JP2000516462A - Modified tomato mottle geminivirus coat protein gene - Google Patents

Abstract

  (57) [Summary] The present invention relates to a modified plant virus gene that confers resistance to a geminivirus infection on a plant. The modified AV1 gene of the tomato mottle geminivirus was cloned into an appropriate expression vector, thereby transforming tobacco. The expression of the modified gene confers resistance to the virus infection on the plant in which it was expressed. The invention further relates to a method for conferring virus resistance on a plant. The present invention also relates to modified coat protein genes that confer virus resistance on plants and fragments thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION          Modified tomato mottle geminivirus coat protein gene   This invention has received government support under USDA CSRSCBAG Grant No. 92-34135-7456. It was done. The government has certain rights in the invention.                         Cross-references for related applications   This application claims the benefit of US Provisional Application No. 60 / 022,680, filed July 26, 1995. Is to be charged.                                Background of the Invention   Geminiviruses are limiting factors in tomato production in tropical regions. Earth In this area above, at least seven different Geminiwis as tomato pathogens Ruth has been identified (tomato mottle, potato yellow mosaic virus, tomato Golden mosaic virus, tomato yellow mosaic virus, tomato leaf club Virus, pepper huasteco virus, and Tomato yellow leaf curl virus newly brought in from the Mediterranean region. This These viruses reduce yield by reducing fruit number and size . Their presence increases the use of pesticides to control vector populations This increases administrative costs and therefore reduces profits.   In many parts of the world, reliable measures to control these viruses are Does not exist. Tomato export production using tomato hybrids produced in the United States and Europe In the industry, chemical control has been established to reduce the population of whiteflies carrying the virus. It is done periodically. The effectiveness of this approach depends greatly on a number of factors. General In some cases, pesticides may reduce the incidence of the virus, but often The extent of the decline is insufficient. Viruses are infected on farmland where large amounts of pesticides are used. It is not rare that many plants are found.   One such case is a serious problem with tomato production in Florida. Tomato mottle geminivirus (TMoV) (Polston et al., 1996, Plant Disea). se 80: Printing). The virus is highly infected on tomato farms in Florida Cause the plague in the other three states and Puerto Rico. (Polston et al., 1995). The host range of this virus is narrow, and it is transmitted from tomato to tomato And the Lord Infects tomatoes from old farmland to new farmland. Current extermination method is penetration After using the pesticide for about 8 weeks, the season (15-18 week season) ends. To perform the leaf surface treatment of various insecticides.   The costs associated with frequent use of pesticides, and reliance on only one control, are Significant reduction by using tomato varieties resistant to minivirus It is considered possible. There are many varieties that have resistance to TYLCV. Sexual range is narrow and has little or no effect on other geminiviruses . Ideally, one gene or two confers immunity to many geminiviruses. Or multiple genes are required. This makes seed handling companies extremely diverse. Varieties with various horticultural qualities that can be grown in It would be possible to introduce a gene or multiple genes.   Plant virus coat proteins play many functional roles in viral pathogenesis And are often involved in vector transmission. Like TMoV The coat protein of the bipartite geminivirus is the virus whitefly Necessary for transmission by mi and transmission of viral plague. Gemini virus Coat protein is constant in systemic virus movement (Pooma et al., 1996) and has multiple nuclear translocation signals. Ko These domains are included in the domain of the Not in. Other studies (eg, vector transmission and conservation near the amino terminus) Of potyviruses that map to identified Asp-Ala-Gly residues Virus coat protein or other Many of the viral protein functions map to amino acid residues near the exposed surface of the protein. Is performed. The terminal residue at either the amino or carboxy terminus of the protein is Often found near the exposed surface of the protein, which has a functional domain Means that.   To date, genes of conventional origin include a wide range of None has been found to impart a spectrum. Resistance from pathogens Has been successful in identifying genes that confer resistance to viruses. It is a technique that is. The viral sequence or a modification of the viral sequence is located on the plant chromosome. It has been shown to interfere with the viral infection process when inserted. This is a lot Plant virus families. Potyviridae (Potyviri dae) a modified coat protein gene derived from a virus belonging to the family , Especially those with a cut at the carboxy terminus, are excellent against potyvirus It has been shown to impart resistance. This resistance is produced constitutively Causes some specific reactions to those sequences when they occur Seems to occur at the mRNA level. Their specific response depends on their plant When inoculated, they confer resistance to potyvirus. However, Which genes from eminivirus can be used to obtain resistance It is almost unknown.   Other reports on the use of geminiviruses to obtain resistance or resistance include: , A movement protein gene from our laboratory (Duan et al., 1996, The Plant Cell, submitted), a specific mutation to the TMoV rep gene (private communication J. Stout, Seminis Seed Co.), of Tomato Golden Mosaic Virus Antisense rep gene (Day et al., 1991), African cassava mosaic virus There is a subgenomic sequence of the A component (Stanley et al., 1990). Jemi Only one report has been published on the use of the divirus coat protein Absent. In other words, tomato housefly, a whitefly-borne virus derived from Israel Toma by unmodified coat protein gene obtained from low leaf curl virus Have been transformed (Kunik et al., 1994). These transformed plants Can recover after a susceptible response, or delayed onset of symptoms It was reported that either one of these reactions was shown. Recovered plants respond to repeated inoculations The first time it exhibited increased resistance, but did not reach an effective level of resistance.                                Summary of the Invention   The present invention provides, in part, TMoV in plants transformed with the modified gene. With the aim of obtaining a modified gene useful for inducing resistance to And a method for modifying the gene. One object of the present invention is to provide TMoV and other Identify genes for TMoV that can be used to induce resistance to minivirus That is. Tobacco (Nicotiana tabacum) is resistant to TMoV infection Trigger The evaluation of the modified coat protein sequence for the evaluation is exemplified.   The present invention also provides tobacco mosaic tobamovirus and tobacco In addition to resistance to infection with tomato mottle geminivirus, other related A modified plant virus residue that also confers resistance to eminivirus infection Also related to the messenger. Appropriate modified AVI gene of tomato mottle geminivirus Subcloned into an expression vector, thereby transforming tobacco . The expression of the modified gene allows plants expressing the modified gene to respond to viral infection. Resistance.   The invention further relates to a method for conferring virus resistance on a plant. .   Further, the present invention provides a modified coat protein for imparting virus resistance to a plant. It also relates to white matter genes and their fragments.   One further aspect of the present invention is a novel transgene with enhanced virus resistance. Related to genic plants.   Other objects and advantages of the present invention are set forth in the Summary of the Complete Invention Disclosure and It will be clear from the claims.                             BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows the TMoV modified coat protein (AV1) constructed in the binary vector pBI121. FIG. The modified coat protein gene of TMoV is cut using restriction enzymes. Refused. The coat protein sequence fragment was converted to cucumber mosaic virus (CMV) coat protein. It was ligated to the 5 'untranslated region (leader sequence) of white matter. The resulting CMV code Proprotein (AV1) was prepared in a caulifla in Promega's pGEMEX vector. Warm Mosaic Virus (CaMV) 35S promoter and Agrobacterium T Polyadenylation signal derived from the nopaline synthase gene (NOS-ter) of i-plasmid Next to the hotel. Cut the resulting cassette and place it in Phosphotransferase II (NTP II) gene and reporter gene β- It was inserted into the binary vector pBI121 containing lucuronidase (GUS).   FIG. 2 shows the results of PCR amplification and agarose gel electrophoresis of tobacco transformants (T₀ ) Is the determination of the presence of the transgene. From transgenic tobacco lines The size of the CMV: TMoV AV1 sequence amplification product was estimated to be 275 bp. Lane From left to right:   Lane 1: M = 1KB ladder size marker.   Lanes 2-11: T transformed with pBI121 containing the CMV: AV1 sequence₀tobacco.   Lane 12: T transformed with pBI121 alone₀tobacco.   Figure 3 shows the modified coat protein (AV1) sequence of tomato mottle virus (TMoV). Transformants containing (T₁5) is a Northern blot analysis of transcripts from the same generation. E Separation of total RNA by electrophoresis on a 1% agarose gel containing lumaldehyde did. RNA was transferred to nylon membrane and prepared from TMoV coat protein sequence³²P sign Hybridized with DNA probe. Plant ribosomal RNA 26S and 18 illustrates the position of 18S. The arrow indicates the location of the transcript of approximately 1.0 kb. Leh Is the total RNA from the transformed tobacco, from left to right: is there;   Lane 1: strain T with binary vector pBI121 only₁-cp0.   Lanes 2-7: strain T by pBI121-AV1₁-cp7.   Lanes 8-9: strain T by pBI121-AV1₁-cp9.   Lanes 10-11: strain T by pBI121-AV1₁-cp6.   Lanes 12-13: strain T by pBI121-AV1₁-cp2.   FIG. 4 is a nucleotide sequence encoding a modified TMoV coat protein. Nucleotides 1 to 55 are the leader sequence of cucumber mosaic virus, Otides 56-890 encode the modified coat protein.   FIG. 5 shows the derived amino acid sequence encoded by the nucleotide sequence of FIG. Column.Detailed description of the invention   The present invention provides a plant against the infection of a plant pathogen when expressed in the plant. It relates to a modified plant virus gene that confers resistance. Smell in one aspect And the modified viral gene is a geminivirus encoding a coat protein. AV1 gene. One of the functions of migration proteins, such as the virus coat protein, is Interaction with host components is included. Perhaps a variety of different viruses Transfer It appears that the same host components are used for the lines. Because of this, a normal virus -A broad spectrum virus by preventing interactions between host components It is believed that resistance is provided. Other viral genes, especially virus Those involved in production tend to be highly virus-specific, and The spectrum of virus resistance obtained by any interference with the virus is narrow. Departure The application of Ming will advantageously provide broad spectrum virus resistance in plants. Offer.   The present invention relates to a modified coat protein of the present invention or a variant or fragment thereof. It also relates to the polynucleotide molecule to be loaded. In one exemplary embodiment of the invention, The 10 amino acid residues at the amino terminus of the TMoV coat protein were removed. This poly The nucleotide molecule comprises the nucleotide shown in FIG. 4 or a degenerate variant thereof. This modification leaves the core intact and still forms virions Provided is a coat protein that appears to have the ability to   The present invention also relates to infection by a plant pathogen such as, for example, a geminivirus. The invention also relates to a method for conferring resistance to plants. The method comprises the steps in which Plants that have been modified to confer resistance to viral infection Transforming a plant with a virus. In one embodiment, the modification The isolated plant gene encodes a protein containing the sequence shown in FIG. 5 or a fragment thereof. The polynucleotide sequence.   In one embodiment, the method of the present invention comprises, for example, tomato mottle geminivirus, Cabbage tree curl gemini virus, potato yellow mosaic virus, Mato golden mosaic virus, tomato yellow mosaic virus, tomato leaf Crumple virus, Capsicum astecovirus, Tomato yellow leaf mosquito Tobamoviruses and Geminiwis, including virus and others Can be used to confer plant resistance to viruses such as ruth .   The present invention further provides a modified coat protein of the present invention, which can be expressed in a suitable host or A combination comprising a vector into which a polynucleotide sequence encoding the fragment has been inserted. Pertaining to a replacement polynucleotide sequence. Therefore, this vector is a novel coat Encodes a protein and / or a fragment of this protein having substantially the same properties . Specifically, the vector may be a plasmid, phage DNA, or derivative thereof. Or fragments, or a mixture of plasmid and phage DNA and yeast plasmid You can choose from things. The polynucleotide encoding the modified coat protein is optional. Restriction ends compatible with insertion into desired expression or integration vectors for plants A commercially available pUC vector or Or can be inserted into multiple cloning sites of a vector such as a pGEM vector. Wear. For this purpose, strong promoters such as viral gene promoters Any vector operably linked to the coding sequence of the polynucleotide of the invention Could be used. For example, the powerful 35S of cauliflower mosaic virus A promoter can be used for this purpose. Other known plant expression vectors Can be used for this purpose.   The present invention also provides a desired protein or a fragment thereof that can be expressed in a suitable host. Recombinant polynucleotide comprising a vector into which the encoding polynucleotide sequence has been inserted Infected, transformed or transfected hosts with leotide sequences Also concerns. Preferably, the polynucleotide sequence is inserted into a suitable vector, This recombinant vector can be used to transfer bacteria or other bacteria capable of introducing the gene into plant cells. It can be introduced into a host by transformation. Inserted polynucleotide Means that the nucleotide sequence is altered coat protein and / or substantially It encodes fragments of this protein having the same properties. According to the invention Infection, transformation or transfection with a recombinant polynucleotide molecule Many proteins capable of expressing this protein or a fragment thereof Suitable hosts include Gram-negative or Gram-positive bacteria, such as E. coli, Bacillus subtilis, and kelp. There are insect cells, plant cells and yeast cells. In one illustrated embodiment, the Apitent Agrobacterium cells are used for this purpose and carry the recombinant vector Plant sections are exposed to Agrobacterium. Genes are efficiently incorporated It is preferable to regenerate the plant cells in a selective medium to confirm that Following this, the regenerated plants are grown under conditions optimized for survival. Or It is also possible to introduce recombinant vectors into plant cells by the biolistic method. (Carrer, 1995).   The present invention provides a polynucleotide encoding the modified coat protein of the present invention. Transgenic plants and plant tissues, including transgenic plants.   The present invention also relates to a polynucleotide encoded by a polynucleotide molecule described herein. Modified coat proteins and fragments and variants thereof. For example , Site-directed mutagenesis, digestion with Bal31, and up to about 50 amino acids from the N-terminus Any modification of the N-terminus of the coat protein, including deletions to residues, It appears to have a similar effect in that the core remains intact. The invention is other Geminiviruses, which, given the teachings herein, will be useful to those of skill in the art. Using any well-known standard plant transformation method, any geminivirus coat protein Modified genes useful for conferring resistance by similar modifications to the quality gene Is likely to be produced.   One of skill in the art will appreciate that to make the coat proteins described herein, the genetic code It is understood that many different nucleotide sequences can be used based on degeneracy. I think that the. Accordingly, it encodes the modified coat protein described herein. Any nucleotide sequence is included in the present invention and the appended claims. Also, As described herein, a gene encoding a modified coat protein Nucleotide fragments may also be used in plants where such fragments are used in the methods described herein. Of one aspect of the present invention as long as the resistance to is there. Such polynucleotides are readily and readily determined by techniques well known to those skilled in the art. It can be manufactured in a mold. For example, over a period of time, Bal31 A exonuclease digestion was performed, followed by expression and expression of the resulting fragment. Performing a routine screen (Wei et al., 1983) uses the polynucleotide Capable of conferring resistance to virus infection on plants transformed with The polynucleotide fragment of the modified coat protein of the present invention. Can be used for   The polynucleotide of the present invention includes a mutated sequence (variant ant sequence). For these mutations, the mutated sequence is Plants capable of inducing resistance to viral infection in plants transformed with As long as the modified coat protein encodes, for example, nucleotides Substitutions, insertions and deletions are included. Those skilled in the art will appreciate that the mutant polynucleotide The rows can be easily prepared and used.   The amino acid sequences disclosed herein use the standard single letter sequence for amino acid residues. Based on abbreviation.   The references cited in the present disclosure are hereby incorporated by reference.   The following describes materials, methods, and procedures for practicing the invention, including the best mode. FIG. These examples are for illustrative purposes only and are not limiting. Should not be considered as a matter of course.Example 1 Preparation of Transformation Cassette for Modified TMoV Coat Protein Gene   Modifications were made to the N-terminal coding region of the TMoV coat protein. Was. From a clone of the A component of TMoV (Abouzid et al., 1992), a restriction enzyme NcoI was added to the 5 ′ end ( nt 355), XbaI at the 3 'end (nt 1192), and 30 at the 5' end of TMoV Nucleotides (nt) were deleted. NcoI restriction enzyme compatible sequence located at the end 5 'untranslated region (leader sequence) of uri mosaic virus (CMV) coat protein (C orne 11 University D. (Obtained from clones prepared by Gonsalves) It was ligated with the TMoV coat protein gene at the NcoI site. The construct is shown in FIG.   A ligated CMV leader sequence having a modified TMoV coat protein sequence The construct as a plant constitutive expression vector, Neomycin phosphotra for selection, a regulatory element of the water mosaic virus 35S gene Transferase II gene and reporter gene β-glucuronidase (GUS) Ligated into plasmid pBI121 (CLONTEC HLaboratories, Palo Alto, CA) containing I let you. This binary expression plasmid with the sequence of the modified coat protein Agrobacterium LB4404 strain (CLON TECH Laboratories).Example 2: Transformation of tobacco   Agrobacterium tumefaciens with modified TMoV coat protein ( A. tumefaciens) and tobacco (N. tabacum) "Xanthi" Transform Fudisk and follow the method of Horsch et al. (1985) MS salt, sucrose (30 g / l), vitamin B5 (1 ml / l), indole-3-acetic acid (0 .3 mg / l), 2iP, N6- [2-isopentenyl] adenine (10 mg / l), kanamycin ( Shoots on a medium consisting of 100 mg / l) and mefoxin (200 mg / l) Was played. Subsequently, the transformed shoots contained 100 μg / l kanamycin. And planted on a plant-free hormone medium and switched to soil.Example 3: Detection of transgene by PCR amplification   Primers selected for amplification of the transgene (CMV: TMoV-AVI) Specific ply to the 5 'untranslated region of remosaic virus (CMV) coat protein (EH55, 5'-GTTAGTTGTTCACCT-3 ') and tomato mottle virus (TMoV) Internal specific primers (EH82, 5'-CCTTACCGATATGTGA-3 '). The PCR reaction was performed using a Biometra thermocycler. Performed as follows: 35 cycles of 94 ° C for 2 minutes, 45 ° C for 2 minutes and 72 ° C for 3 minutes. Finally, a DNA extension cycle was performed at 72 ° C. for 10 minutes to complete the amplification.Example 4: Southern and Northern blot analysis DNA extraction from tobacco leaf tissue   DNA was obtained from Soni and Murray (19). 94). Fern Mosaic Geminivirus DNA Leaves from agro-inoculated transformants Alternatively, tissue (0.2-0.5 g) collected from the leaf disk of the transformant was The mixture was ground in a mortar and pestle into fine powder. 1.5 ml of extraction buffer with this powder Solution (50 mM Tris, pH 8.0, 10 mM EDTA, 2% SDS, 100 mM LiCl and 10 μg / ml The mixture was homogenized with Rotinase K). The mixture is stirred for 15-30 minutes, followed by And centrifugation at 600 rpm for 5 minutes. Add equal volume of isopropanol to the supernatant Then, the nucleic acid is precipitated by centrifugation at 1000 rpm for 5 minutes, and 20 μg / ml RNAse is precipitated. Was resuspended in 0.5 ml of TE (10 mM Tris, pH 8.0, 1 mM EDTA). 30 minutes at 37 ° C After incubating for 2 hours, the mixture is phenol: chloroform-isoamyla Extracted once with alcohol (25: 24: 1) and once with chloroform . With 1/3 volume of 7.5M ammonium acetate and an equal volume of isopropanol. To precipitate the nucleic acid, followed by H_TwoResuspended in O. PCR and resuspend the resuspended DNA Used for Zan blot analysis.Southern blot   Southern blots were obtained according to standard procedures.RNA extraction from tobacco leaf tissue   Cosciolone and Cone ) (1993) and modified by Duan et al. (1996). A was isolated. Young tobacco leaf tissue (0.2 g) is mortared and pestled in liquid nitrogen It was ground to a fine powder. This powder was mixed with 1.0 ml of extraction buffer (7 M urea, 0.35 NaCl, 50 Homogenization with mM Tris, pH 8.0, 20 mM EDTA, 1% sarkosyl). Mixed The compound is phenol: chloroform-isoamyl alcohol (25: 24: 1) with 1 Twice, and once more with chloroform-isoamyl alcohol (24: 1), Subsequently, centrifugation was performed at 600 rpm for 5 minutes. An equal volume of isopropanol in the aqueous phase Heavy nucleic acids were precipitated by adding and cooling the mixture on ice for 5 minutes. -20 Incubate at 1 ° C for 1 hour, then centrifuge at 14,000 rpm for 10 minutes RNA was obtained from the soluble fraction. Wash the pellet with 70% ethanol, TE (10m MTris, pH 8.0, 1 mM EDTA), resuspended in 0.4 ml, chloroform-isoamyl alcohol Extracted by the 0.1 volume of 3M sodium acetate and 2 volumes of RNA was precipitated from the aqueous phase by adding 100% ethanol.Northern blot   Total DNA was run on a 0.9% agarose gel in TAE buffer Electrophoresis and transfer to nylon membrane using alkaline transfer solution (0.4M NaOH, 0.6NaCl) Migrated. Total RNA was purified using 1.0% agarose in MOPS buffer containing formaldehyde. Electrophoresis on Sgel and transferred to nylon membrane using 10 × SSC solution . Using a Prime-a-Gene® labeling kit (Promega), the probe was A component of VAV1 DNA and SiMV DNA was randomly primed³²P sign It produced by performing. Blot hybridization (overnight at 65 ° C) Is a 6 × SSC, 5 × Denhardt solution according to Sambrook et al. (1989). The test was performed in liquid and 0.5% SDS, 0.1 mg / ml salmon sperm DNA. Blot is Blair (B lair) et al. (1995).Example 5: Western immunoblot analysis   Expression of the modified coat protein of TMoV in transgenic tobacco, Grind young leaf tissue in 1.5% SDS in sterile water, then add 1 volume of tissue extract to 1 volume Were examined by mixing with the same product of Laemmli dissociation buffer (Laemmli, 1970). Mixed The mixture was boiled for 2 minutes and the sample was subjected to 10% SDS-PAGE. Transfer protein to Bio-Rad Mini- PR Electrophoresis in OTEAN II, Bio-RadTrans-Blot® electrophoresis transfer The separated proteins are nitro-treated using a tank (Bio-Rad Laboratories, Richmond, CA). Transferred to a rocellulose membrane. Chemiluminescence detection system Western-Light (Tropix, I nc., Bedford, Mass.) and procedures previously described (Cancino et al., 1995). Immunoblots were prepared against TMoV coat protein expressed in E. coli. Polyclonal antiserum (Abouzid et al., Unpublished), and goat anti-rabbit IgG alkaline Labeled with phosphatase conjugate.Example 6: Inoculation of tobacco leaf disk with SiMV   T on MS rooting medium containing 100 μg / ml kanamycin₀Germination of seeds of individual lines Let the descendants (T₁Ag) by SiMV DNA (Abouzid et al., 1995) on leaf disk Loin calculations were performed (Klinkenberg et al., 1989).Example 7: Inoculation of TMoV to tobacco using whiteflies   T on MS rooting medium containing 100 μg / ml kanamycin₀Germination of seeds of individual lines And the resulting T₁Offspring were transplanted to soil for whitefly inoculation. Four The seedlings at the 6th leaf stage were infected with TMoV and fed with a tomato strain grown in a growth room at 30 ° C. White lice were inoculated. 0.25m in growth chamber at 30 ℃^ThreeWhiteflies in a shielded cage Adults were added at a concentration of 20 animals per strain. Inoculation of whiteflies on tobacco seedlings The inoculation access period was set to 5 days. Insecticide on inoculated plants The inoculatable period was ended by spraying with soap.Example 8: Evaluation of tobacco inoculated with TMoV   After inoculation, symptoms were evaluated approximately every 30 days. Systemic feeling of TMoV 70 days after inoculation Dot spot hybridizer for B component related to systemic infection The youngest leaves for analysis by PCR and / or PCR primers A sample was taken from A sample was taken again 100 days after inoculation, and TM was The systemic infection by oV was examined. Dot spot hybridization using probe Digestion was performed under high stringency conditions and included the cloned TMoV B component ( Polston et al., 1993). Amplification of viral DNA amplifies component B but does not amplify component A. Using degenerate primers (pBL1v2042 and pCRc154) (Rogas et al., 1993). ). The amplification procedure involves denaturation at 94 ° C for 5 minutes, followed by 35 cycles of: Temperature: 50 ° C Anneal for 1 minute, extend for 3 minutes at 72 ° C and denature for 1 minute at 94 ° C.Example 9: Analysis of tobacco transformants   T using primers for CMV leader sequence and coat protein₀Cigarette shape Of the CMV (leader sequence) and the modified TMoV AVI sequence It was shown that 10 tobacco plants were transformed by both (FIG. 2). Sa In addition, as a control, one line was successfully transformed with pBI121 alone . Take samples from each strain before transferring the transformed plants to soil and transform GUS assay for efficiency, Southern blot for presence of transgene, and Western blot analysis of the expression of modified and altered coat proteins. Was. All the transformants examined were found to have the GUS gene. Southern blot analysis shows multiple copies of transgene in plant genome It has been shown.Example 10: Presence of mRNA for modified coat protein   Selected tobacco transformants not inoculated with virus (T₁Generation) Noza Blot analysis showed only one transcript, approximately 1.0 kb in size (FIG. 3). . This was larger than the expected CMV leader-coat protein transcript (1.0 kb 0.9 kb compared to the presence of the NOS-ter sequence (260 bp) of pBI121 in the transcript. It is thought.Example 11: Expression of a modified coat protein   In a standard Western blot procedure using a chemiluminescence detection system, No expression of the modified coat protein was detected in any of the transformants. This This is probably due to the instability of the amino-terminally modified coat protein, or It is considered that the current level was lower than the detection sensitivity.Example 12: Inoculation of tobacco with TMoV by whiteflies   10 T₀T from 8 strains of tobacco strains₁T against the offspring by whiteflies MoV was inoculated. As a result of analysis of the plant strain 70 days after inoculation (Table 1), Despite the separation, the infection rate was control (T₁-cp0) It became clear that. Overall, no matter which assay you measure, There was good agreement between the onset of symptoms and the presence of viral DNA, Do Good agreement between the results of spot spot hybridization and PCR analysis Because it was seen. We believe that TMoV replication levels are so low that PC Detection by R is possible, but detection by dot spot hybridization I couldn't find a case that couldn't be done. The ratio of resistance to TMoV depends on the system. It was different from one another. Two systems T₁-cp10 and T₁-cp11, 100 days from the first inoculation No later plants became infected. Variation between strains depends on the copy number of the transgene, Location of the transgene and / or isolation ratio of resistant and sensitive plant strains Due to their influence on   Infection rates generally increased gradually over time. This is, in part, the first contact Due to intermittent inoculation by whiteflies generated from whiteflies eggs used for seeds It is believed that there is. Overall, the presence of the transgene assessed by PCR Good immunity against systemic infection with TMoV 100 days after the first dose Significant agreement was observed (Table 2). This varies between strains, and some strains (T₁- cp4, T₁-cp10 and T₁-cp11) showed a 90-100% correlation.Example 13: Transgenics for Resistance by Agroin Occlusion Evaluation of cigarettes   In a preliminary study, the inventors found that geminiviruses closely related to TMoV DNA replication of one of the fern mosaic virus (SiMV) (Abouzid et al., 1995) The effect of transgenic tobacco resistant to TMoV was investigated. The present inventors have In vitro leaf disc agroinoculation assay (K1inkenberg Et al., 1989). Transgenic T₀Stock T₁Leaf leaf from 10 to 15 offspring Against the disc, the cloned genome of SiMV (DNA A and DNA B) (Abouzi Agrobacterium carrying the head-tail dimer of d. et al., unpublished) To improve their ability to suppress viral DNA replication. Say. Transformed leaves 10 days after agroin occlusion Assays for viral DNA were performed using DNA extracts from the disks. SiMV DN After Southern blot hybridization with AA probe (full length) , The presence of single-stranded (ss) SiMV genomic DNA and its replication intermediate Formation of oc and sc in the virus-susceptible leaf disks MV DNA It has been released from the G. bacterium Ti plasmid and replicated in the inoculated tissue. Was shown. Viral DNA levels in tobacco transgenic lines There was a sudden. SiMV DNA detected in one transgenic line is extremely low Quantity.   Tobacco transgenic for a modified TMoV coat protein gene Showed resistance to systemic infection by TMoV across plant strains. Introduction The presence of the gene was suppressed in the leaves that occurred 60 days after the initial inoculation with TMoV and symptom suppression. Correlated with undetectable viral DNA. For the modified TMoV coat protein gene One transgenic tobacco strain is In this case, the replication of the fern cyclin gemini virus was suppressed. Modified TMoV coat Plants transformed with a protein gene are shown to produce mRNA transcripts However, the expression of the modified coat protein was not detected by Western blotting Was. The modified TMoV coat protein gene can be used in accordance with the teachings herein to make Useful for gaining resistance to irs.   The above description and examples provide details on how to make and use the invention. Although good aspects are provided, obvious variants and functional equivalents are not Should be considered part of the statement and therefore fall within the scope of the following claims. .

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, S D, SZ, UG, ZW), EA (AM, AZ, BY, KG) , KZ, MD, RU, TJ, TM), AL, AU, BA , BB, BG, BR, CA, CN, CU, CZ, EE, GE, HU, IL, IS, JP, KP, KR, LC, L K, LR, LT, LV, MG, MK, MN, MX, NO , NZ, PL, RO, SD, SG, SI, SK, SL, TR, TT, UA, UZ, VN, YU, ZW (72) Inventor Hibert Ernest             United States Gainsby, Florida             Le 36 Stellars N. AW.             2201 (72) Inventor Avoged Ahmed             United States Gainsby, Florida             Le 20s Avenue S. AW.             3520 # 1

Claims (1)

[Claims] 1． The polynucleotide encoding the modified plant virus coat protein The plant against plant virus infection, including transforming the plant with A method for conferring resistance to a plant, wherein the polynucleotide is a plant plant. A method for imparting resistance to ills infection. 2． The polynucleotide has the nucleotide sequence shown in FIG. 4 or a fragment of its degeneracy. 2. The method of claim 1, comprising a piece or a variant. 3． The polynucleotide comprises nucleotides 56-890 of the nucleotide sequence shown in FIG. 2. The method of claim 1, comprising otide or a degenerate fragment or variant thereof. Four. A polynucleotide encoding a modified plant virus coat protein; To impart enhanced viral resistance to a plant having the polynucleotide. Renucleotide. Five. Including the nucleotide sequence shown in FIG. 4 or a degenerate fragment or variant thereof The polynucleotide according to claim 4, wherein 6． Nucleotides 56 to 890 of the nucleotide sequence shown in FIG. 4 or truncations thereof 5. The polynucleotide according to claim 4, comprising a heavy fragment or mutant. 7． A modified plant virus coat protein that expresses said protein. A coat protein having the ability to confer enhanced virus resistance to an object. 8． Claim 7 comprising the amino acid sequence shown in Figure 5 or a fragment or variant thereof. The modified plant virus coat protein according to the above. 9． Coding by nucleotides 56 to 890 of the nucleotide sequence shown in FIG. The modified amino acid sequence according to claim 7, which comprises the amino acid sequence to be Modified plant virus coat protein. Ten. Transgenic plants or plants with increased resistance to viral infection A modified plant virus coat protein according to claim 7, wherein the plant virus coat protein is a plant tissue. Transgenic for a polynucleotide encoding Nick plant or plant tissue. 11． A cell transformed with the polynucleotide according to claim 4.