treated with Flenow. The overhang ends were blunted and finally digested with Pstl.The insert was l/S■a1 digested pBS (-), resulting in the deletion of the TMV RNA sequence. Plasmid pR containing TMV sequences derived from cleotide residues 3472 to 4914 It became TT-1. The orientation of the insert is shown in Figure 1, with transcription from the T7 promoter. It provides a (+) sense transcript so that the

Sequencing revealed that all clones tested contained sequence from position 3332, but It lacked the Ba-bound H1 site provided by the imer. This results in a 54kDa termination. The result is a codon deletion and five amino acids derived from vector M13mp18. This resulted in a 54 kDa protein extension at the C-terminus. The presence of an intact open reading frame indicates that T This was demonstrated by insertion of the MV sequence into the T7 transcription vector. synthesize T7 transcript, Translated with a reticulocyte lysate system. In vitro translation shows that AUG opens at position f13495. The desired 54 kDa product was obtained which was confirmed to function as an initiation codon. Living The product was isolated with the desired 54kDa protein by immunoprecipitation using the 54kDa antiserum. It has been proven that there is.

The TMV 54 kDa sequence insert of pRTT-1 was generated by Hindm and S acl. removed by digestion, treated with Flenow to terminate the plant, and pMON31 [S, G, Rogers et al. , Mary's in Enzymology 118, 627 (1986)]. p MON316 is cauliflower mosaic virus (CaMV) 35S promoter The untranslated portion of the polylinker located between the 3′ untranslated portion of tuberin synthase Contains a unique Xho1 site. The 51a1 site is part of the polylinker and pM Found within the Ti plasmid homologous portion of ON316. Plasmid pTs541 A is the deletion of part of the untranslated part of nopaline intase 3 and the homologous part of T1. was generated by insertion of the TMV sequence into the 5ea1 site. Xho1 part of TMV sequence pTs541 was formed as a result of the insertion into the position. sense or antisense localization Clones containing any 54 kDa sequence were characterized and isolated. each construction Agrobacterium carrying pTiB6S3-8E by means of a double mating system. Introduced into Umm tumefashens GV3111 [R,T.

[Refer to F. et al., Bio/Chikunoroshii 3: 629 (1985)], lance conshugant due to resistance to kanamycin and streptomycin. I selected it.

The 54 kDa coding sequence was sequenced as shown in Figure 2. After the It was subcloned into the expression vector pMON316. Finally, this construct Bacterium tumefaciens was transferred to tobacco plants by leaf disc transformation. I entered. Transformants are based on kanamycin resistance and novalin nonciter. It was selected based on the production of ze. Four transformed plants were produced with pTs541 and four other As soon as the plant leaves the 3' nopaline synodase untranslated part and the 54 kDa open reading frame, was produced in pTs541A, which lacks part of the T1 homologous portion located downstream of. this The deletion does not interfere with the integration of the chimeric TMV 54kDa gene sequence into the plant genome. won.

Progeny seeds were collected from each self-fertilized plant. Furthermore, plants can be shaped with chimeric TMV genes. Transformation was performed to produce 54 kDa antisense RNA. two independent Antisense transformants were selected and regenerated into mature plants.

Example■ plant transformation Sections of sterile TMV-susceptible Nicotiana tabacum CV As described by [Science 227: 1229 (1985)] , a modified Agrobacterium strain containing the TMV 54kDa coding sequence. The cells were transformed with S. tumefaciens GV3111. 300u of transformed callus Selection was made on regeneration medium supplemented with kanamycin at a concentration of g/ml. resistant callus Shoots and roots were induced to regenerate, transferred to soil, and maintained at room temperature.

Example■ Nucleic acid analysis DNA was isolated from plant leaves by the Murray and Thompson modification method [Nuclei Tsuku Ann's Research 8: 43201980)]. DNA 1.0 % agarose gel, transferred to a nylon membrane, and TMV54 3 specific for kDa sequences! Hybridized to P-labeled probe. RNA leaves group RNA was separated from the tissue and placed on a 1.2% agarose gel containing formaldehyde. It was divided and transferred to nitrocellulose filter paper. Plot 54kDa Hybridized to a 32P-labeled probe complementary to the coding sequence. six Germans The vertically transformed plants were analyzed for the expression of the chimeric gene. Transform genomic DNA and isolated from untransformed N, tabacum CV, xanthi nn. genomic DNA The Ba■H1 digest of It was soybean. 3. Hybridization to the full-length 54 kDa Qkb fragment Proved the existence of a coding sequence. The 54kDa sequence insert is 1.44kb and the other 1.59 kb is provided by flanking vector DNA. to The copy number of the 54kDa protein gene in transgenic plants was determined by Southern analysis. Diploid genome equivalence between different transgenic plants, as determined by It was proved that there were 1 to 5 copies of the original. A copy of the 54kDa sequence is non-transforming. Plants were also transformed with pMON316, which lacks the 54 kDa sequence insert. It was not detected in any plants.

The TMV54kDa transcript extracted from transformed plants was also analyzed by Northern RNA analysis. Tested by analysis. The expected size for the chimeric mRNA of 1.5 kb was Total RNA derived from each transgenic plant was confirmed. 3° tubalin synthase Plants containing integrated plasmids lacking untranslated and Ti homologous portions1, A 6kb transcript is synthesized. Furthermore, the termination normally conveyed by the nos 3° sequence Larger transcripts were synthesized as a result of the sequence deletion. in all plants A small, unidentified transcript of polymorphism was also detected. Plants transformed with the vector alone , did not produce transcripts that hybridized with the TMV 54 kDa sequence probe. Ta.

Regarding the expression of TMV54kDa protein in transgenic plants according to Example ① analyzed. Analyze using Western blotting or the immunoprecipitation method described. Then, the 54kDa protein is derived from the 54kDa) transgenic protein or from the 54kDa protein. a From protoplasts prepared from transgenic plants or controls Could not be detected.

Example V immunoassay Antiserum against the 54kDa protein is directed against internal parts of the 54kDa protein, especially amino acid residues. Groups 164-179 were prepared by injecting rabbits. 54kDaT7 In vitro translation products of transcripts are obtained by antisera raised against synthetic polypeptides. It was immunoprecipitable. Transformed and untransformed for Western blotting The total extract of the plant was prepared in 50mM) Lis-HC1, pH 7.5, 1% sodium dodecyl sulfate. Equalize the leaf samples in thorium (SDS), 10 μM2-mercaptoethanol buffer. Prepared by combining and electrolyzed in a 12.5% 5DS-polyacrylamide gel. It was subjected to pneumophoresis and transferred to a nitrocellulose filter. filter first Specific antibody was followed by incubation with gold-conjugated anti-rabbit antibody and silver enhancer.

In a study to detect a 54kDa protein, 11-2X50 TMV-infected Turkish Sam Tobacco leaf strips were mixed with 10+M containing 1 mg/ml chloramphenicol. Vacuum filtered with 353-methionine at a concentration of 10 μCi/ml in KH2PO4.

These were then incubated for 20 hours at 25°C in dim light. protoplus was also labeled with 5sS-methionine. They are Nicotiana tabacum CV xan It was prepared from ChiNN leaves. Protoplasts (5-10 IICi/ml of 5s5-methane) 150.000/+1) containing thionine/ml) at 25°C under light. Incubated for 0 hours. These were then collected by slow filtration and 2mM E DTA, 0.5% 3DS, 0.2% β-mercaptoethanol and 10 μg/m Contains phenylsulfonyl fluoride as a protease inhibitor 2 Dissolved in 0mM Tris-HCl, pH 7.5 buffer. Place the leaf sections in the same mortar. solutions, one of which was extracted without inhibitor. Then the extract Purification was performed by microfuge centrifugation, and the supernatant was purified by microfuge centrifugation. Tested on white. Extraction of leaves or protoplasts labeled for the presence of 54 kDa protein were determined by incubating with the antisera described above. Immunoprecipitation, polyac Rylamide gel and autoradiography assays were also performed.

This antiserum is highly active against the in vitro translation product of the 54 kDa gene transcript. It was confirmed that T A 54 kDa protein was derived from the in vitro translation product of RNA prepared from M. could be easily precipitated. The protein was obtained from TMV-infected plants or 54kDa transformed plants. It could not be detected in any leaves of the plants.

Example■ Inoculation of transformed plants R1 seedlings from self-fertilized transgenic plants were treated with serum added as an abrasive. 100g per liter of 50% M phosphate buffer containing Lite (trademark), pH 7.2 TMV-U, or 300 μg in pH 8,6,50 IIM trisphosphate buffer TMV-1J and RNA were inoculated as usual. Connect two leaves of each plant. I planted a seed. The inoculum concentration is increased until there is sufficient volume for proper application. , was not standardized as it is a critical determinant. In subsequent experiments, we will use closely related TMs. V Mutant, F Garn Ah Arenal, etc., Pyrology 132: 131 ( (1984), it is recorded as a result of a bright yellow symptom. Mutant b6, which is easy to isolate, is induced in leaves. Plants develop symptoms every day. Recorded by visual observation. In some instances, marking the presence of virus in inoculated plants It was determined by probing the leaf extract with cDNA to TMV.

First fruits to determine the susceptibility of transgenic plants to infection by TMV In experiments, plants were inoculated with 50 μg TMV-Ul/111. 54kDa Cody 4 root seedlings from each of 8 independent transformed plants containing the Controls transformed with vector only and some non-transformed Kinsachi nn inoculated with the mutant. Plants were kept in one room and monitored daily for symptom development. Inoculation 5 At the same time, transgenic controls and non-transformed controls were Zyk symptoms clearly developed, but the transformed plants showed no signs of symptom development. It was. Symptoms do not develop in transgenic plants until 48 days after inoculation at the end of the experiment. I didn't reach it. Inoculations and upper leaf averages of these plants were applied to local lesion hosts N, Ta. used to inoculate Bakum CV, I measured it. Local lesions showing the absence of detectable virus on these plants are I didn't reach it. All regenerated plants were transferred to the complete pMON316 vector. pTs541 with the inserted TM■ sequence, or nos 3' untranslated part regardless of whether it was transformed with pTs541, which lacks the minute and Ti homogeneous portions. It was highly resistant to TMV. The constant that is the result of the synthesis of 54 kDa antisense RNA Plants transformed with the chimeric gene at the position are not resistant to infection by TMV. Ta. However, these plants are less functional compared to vector-transformed controls. showed a delay in structural development. This was simply a delay in mechanism development, so these Plants were not tested further.

Progeny seedlings derived from self-fertilized transgenic plants show that the resistance phenomenon is heritable (inhe ritabiFty) was also analyzed. 300g of R1 producing seeds per ml Germination was performed in tissue culture medium containing kanamycin. Kanamainon sensitive seedlings are It was chlorotic and did not grow beyond the cotyledon stage. Kanama The splitting ratio of seedlings expressing resistance to isin versus those susceptible to kanamaynon was In each of the transformants, the NPTll gene was integrated at multiple gene positions. shows. Seeds derived from self-fertilized plants containing 300g kanamaynon per ml. 95% of seedlings are resistant to kanamycin when germinated in 5% of the seedlings became chlorotic. 100 transgenic seedlings When inoculated with TMV-Ul at a concentration of gg/ml, 24% of these plants showed no symptoms. However, the remaining 76% were resistant to viral infection. Therefore, for TMV Seedlings were separated at a ratio of approximately 3:1 (resistant; susceptible), but the seedlings were They separated in a ratio of approximately 19=1 in terms of resistance to chlorine. Many kanamycin-resistant 'Fugitive' screened progeny seedlings for expression of the integrated chimeric TMV gene. use an uncertain means of All subsequent infection experiments were conducted using line 541A11-induced R1 fruit. This was done by separating live populations.

In experiments to determine the level of resistance, seedlings were inoculated with varying concentrations of TMV. . Resistance was observed at increasing concentrations up to 500g TMV/l. resistant plants are There was no continued development of symptoms for 30 days after vaccination. Leaf samples were taken from the inoculated plants. and assayed for virus replication and virus spread. Extracts of leaf samples are It was probed with cDNA prepared from purified TMV RNA. Virus is inoculated leaves However, it could not be detected in the entire collection of resistant plants, indicating that there is no virus replication in resistant plants. Also, resistance is complete and the virus can spread asymptomatically in plants by suppressing the development of symptoms. Indicates that there is no A transgenic product containing only the vector without TMV sequences. Transformed plants and untransformed plants were used as controls, and the virus segregants in control plants and the progeny in which symptoms have developed could be easily detected.

As a final evaluation of the resistance of transgenic plants to virus infection, several Immediately after inoculation, the plants were transferred to a growth chamber maintained at 31°C and tested for 54k against TMV. We determined whether Da-induced tolerance is temperature sensitive. 54kDa gene sequence Of the 7 inoculated plants retained, 5 did not develop symptoms at 31°C, but all controls Roll plants developed specific symptoms when kept at 24°C.

In conclusion, the above description shows that the viral genome related to the replicase part of the virus A transgenic plant containing the protein coding sequence of the A novel aspect of the present invention is that it is resistant to infection by the first available virus in white. showed that.

There are many advantages to the present invention compared to viral envelope-induced resistance. For example, Viral infections that utilize replicase-associated coding sequences as described in Resistance to tegument protein-induced resistance breaks down when high concentrations of inoculum are used. It is not "fragile" like. Conversely, according to the present invention, complete tolerance is It is not observed in non-inoculated leaves of plants challenged with virus or viral RNA. on the other hand, Protection conferred by the coat proteins of TMV and AIMV can be conferred by inoculation with viral RNA. In the present invention, which utilizes a 53 kDa code sequence, This induced resistance remains intact when challenged with viral RNA. 54k The level of resistance in Da transgenic plants appears to depend on the level of expression. do not have.

Plants with only one copy of the gene sequence have reduced resistance to intact virions. Didn't show it. A single copy of the TMV coat protein is also sufficient to protect plants; In contrast, one copy of AIMV coat protein does not.

In addition to the above, the viral life cycle is part of the replicase genome according to the invention. We also conducted research aimed at finding the stages that are broken down using .

Example■ Cotiana tabacum L) cv, xanthi tune and 54kDa tiger in tobacco plants Sunoenisoku xanthin nn, and TMV local foci indicator-host xanthin ChiNNs were maintained under greenhouse conditions. The plants used for protoplast preparation were (Both were transferred to a growth chamber at 24°C with a 14-hour light/10-hour dark cycle for one week.

125-150gE・2・s−1 by darkening the light intensity with cheesecloth decreased to

TMV strains U1 and U2 [see Phytopathology 44:277 (1954)] were purified. Manufacture [see Pyrology 91, 133 (1978)]. Some of the examples below The TMV strain U1 used was a transcript produced from a full-length cDNA clone of the virus. Induced from things. Viral infection of whole plants is 0.05 with Celite as an abrasive. M potassium phosphate, pH 7.0 buffer 005.0.5 or 1. Omg/ml T Completely expanded xanthi or 54kDa) transgenic tobacco by MV strain U1 This was achieved by inoculation of the upper and lower surfaces of the leaves. Viral RNA was obtained from TMV strains U1 and U. 2 by phenol extraction and ethanol precipitation.

Example■ Protoplast preparation and infection Protoplasts were isolated from 54kDa transgenic plants and control, nontransgenic plants. Obtained from the leaves of the Nicotiana tabacum plant. Protoplast (0,5-1,0X1 0@cell/■1) is an element with viral RNA extracted from TMV strain U1 or U2. infected by chotroporation. Electroborane Jin has two 300 Brogenator 1 electrovolesis by applying pulses of V Using a single ring electrode (2.5 steps high, 1c gap) coupled to a condenser device, A final volume of 0.7 μl of sterile 0.7 M mannitol was achieved. In customary terms 10ag/■l is used, but the viral RNA concentration is 10 to 10(bg/■l). It reached 1. Additionally, all experiments were performed using mock inoculations electroporated with buffer only. Contains a set of protoplasts.

After electroboration, protoplasts were incubated with incubation medium for 50 m Buffered with M citrate, 1mM KNOs, lmM MgS Oa, 0.1 mM CaC1*, 1uM Kl, 0.01#M CuSO4, 10gg/ml 0.7M Mannitol with Rimosidin and 100 gg/ml Lubeninoline , resuspended in pH 5.5 buffer. Protoplasts (3 x 1) were placed on an agar plate ( Transfer to 1% Super Agar in inoculum medium prepared in a 60x15mm dish and incubate for 25 minutes under low light. [See Pyrology 161:488 (1987)].

Example■ Analysis of protoplast proteins Accumulation of TMV coat protein in protoplasts was detected by Western blotting. I put it out. Protoplasts were collected by low-speed centrifugation and 50-100 μg of 5DS-polymer Lyacrylamide gel electrophoresis (PAGE) Dissolved in sample buffer [Nature See 277:680.

Released proteins were separated by 5DS-PAGE and electroproduced onto nitrocellulose. and rabbit ports for strain UITMV coat protein and [128■] protein A. Probed using reclonal antiserum (1+1000 dilution). protoplast To monitor the synthesis of virus-encoded proteins in L[363]methionine was added to the incubation medium at a concentration of 10 g C1/ml. continuous labelin After washing, protoplasts were washed with 0.7×1 mannitol and disintegrated in buffer. . [s6S]-labeled protein was subjected to 5DS-PAGE [Nature 227:680 (197 0) Analyzed by reference and autoradiography.

Example X Analysis of RNA At various times after electroboray, protoplasts were collected and kept at sterile 0°7. Wash with 501M Tris-HCI, pH 8,0 buffer 10M E DTA, dissolved in 2% SDS, phenol/chloroform/isoamyl alcohol Extracted with a 50:50:1 ratio. In some instances, following ethanol precipitation, Lithium chloride soluble (dsRNA rich) and lithium chloride insoluble (ssRNA rich) Fractions were prepared [Molecular and Cellular Biology 5] : 2238 (1985)]. Leaf RNA is obtained from leaf tissue pulverized in liquid nitrogen. Prepared in a similar manner starting with .

RNA was separated on a formaldehyde-containing 1.2% agarose gel and nitroseparated. This was then synthesized in vitro, [“produced with pco-labeled transcripts]. Robed. Northern blot was performed using 5xSSC (1xSSC-0,15M NaCl). Thorium, 0.015M sodium citrate, pH 7.0), 5x Denhard's solution Solution, 505M sodium phosphate, pH 7, 0.0, 1% SDS, 250 μg/mare 1 yeast RNA and prehybridization in 50% formamide for 24 hours at 45°C. and hybridize to 0. lX5SC, 0.2% SDS, 5 at 65°C Washed twice. The relative amounts of specifically hybridizing RNA bands are determined by Cut the nitrocellulose filter in the appropriate area using an autoradiograph. and coupled radioactivity using a liquid non-tyrene spectrometer. Quantification was done by measuring the amount of sex probe.

In vitro synthesized RNA probes were prepared from two DNA templates. 1) 126 nucleotides 1-3.785 of strain UITMV, including all of the kDa protein reading frame. A derivative of pBSM13 containing the corresponding insert, the T3 polymer of pBs126. [Molecular Cloning, Cold Spring Harbor] Laboratories (1989) @] corresponds to the main part of Thi~' genomic RNA. and a complementary (+) sense transcript to the 3' part of the full length (-) sense TMV RNA. arise. 2) TMV coat protein gene (nucleotides 5.663 to 3 SP6 volumera of pSP64 derivatives containing inserts corresponding to [Molecular Cloning, Cold Spring Harbor Laboratory] See Volatries (1989)]. This means that the (+) sense total length and all (-) sense transcript complementary to subgenomic TMV RNA with identical 3' end will occur. In addition, strain UITMV contains a sequence encoding a 54 kDa protein. The T7 transcript of pRTT-1 was prepared and transduced into wheat germ [PNA] using an in vitro translation system. (USA) 70:2330 (1973)] and reticulocyte lysate derivatives [Yo Lopian Journal of Biochemistry 67+247 (1976) Used to program Teruko.

54k electroborated for 24 or 48 hours fast with strain UIThiVRNA Protoplasts derived from Da transgenic plants [see Example ■] can be used to treat local diseases. Nest indicators - infectious viruses that can be detected by bioassay on plants However, under the same experimental conditions, these protoplasts did not contain any infected strains. U2TMV was cloned. Conversely, control protoplus derived from non-transformed plants Both strains of TMV were cloned. According to bioassay data, 54kDa) Protoplasts derived from susgenic plants can be produced even if the inoculum concentration is between 10 and 1 remained resistant to strain UITMV RNA even when increased to 00 μg/ml RNA. Ta. Consistent with the bioassay data, Western profiling of protoplast proteins Analyzes showed that under the same conditions, these cells produced strain U2TMV coat protein and control protein. , an amount that accumulated, similar to that in non-transgenic tobacco protoplasts. However, the 54 kDa transgenic protoplasts are detectable strain UITM. did not accumulate V tegument protein.

These results were confirmed by the whole intact 54 kDa transgenic plants according to the present invention. The resistance shown can be maintained by protoplasts prepared from these plants. and that resistance mechanisms operate at the single cell level. This is because the resistance is Primary in preventing cell-to-cell or long-distance virus spread at the whole plant level This may be due to the prevention of the onset of virus infection, rather than the This means that it must work by blocking the replication of the virus that caused the infection. Ru. Our conclusion is that plasmodesmata (cell virus) in transgenic plants (54kDa) cellular pathways to spread) appear to be unmodified and have normal molecular exclusion limits. Matches data showing that

The 126 kDa protein is the more abundant of the two known virus-encoded TMV replicas. component of the TMV genome, which is directed by the 5′ proximal open reading frame of the TMV genomic RNA. synthesis is probably the first step in replication after (or during) virus uncoating. .

The 126 kDa protein was expressed in a 54 kDa transgenic protein infected with strain UITMV. There was no obvious difference between [36S] and labeled proteins extracted from protoplasts. deer However, under the same conditions, the 126 kDa protein present in extracts of [”S]-labeled proteins derived from susgenic tobacco protoblasts. Ta. The equivalent, faster migrating protein encoded by strain U2TMV, Both transgenic and non-transgenic protoplasts infected with the strain It was synthesized by Similarly, synthesis of strain U2TMV coat protein was observed in both cell types. Ta. Synthesis of type UITMV tegument protein is inhibited because it lacks methionine. It could not be observed by law. 126 and 183k by immunoprecipitation with appropriate antisera. Attempts to improve the sensitivity of detection of DaW white were unsuccessful.

The direct method uses a 54kDa) transgenic tobacco prototype infected with strain UITMV. Synthesis of virus-encoded precase protein in the plast was unsuccessful. However, indirect methods still existed to detect the presence of these proteins. especially the replica All products of enzyme activity reveal the presence of all replicase components of lj lebell. will. The first product of replication is the (-) sense RNA produced from the knife. Therefore, (+) sense, genome TMV RNA, strain UITMV infection 54kDa Convert l-transgenic tobacco protoplasts into full-length (-) sense TMV R The presence of NA was investigated. After 21 hours post-incubation, the Although the ds form could not be detected, trace amounts of SS, (-) sense, and full-length TMV R NA strain UITMV infection (54 kDa) transgenic tobacco protoplasts. It turns out that it exists. Therefore, some small amounts of virus-encoded precursors The components must have been synthesized after infection and functioned to some extent in these cells.

(-)Sense TMVR in 54kDa transgenic tobacco protoplasts Detection of NA measures whether replication has progressed beyond (-) strand synthesis. This led to research into all (+) strand synthesis. TMVRN A (+)-sense, 3′ sequence-specific probe of protoplast RNA The Northan assay detects low levels of (+)sensitivity with a 5-hour post-incubation. Presence of UITMV RNA and strain UITMV infection (54 kDa) transgenic vector Completion of complete components by 21 h post-incubation in Bacoprotoplasts Long subgenomic TMV RNA was detected. Full-length subgeno of the complete component ds form MuTMV RNA was observed in transgenic protoplasts infected with TMV. It was done. However, plot analysis showed that TMV-infected 54kDa transgenic was not sensitive enough to detect TMV dsRNA in cuprotoplasts. . Calculations of radioactive probes bound to specific 5sRNA bands were performed using strain UITMV. Full-length (decades) accumulated in infected 54kDah transgenic tobacco protoplasts. ) Sense TMV RNA is isolated from infected non-transgenic proteins depending on the specific experiment. They showed that they were 20 to 80 times smaller than those of toplasts. Similar results Obtained using laboratory UITMV RNA or clone designated UITMV RNA Ta. Therefore, the results indicate that low levels of strain UITMV replication We show that this can occur in tobacco tobacco protoplasts.

The above examples, in particular the 54kDal-transgenic tobacco plant with strain UITMV. The studies described in Toplast Examples It was shown that this method enables the synthesis of different RNAs. The next experiment was performed using protoplasts. The results show that the resistance phenomenon in leaf cells of transgenic plants (all 54 kDa) The question was whether it truly reflected its characteristics. To accomplish this, a 54kDah Lancer Leaves of tobacco plants have 0.05 and 0.5@g/g on their upper and lower surfaces. Strain UITMV particles were inoculated at a concentration of wL. These highly concentrated inocula (non-tran 2 and 3 times that typically used to infect genetic tobacco plants. size) to maximize the number of leaf cells and detect virus-specific RNA. Increased opportunities.

54kDa) transgenic plant inoculated with UI TMV strain 0.05mg/a1 Northan blot analysis of RNA from tobacco leaves will detect any viral RNA. Didn't make it clear. However, increasing the inoculum to 0.5 mg/ml , generation of detectable concentrations of full-length and subgenomic (+) sense TMV RNA. , which increased over the period. Once again, all the TMV2 that could have existed Full-stranded RNA was too low in concentration to be detected using this method.

Radioactive probe bound to specific RNA band in Northan plot Comparison of amounts shows the complete amount accumulated in densely inoculated 54kDa transgenic tobacco leaves. Although long (+) sense TMV RNA is found in non-transgenic tobacco tissues, It was shown to be 17-20 times lower than that of Similar results were obtained for laboratory virus isolates and and virus propagated from infectious TMV cDNA clones (at a concentration of 1 mg/ml). (applied in ). Densely inoculated with 54kDa transgenic Nicotiana tabacum leaves Local lesion indicator - source of inoculum for plant assay Small amounts of physiologically active virus were often detected when used as Black When a virus derived from a plant is used for inoculation, a pond leaf of the same plant or an uncontacted leaf of the same plant can be used for inoculation. No virus was detected in the seed part.

Overall, the results obtained with densely inoculated 54 kDa transgenic tobacco leaves appears to be consistent with that obtained using protoplasts, where U. It appears that virus copying is being inhibited by the king, but it has not been completely stopped.

How inhibition of the replication of the UI TMV strain is achieved by the present invention There are two possible descriptions of how this can be achieved in transgenic tobacco plants. Firstly , the 54 kDa protein or its RNA directly inhibits rebricase activity, or Second, the 54 kDa protein or its RNA is e.g. a virus-encoded precursor. indirectly by inhibiting the synthesis of enzyme components, 126 and 183 kDa proteins. act. A second possibility is that an in vitro synthesized TNA encoding a 54 kDa protein Utility in in vitro translation systems programmed or preprogrammed by transcripts. We dealt with TMV RNAg translation in heron reticulocytes or wheat embryos. both of these In a cell-free translation system, the synthesis of 126- and 183- and 54kDa proteins is Occurs without any indication of specific inhibition of 126- or 183-kDa protein synthesis. did. Therefore, the 54 kDa protein of the present invention or its RNA may be There is no evidence that it affects precase components. Therefore, the result is the first possible activity, i.e., the 54 kDa protein or its corresponding RNA directly activates replicase activity. It is consistent with having an influence.

In addition to TMV, research was also conducted on cucumber mosaic virus (CMV), and the same The results were as follows. In these experiments, Nicotiana tabacum (V. Replicase elements of Nyu Samsan NN plants, CMV subgroup ■ strain Fny A modified cDNA clone of RNA-2 encoding the transcript was transformed. genetics The child was modified by deletion of a 94 bp portion ranging from nucleotides 1857 to 1950. (Figure 2). More specifically, modified as described in Example XII, Contains Ncolt and BstEII sites used to generate a 94bp deletion. The partial nucleotide of the Fny-CMV RNA-2 cDNA clone is: AA IJACCA[X:GLX:I(shi(i-1 to knee 1νL)](1jGAG[XJ GCC [XEGUGJUAUGAC42AO: GACCM tJtc GM AAGαJJ tJIJA IJ[JCtJcA GGCGAIJ GAIJ LCtl　E14 [In the formula, the first underlined part corresponds to the Ncol restriction site, and the bold “G” corresponds to nucleotide 1856, second underlined part is BstEII restriction The bold "G" corresponds to nucleotide 1951. ]　1 and What is the amino acid sequence?

Once the 94 nucleotide deletion is made as described in Example XIf, the modified 1'yn- The nucleotide and amino acid sequences of the carboquine terminus of the cDNA clone are:

[In the formula, the underlined part corresponds to the first underlined restriction site, and the bold “G” corresponds to the original nucleo 1856 and 1951]; and the amino acid sequence is The region around and including this 94 bp is present in many positive sense RNA plants and Closely conserved among putative replicase encoding sequences in animal viruses Contains 4 domains. This deleted 94 bp region also contains the third domain, Gly-A It contains an sp-Asp domain, which is required for bacteriophen Qβ replication. It has been shown that As shown in the sequence above, this defect also truncates the tip. In an open reading frame due to the translated product taken / raised. Origin of original transformant The previous R1 generation plants were tested for resistance to virus infection. 18 transformations Plants from two of the lines contain either Fyn-cDNA pillions or RNA Even when inoculated at a high concentration of 500μg/IN1, it was resistant to systemic disease, “!” there were. Resistance is achieved by inoculating a plant with a virulent aphids and inoculating the plant with a highly related subspecies. This was observed when Glue 11 strain 0.5ynY and Ve85 were inoculated and maintained. , LS, was not observed when subgroup ■ strains were inoculated.

Example X cucumber mosaic virus strain Cucumber mosaic virus strains Fny, O, 5nySVe85, Y and LS, Nicotiana Tabakum (from V Turkino/Yu Samsan, Parkaitis [Meso 's for Plant Biology, Wine Yubatuba and Wine Yubatuba ed., Academic Press, New York (1988)]. It was purified as follows. Viral RNA intact cucumber mosaic virus, pheno It was isolated by chloroform/chloroform extraction and ethanol precipitation. normal to the plant , 50 wM sodium phosphate buffer, pH 7.2 or 50+e Inoculate RNA in M Tris phosphate buffer, pH 8,9 using polished Celite. did. Plant transformation of Nicotiana tabacum CV Turkish Samsan NN and cucumber mosaic virus was used as a susceptible general host. 24 plants The cells were grown in a greenhouse or growth room at 16 h/8 h light/dark cycle at 10°C.

Example■ Modification and subcloning of cucumber mosaic virus RNA-2 full-length insensitivity Fny-CMV cDNA clone pFny206 encoding dye RNA-1 [ Modified from J. Yin, Pyrology, 69: 1777 (1988), Figure 2 The vector was subcloned into a two-component plant transformation vector as described in . R.N. A-2 clone was digested with pFny206 with restriction endonuclease Ncol. These enzymes were removed by phenol/chloroform extraction. Ethanol precipitated. The DNA was nucleated in an attempt to obtain plant terminal molecules. Treated with Klenow fragment in the absence of cleotide. The DNA is then subjected to restriction endonucleation. Digested with Klease BstEII and treated with Klenow fragment in the presence of nucleotides. Plant terminal molecules were obtained, phenol/chloroform extracted, and ethanol precipitated. . This linear molecule is then recircularized with T4 DNA ligase and the nucleotide 1 Fny lacking nucleotide 94 from 857 to nucleotide 1950 (inclusive) -Plasmid containing the cDNA clone of CMVRNA-2 (pFnyN/B 4 ) was produced.

This 94-nucleotide portion is present in the replicase proteins of many positive sense viruses. It contains a highly conserved Gly-Asp-Asp domain. book loss and an open reading frame resulting in an in-frame translation stop codon 15 codons downstream from the deletion site. This caused an internal rift. Therefore, this deletion is not only a deletion of the 94bp portion. , resulting in a truncated open reading frame. Cloning also in the RNA2 gene, resulting in efficient translation of the terminal 29 additional amino acids. 89 upstream of AUG in AUG as an effective translational ininoator. I did it. Therefore, the protein encoded by this modified gene is a 96.7 kDa wild protein. The size is approximately 75 kDa compared to the native protein.

This modified Fny-CMV RNA-2 cDNA clone was transformed into a two-component plant transformation vector. For subcloning into vectors, pN/B-4 was cloned with RNA-2 cDNA. 5ph1 cuts this plasmid at the N-terminal 5° site of the plasmid of the damaged clone. I digested it. An adapter containing a 5'Ba ridge 11 overhang and a 3'5phl overhang, ligate into this site and then digest with BamHl which cuts at the C-terminal 3° site. and thereby liberate a fully modified cDNA molecule. 3kb fragment, The BamH1 portion of the two-component plant transformation expression vector pRok2 was transformed using standard techniques. [See Nature, 321: 446 (1986)] , pCMV N/B-23 was produced. The construct was transferred to Agrobacterium tumefaciens. to E. faciens strain LBA-4404, to E. coli strain MM294-pRK. Biparental crosses mediated by 2013 [Methods in enzymes, 11 8:627 (1986) v.]. Transconsumegate, kanamycin and streptomynon at 50 and 1254 g/ml, respectively. I chose more. pFny206 and pCM~' Fny-C in N/B-23 The sequence of MV RNA-2 is shown in Figure 2.

Example■ plant transformation Examples of the transformation of Nicotiana tahakum C to Turkyosa samsan NN Agrobacterium fumefanense vectors described by Hornyu, such as in 11T. This was done by leaf disc transformation. Leaf disc and pCMV N/B-23 After 2 days of co-cultivation, leaf discs were treated with carbeninoline 500I 1g/ugly 1, Kanamai Thin 300 gg,/ml, 6-pencylamitubulin (BPA) 1 m g/l and naphthylic acid (NAA) O, 1 mg/l containing germination regeneration medium. . Leaf discs grown in medium were transferred to fresh medium every 7 to 14 days. Approximately 4 weeks Buds are formed between them, and they are treated with kanamayone 300 Mg/mL BAPo, 2 mg /l and NAAO, 1 mg/l containing root induction medium. These buds are about 2 - Form roots in 3 weeks, at which time transfer them to vermiculite for 2 weeks and soil I potted it again and moved it to the growing room. 18 independent kanamycin resistance transformations Transfections were selected for further analysis.

Example XIV Evaluation of resistance Eighteen independent transformants were initially isolated for resistance to Fny-CMV infection. Screening was carried out using the Facility Test. Is each leaf a RO generation transgenic plant? The cells were separated and 50 μg/ml of Fny-CMV was inoculated into the upper and lower epidermis. leaf The stems were soaked in water and the leaves were incubated at 25°C for 6 days, then 2c in diameter. The leaf discs were removed. The extract of this tissue was converted to Fny-C using 52P-labeled cDNA. tested against MV to determine the presence of viral RNA (Parkite Isu, 1988). The absence and presence of viral RNA are indicative of viral infection, respectively. suggested resistance or susceptibility to infection. These transgenic plants After blooming, seeds of the R1 generation were collected and planted.

This assay showed that seven of these transgenic lines were infected with Fny-CMV. Each of the seven transformed lines that showed resistance by isolated leaf assay Next R1 generation seedlings (N/B1-1.1-2.1-8.2-3.2-5.2-6 and and 2-7) and one transgenic strain that showed no resistance in isolated leaf measurements. (N/B2-1) and untransformed Nicotiana tabacum eV, Turkish To the Shu Samsan NN plant, Fny-CMVo, 2.1.0.5.10 and and 1100a/111 were inoculated. All plants of putatively resistant lines are R1 generation tiger Evaluation of Rebricase-Mediated Resistance to CMV Infection of Genetic Tobacco Plants Inoculum doses of 0.2 and 10 μg/ml as described in Table 1 below provided. Tolerance or delayed symptoms in the range of In Table 1 above, 0 initial separation assay, all of this system (other than N/B 2-1 and N/N) It was shown to be resistant to Fny-CMV infection; one plant was treated with 50mM Na Fny-CMV in PO4 buffer, pH 7.2, with Celite as polishing agent. was inoculated. These data estimate the number of plants showing symptoms two weeks after inoculation, These numbers are shown as a function of the total number of seed plants: ° these numbers represent the spread of plants exhibiting whole symptoms. indicates the total number of inoculated plants; 'ND±not measured; To determine whether temperature affects tolerance, plants were grown with Fny-CMV20 gg/ug1 and incubation at 30°C for 2 weeks; and the plants were , 10mM) Fny-CMV RNA in Lis-HCl buffer, pH 8,9 50 ag/1 irises were inoculated.

Two lines in which individual plants were tolerant at these virus concentrations (N/B 1-2, N /B1-8) as Fny-CMV inoculum with a virus concentration of 500 pg/ml. Fny-CMV RNA5 was added to system N/B 1-8. 0++g/garden 1 was inoculated. All R1 populations from these systems are This does not mean that the resistance is resistant due to Mendelian segregation of the resistance trait in the population. Plant Items were scored by daily observation of symptom progression. In some cases, in inoculated plants The presence of viral RNA in the leaf extract s! P-labeled Fny-CMV cDNA It was measured by a test by. System N/B 1-2.1-8.2-3 and two pairs R1 seedlings of the control line were inoculated with 500 μg of Fny-CMV as described in Table 1, and 2 inoculated N/B 14 out of 1-8 plants and 26 inoculated N /B 21 of the 1-2 plants had advanced disease symptoms. Therefore, very high The inoculum dose may be such that it destroys some plants that would be resistant to lower inoculum doses. It seems to me. This process was applied to some plants (3 3 out of 5) R2 generation who showed symptoms when superinfected with 500I1g/111 This was confirmed in experiments using substitute plants.

However, both viral RNA and progeny virus are present in uninoculated leaves of resistant plants. Detected using Tuto-plot hybridization and bioassays These plants have shown true resistance and are simply suppressing symptoms. It suggests that something is wrong.

Two lines (N/B 1-2 and N/B 1-8) showed resistance in the isolated leaf assay. A transgenic line that showed no sex (N/B 2-1) and a non-transformed strain Cotiana tabacum Cν, R1 generation seedlings from Takkinyu samsang NN plants is also susceptible to Fny-CMV infection by the virulent aphids (Misas bernchae). tested for resistance to Nicotiana freys infected entirely by Fny-CMV. Brandi is a virus source plant that these aphids eat before eating the experimental plants. It acted as a thing. After a 30-minute feeding period on the source plant, the aphids were transcribed. Szhenitsk and control plants were infected with 10 aphids on each plant. Moved.

Let the aphids feed for about 15 hours, then insecticide the plant to kill the aphids. Disinfected with disinfectant. The resulting plants develop Fny sensation as soon as they are transmitted by aphids. It was resistant to staining.

Example XV Genomic DNA analysis Genomic DNA was extracted from leaf tissue using a modified method of Murray and Thompson (1980). isolated from High-molecular-weight DNA was digested with anti-enzyme and analyzed on a 0.8% agarose gel. Separated and transferred to a /-N Screen Plus (Dupont) nylon membrane. 5 membranes Fn in XSSC15x Denhard, 5% dextran sulfate and 2% SDS. y-CMV RNA-2 gene sequence-specific 32p-labeled DNA probe and ) was hybridized. All DNA probes are random hexamer primers reaction [see Analytical Biochemistry 132:6 (1983)] DNA-specific activity of at least 5 x 10'cps/IIg It is gender.

These seven transgenic lines and lines N/B 2-1 and pROK2 Genomic DNA derived from a control line (ROK9) transformed with the vector alone was transformed into the above It was tested as follows. Sasanbrosoto hybridization analysis revealed that these seven lines were shown to contain approximately 1 to 7 cobies per haploid genome. the tallest The lines N/B 1-2 and 1-8 contain approximately 3-5 and 5-7 copies. each included. Line N/B 2-1, which is not resistant in the detached leaf assay, is contained one copy per id genome. The control ROK9 system is Fny-CMV RNA -2 did not contain homologous sequences.

To determine whether resistance is temperature sensitive, resistance lines N/B 1-2 and 1-8 and control line N/B 2-1 and non-transformed Nicotiana tabacum CV, Yuichi R1 seedlings of Kinyu Samsan NN were inoculated with 20 gg/ml of Fny-CMV. The cells were immediately transferred to a growth room maintained at 30°C. During the 4 days after inoculation, all control plants Although the physical symptoms progressed, many N/B 1-2 and 1-8 resistant plants were obtained. , showed that this resistance was not temperature sensitive (see Table 1).

Previous research [EMBO Journal 6; 1845 (19g?); Bio/Technology Technology 8: 127 (1990) and Pyrology 159: 299 ( (1987)] showed that the coat protein mediates TMV protection and that alfalfa moss Zyke virus may be able to overcome viral RNA inoculation It shows. Therefore, line N/B1-8, non-resistant N/B1-2 control line and non-resistant Transformed Nicotiana tabacum cv, R1 seedlings of Turkish Samsan NN line. , Fny-CMV RNA 5011g 7ml. The control system had no overall symptoms. Progressed in 100% of plants within 4 days, but isolated from N/B 1-8 plants. Only 36% had progression of symptoms even after 14 days (Table 1). From these data, NB 1-2 and 1-8 lines have high contact with both viruses and viral RNA. Tolerance is evident at the seed material level.

The specificity of resistance was determined by resistant N/B 1-2 and non-transformed Nicotiana tanokucum. CV, 1 subgroup of 3 types of R1 seedlings of Takkinyu Samsan NN (0, S ny.

)', Ve85) and one subgroup II strain (LS) at 20 pg/ml. It was measured by shooting. The control lines were all four strains tested, 7 days after inoculation. Symptoms developed in 100% of the plants. Resistance N/B 1-2 and 1-8 plants are , obtained when attacking with one strain of the three Zabu groups, but when attacking with subgroup II strains. You couldn't get it if you shot it. This resistance is obtained as a result of carrying out the present invention. Modified chimeras that provide resistance to infection by certain group ICMV virus strains This shows that plants transformed with the Fny-CMV RNA-2 gene are obtained.

The level of resistance depends on the number of copies of the defective CMV replicase gene inserted into the genome. It seems to be influenced by the number. Transgenic plants of origin tested in isolated leaf assay The number of copies of the product is 1-3 copies per hubroid genome in the 5 non-resistant lines; For gender N/B 1-2 and 1-8 lines, 3-5 copies and 5-7 copies, respectively. It was within the range of These two resistant lines also produce different numbers of resistant R1 plants. At various inoculum concentrations (Table 1), it is possible to determine the resistance level of all plants given If the inoculum concentration is particularly high, 100 to 500 μg/■1, the plant germs The number of copies of the modified RNA-2Fny-CMV RNA 2 sequence present in the genome It again suggests that it can be influenced more.

To assess the possible role that copy number may have in determining resistance levels , both non-resistant and resistant R1 generation N/B 1-2 and 1-8 plants were analyzed.

The presence of virus, copy number and transcripts in non-inoculated leaves were determined by these methods. and two control plants. This is expressed as the replicase gene copy number and its Injected with Fny-CMV virus 100 gg/ml and 500 gg/ml, respectively. Resistance to CMV in R1 generation plants of seeded N/B1-2 and 1-8 isolates The interaction with sex is shown in Table 2 below.

Table 2 General symptoms: “Virus Incomplete Revuli Presence of resistant genetic system 5 Case copy number Child transcript 61-2-2 ND + + 0 or IND “Three symptomatic (susceptible) and three asymptomatic (resistant) plants, N/B 1 -2 and 1-8 systems were tested.

The presence of viral RNA in uninoculated virus leaves was determined using Fny-CMV cDNA. Dot plot hybridization using A probe [Vulkaite Systems, Methods for Plant Molecular Biology, Academic (New York Press), pp. 487-506 (198g). We calculated the number of incomplete replicase gene copies per haploid gene, respectively. Probe the 35S CaMV promoter of genomic DNA isolated from plants of Determined by Sasan plot hybridization analysis used as . These genomic DNAs were purified using Hindl [[/E Incomplete digestion of the coR1,4,1 kb fragment and methylated DNA resulted in Pstl and modified replicas in the genome that release higher molecular weight fragments Hinase releases fragments of various molecular weights depending on the insertion site of the enzyme gene. 1 total RNA was isolated from each plant and 40ag was digested with form Pour onto aldehyde 1.2% agarose gel and use SceneScreen Plus (Dupo hybridization using the NOS terminator sequence as a probe. 'NN control lines were Nicotiana tabacum CV, Turkish Samsun. NN was: 1 uninoculated plants; and “100 gg/mtFny-CMV inoculated plants.

As shown in Table 2, the virus is present only in plants showing systemic symptoms. did. The copy number in susceptible N/B 1-2 plants is 0 or 0 per hubroid genome. or 1, while resistant N/B 1-2 plants each have 2 per haploid genome. Contains a copy. This requires a copy of the gene but does not ensure resistance. , the presence of multiple copies of a gene does not guarantee that a specific plant will be resistant. suggested that. This means that all of them and 3 or 4 per haploid genome Analysis of N/B 1-8 resistant and susceptible plants showed that was demonstrated (Table 2). In addition, most of these inserts can be used in resistant or susceptible plants. Regardless of their origin, they appear to be located in similar locations within the genome. these feelings Northam plot analysis of susceptible and resistant N/B 1-8 plants to this modified RN -2Fny-CMV RNA 2 was shown to exist only in resistant plants (Table 2). These results indicate that resistance depends on the presence of gene sequences in the tobacco genome and their activity. This is shown to be caused by sexual transcripts.

Many resistant plants have chlorotic lesions on the inoculated leaves, or levels of viral RNA in dot-plot hybrids compared to non-tolerant plants. oxidation is very low or unanalyzable; Viral RNA was detected in seed leaves by dot-plot hybridization. Not possible. These observations suggest that resistance may be due to inhibition of viral replication at the site of infection. This suggests that CMV - Judging from the spreading symptoms seen on the inoculated leaves, Some viruses can move from cell to cell, but cannot invade the vascular system of plants or Cannot survive in ducts.

In conclusion, the data presented herein contain a modified CMV-replicase gene. The transgenic tobacco plants in the plant are highly resistant to infection by CMV. clearly indicate that Furthermore, the concentration of CMV replicase that mediates resistance is 50 Maintaining an inoculum dose of 0 gg/ml, it is consistent with CMV coat protein-mediated protection [by O/Technology 6: 549 (1988). 10 times more expensive. The data also encode reads via the replicase protein Transgenic tobacco plants transformed with the 54kDa gene also have TMV Indicates resistance. In both cases not described herein, the replicase The columns are used for plant transformation. Both traits are determined by viral replicase sequences. Although the two systems exemplified herein involving conversion may operate by different functions, , replicase-mediated resistance is common against other plant and animal RNA viruses. It seems to be applicable in

Accordingly, while we have illustrated and described the preferred embodiments of this invention, we have shown that the invention may be modified and described. and modifications are possible and we are therefore limited to the precise terminology described herein. It is neither desired nor intended that the present invention be used in a variety of uses and situations. We therefore wish to take advantage of the changes and modifications that can be made to apply the do! ! This is for illustration purposes only. Therefore, such changes and modifications are equivalent and complete It is of course intended to be within the scope and therefore within the scope of the following claims. Until now The terms and expressions used in the description have been used consistently for the purposes of the description. and not as a limitation, and therefore the use of such words and expressions. is not intended to exclude equivalents of the characteristics shown or described, or any part thereof; It is recognized that the scope of the invention is defined and limited only by the following claims. It will be done.

Among such modifications, for example, plant traits other than those embodied in the examples above. There is a replacement of the conversion vector. Vectors within the scope of substitutions or equivalents, for example, pBIN19, pBllol, pRokl, pAGs135, pARc12, P GA470, pRAL3940 and pcTIT3, etc., the present invention and CMV, but other plant viruses, e.g. Alfalfa mosaic, potex will also contain the ze part in its genome The bromovirus, botivirus and luteovirus groups A host plant transformed with a gene sequence related to the lebricase portion of the virus. Therefore, it is included in the present invention. similarities between certain plant and animal RNA viruses The RNA replicase (polymerase) part of many (unrelated) plant viruses containing Because it is known that sequence similarities exist in Kraitsk Acids Research 17: 9543 (1989)], these are They are of course considered equivalent and therefore included within the scope of the present invention.

All nucleic acid and amino acid sequences contained herein are reproduced in the sequence listing below. .

Sequence list (1) General information (i) Applicants: Milton Zeitlin, Daniel Golemboski and George ・Lomonosov (1. Name of the invention, transforming plants with the replicase part of the plant virus genome. (iii) Number of sequences 8 (2) Information on sequence number 1: (i) Characteristics of array (A) Length: 6 base pairs (B) type/nucleic acid (C) Number of strands: 1 strand (D) Topology linear (ii) Sequence species i: DNA (xl) Sequence description, Sequence number 1゜ CAGGA (2) Information on sequence number 2 (i) Characteristics of array (A) Length: 26 base pairs (B) type nucleic acid (C) Number of strands: 1 strand (D) Toboronow, linear (ii) Type of sequence: DNA (xi) Sequence description: Sequence number 2: CAAAGACUGG UGAUAUUUCU GAUAUG 26 (2) array Information for number 3: (1) Array characteristics (A) Length, 9 base pairs (B) type nucleic acid (C) Number of strands: 1 strand (D) Topology +i! ! chain-like (11) Type of sequence: DNA (xi) Sequence description Sequence number 3 AGUUGULJAA (2) Information on SEQ ID NO: 4.

(i) Characteristics of array (A) Length: 1425 base pairs (B) type: Nucleic acid (C)! Number of II, single strand (D) Topology: linear (11) Type of sequence: DNA (xi) Sequence description 2 Sequence number 4゜ (2) Information on array number 5 (i) Characteristics of array (A) Length: 132 base pairs (B) type/nucleic acid (C) Number of strands: 1 strand (D) Topology linear (11) Type of sequence: DNA (xl) Sequence description Sequence number 5 MIJ KCA [JCGIJ: ACCAIJG GCU GAG UUUα: C (DG LJGIJ UAU GAC42Aa: GACCAA (JtJC GAA AAGαJJ t, ti uuc ucxαZ GAU GAU LC tJ 840 included αsuUUU tJf? Aα heart alpha le α℃α sword 0 le alpha sword hiro CCCG AGU AJ evening 126IJLCACA 132 (2) Information on sequence number 6 (i) Characteristics of array (A) Length: 44 amino acids (B) type peptide (C) Number of strands: 1 strand (D) Topolo/-linear (11) Type of sequence: Peptide (X]) Sequence description Sequence number 6 (2) Information on sequence number 7 (1) Array characteristics (Go to 4) Length: 63 base pairs (B) type nucleic acid (C) Number of strands: 1 strand (D) Topolon-1 linear (11) Type of sequence: DNA (xi) Sequence description Sequence number 7: (2) Information on SEQ ID NO: 8.

(i) Characteristics of array (A) Length: 20 amino acids (B) type peptide (C) Number of strands: 1 strand (D) Topology linear (11) Type of sequence: Peptide (xi) Sequence description Sequence number 8: In this way, engineers in the field to which it belongs or to which it is most closely related can to make and use our inventions and products. describe in complete, clear, concise, and precise terms the operations and methods to be performed and used; .

Continuation of front page (72) Inventor Lomonosov, George UK NRA 24 G.B. 128 Azore Street, Norwich

Claims (4)

[Claims] 1. (1) Viral RNA or D related to the replicase portion of the viral genome Separating the fragments of NA (2) Receiving the separated fragments or DNA copies of the parts into the genome of an endoplant in such a way that the plant is transformed with the inserted fragment. A method of conveying resistance of an RNA or DNA virus pathogenic plant host to a plant, comprising: 2. whose genome or portion is associated with the replicase portion of the viral genome; Contains a DNA copy of a fragment of an RNA or DNA viral genome or a portion thereof. pathogenic plants. 3. whose genome or portion is associated with the replicase portion of the viral genome; Contains a DNA copy of a fragment of an RNA or DNA viral genome or a portion thereof. pathogenic plant seeds. 4. It is resistant to viruses and contains the replicase gene part of the viral genome. A plant and its progeny characterized by having a genome containing the same.