WO1997007217A1 - Procedes et materiaux pour la production de plantes resistant a des agents pathogenes - Google Patents

Procedes et materiaux pour la production de plantes resistant a des agents pathogenes Download PDF

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WO1997007217A1
WO1997007217A1 PCT/US1996/013097 US9613097W WO9707217A1 WO 1997007217 A1 WO1997007217 A1 WO 1997007217A1 US 9613097 W US9613097 W US 9613097W WO 9707217 A1 WO9707217 A1 WO 9707217A1
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gene
plant
bcl
plants
mutated
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PCT/US1996/013097
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English (en)
Inventor
Ernest Hiebert
Ahmed Abouzid
Ping Duan Young
Charles A. Powell
Jane E. Polston
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University Of Florida
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Priority to EP96927402A priority Critical patent/EP0843728A1/fr
Priority to NZ315512A priority patent/NZ315512A/xx
Priority to IL12306296A priority patent/IL123062A0/xx
Priority to AU67233/96A priority patent/AU726197B2/en
Publication of WO1997007217A1 publication Critical patent/WO1997007217A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/12011—Geminiviridae
    • C12N2750/12022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention pertains to the field of conferring pathogen resistance to plants. More particularly, the invention is directed to virus-resistant transgenic plants.
  • Tomato producers suffer significant losses due to tomato mottle geminivirus infection.
  • farmers must purchase chemicals in order to control tomato mottle virus in their tomato fields.
  • losses are experienced by farmers producing tobacco as a result of tobacco crop infection by tobacco mosaic tobamoviruses. Accordingly, there is a need for a solution to this problem which is less costly and less damaging to the environment than the chemical controls currently employed.
  • Transformation of plants with portions of viral genomes may result in plants with virus resistance (Beachy, 1993). This phenomenon is known as "pathogen-derived resistance” (Sanford and Johnson, 1985). The level of resistance obtained is variable. This variability has been attributed to the random nature ofthe transformation process (Lomonossoff, 1995). Independent lines of plants generated from a single transformation experiment may contain different transgene copy numbers inserted in various chromosomes. Phenotypic differences have been noted among plant lines containing a single copy ofthe transgene. Some ofthe variability in transgene expression has also been attributed to tissue culture-induced changes (Phillips et al., 1994). This variability in the phenotype is also observed in subsequent progeny derived from the R ⁇ , plants.
  • TYLCV Yellow Leaf Curl Virus
  • TMV tobacco mosaic virus
  • Nejidat and Beachy (1990) disclosed that transgenic tobacco plants expressing a TMV coat protein have increased resistance against several of the tobamoviruses.
  • Gilbertson et al. (1993) disclosed the reduced pathogenicity of pseudorecombinants of two bipartite geminiviruses, tomato mottle (ToMoV) and TGV-MX1.
  • a mutated plant virus gene which protects tobacco plants against tomato mottle geminivirus and tobacco mosaic tobamovirus infections.
  • This resistance gene has been introduced into tobacco chromosomal DNA by genetic engineering.
  • the transgenic tobacco plants expressing this gene show resistance to tomato mottle gemudivirus and tobacco mosaic tobamovirus infections (lack of or reduction of disease symptoms when inoculated with the viruses).
  • the mutated gene can be introduced into chromosomes of desirable tomato and tobacco lines to develop commercially improved tomato and tobacco cultivars/hybrids.
  • this invention comprises a mutant plant virus gene which confers resistance on tobacco and tomato plants against tobacco mosaic tobamovirus and tomato mottle geminivirus infections, as well as resistance to infections of other related geminiviruses.
  • the known BCl gene between nucleotides 1278 and 2311 of the B component of tomato mottle geminivirus, was subcloned into an appropriate expression vector and transformed into tobacco plants.
  • a mutated gene product was produced which confers resistance against viral infection to the recombinant plant in which it is expressed.
  • One object of this invention is to provide a memod for conferring viral resistance on a plant.
  • Another object of this invention is to provide a mutated BC 1 gene and any fragment thereof which confers viral resistance on a plant.
  • Another object of this invention is to provide novel transgenic plants with enhanced viral resistance.
  • Figure 1 is the sequence ofthe single stranded mutated tomato mottle geminivirus BCl gene except for positions 1742-1766 which initially were not identified; wild-type nucleotides which are different in the mutant gene are shown in lower case text above the mutant gene sequence.
  • Figure 2 is the sequence shown in Figure 1 along with its complementary strand; the translational start and stop codons are underlined; the terrnini are Hindlll restriction sites.
  • Figure 3 is the deduced amino acid sequence ofthe mutated gene product encoded by the nucleotide sequence of Figure 1, except for positions 151-159 which in initial sequencing efforts were not identified.
  • Figure 4 shows a comparison ofthe mutant and wild-type gene products (the mutant protein is the lower sequence).
  • FIG. 5 shows phenotypic comparison of transgenic R, tobacco plants expressing BCl protein of TMoV.
  • Transgenic plants were derived from a R Q plant which contained two copies of BCl gene (see Fig. 6) and which did not show any stunting.
  • A Plants from left to right: a. transgenic plant (BCl -3-11-5) expressing symptomatic BCl protein, showing stunting, mottling, and curling on the leaves. Symptoms are more sever than those induced by TMoV infection; b. transgenic plant (BCl -3- 11-2) which contains one copy of the non-symptomatic BCl and the symptomatic BCl transgene, showing mottling with no stunting; c. transgenic plant (BCl -3-11-6) which contains one copy of non-symptomatic BCl transgene; and d. non-transgenic tobacco.
  • B Plants from left to right: a. transgenic plant (BCl -3-11-5) expressing symptomatic BCl protein, showing stunt
  • Figure 6 shows Southern blot analysis ofthe R, transgenic plant with different phenotypes. Segregation ofthe BCl transgene in R, generation of transgenic tobacco plants which displayed different phenotypes in Fig. 5 (BC1-3-11-1 and -2, mottling only, -4 and -5, severe stunting and mottling, -6 and -7, no visible symptoms). Blots from BCl -3- 16-2 showing stunting and mottling, and BCl -3-6-3 and -4, no visible symptoms are shown for comparative purposes; NT- nontransformed plant; and pKYsBC 1, vector construct used for transformation. Genomic DNA of the transgenic plants was extracted and digested with Xbal. Southern blots were subjected to hybridization with 32 P-labeled BC 1 DNA fragment.
  • Figure 7 shows Western blot analysis ofthe P30 fraction of tissue extracts from transgenic R, tobacco plants expressing the BCl gene. Lanes represent extracts from plants described in Fig. 6 except for TMoV-infect. extract from TMoV infected tissue). The subcellular fractions, P 1, P30 and S30 were prepared (Pascal et al, 1993) and subjected to SDS-PAGE (Schagger) with some modification and immunoblots using the polyclonal antiserum against expressed BC 1 protein. The results of the P 1 and S30 fractions are not shown here.
  • Figure 8 shows Northern blot analysis of transgenic plants which express the BCl gene, probed with labeled-BC 1 DNA. Two BC 1 related transcripts were found in the transgenic plants which expressed the full-length BCl gene, while only one transcript was found in the transgenic plant which expressed a 3 '-truncated form ofthe BCl gene (BCl -3-11-6). The samples indicated are as in Fig. 6.
  • Figures 9A-1 thru 9A-5 and 9B show nucleotide sequences (A) and predicted amino acid sequences (B) ofthe TMoV BC 1 and its transgene mutants.
  • the nucleotide sequence of TMoV BC 1 gene from GenBank Accession U14461.
  • the sequence ofthe PCR amplified BC 1 ORF was verified before and after cloning into pGEM-T vector.
  • the sequence was analyzed from the PCR product derived from genomic DNA (BC 1-3-6-3 A).
  • BC 1 At r sequence determined from the cDNA, the RT-PCR products, amplified from the total RNA (BC1-3-11-6A). The sequence was also verified by sequencing the PCR product from the genomic DNA and from cloned
  • BC 1 S sequence determined from a symptomatic transgenic plant which expressed full length BCl protein The sequence was analyzed after RT-PCR of total RNA (BC1-3-11-5S), after PCR amplification of genomic DNA (BC1-3-11-5S) and after PCR amplification from 3 different lines with a similar phenotype. Note that identical nucleotides and amino acid residues are indicated by (.).
  • the subject invention concerns a mutated plant virus gene that when expressed in a plant confers on that plant a resistance to infection from plant pathogens.
  • the mutated virus gene is a BC 1 gene of geminivirus.
  • the mutated gene of the present invention can be prepared by inserting the wild-type gene into the genome of a plant and identifying those plants transformed with the gene that exhibit increased resistance to viral infection.
  • the subject invention also concerns a method for conferring resistance on a plant to infection by plant pathogens.
  • the subject method comprises inserting a wild-type viral movement gene, such as BC 1 , into the genome of a plant and then identifying those plants that do not exhibit pathogenic symptoms when the inserted gene is expressed but which have enhanced resistance to infection by pathogens.
  • the subject invention also concerns transgenic plants and plant tissue having a mutated gene ofthe present invention inco ⁇ orated into their genome.
  • the following is a specific example ofthe subject invention, a method for creating a virus- resistant plant, using the BCl gene of tomato mottle geminivirus to illustrate the invention. The method is generally and broadly applicable to other plant viruses.
  • the complete sequence ofthe BCl gene of tomato mottle geminivirus is known (Abouzid et ai, 1992, herein inco ⁇ orated by reference).
  • the BC 1 gene of tomato mottle geminivirus ofthe B component ofthe genome is isolated in sufficient quantity for subcloning in an expression vector. This may be accomplished by any of several methods well-known in the art. A simple method is to use a pair of specific primers to amplify the desired segment according to the well known polymerase chain reaction (PCR) technique. For this pu ⁇ ose, a useful primer pair such as: 5 '-CCCAAGCTTCGAGTTCGAAACTGC-3 ' (SEQ ID NO. 1) and
  • 5'-CCCAAGCTTAACGAAGTGTGTTTGAC-3' (SEQ ID NO. 2) may be used. All or portions ofthe BCl gene may be used for this pu ⁇ ose.
  • the gene is cloned into a vector for production of a stable source for mass production of the gene.
  • Any vector known in the art can be used for this pu ⁇ ose, and mass quantities of the vector may be cultured, for example, by transformation of competent bacterial cells such as E. coli followed by harvesting ofthe plasmid DNA.
  • the gene is inserted into the multiple cloning site of a vector, such as the commercially available pUC vectors or the pGEM vectors, which allow for excision of the gene having restriction termini adapted for insertion into any desirable plant expression or integration vector.
  • any vector in which a strong promoter, such as a viral gene promoter, is operatively linked to the coding sequence ofthe mutant gene of this invention could be used.
  • a strong promoter such as a viral gene promoter
  • the powerful 35S promoter of cauliflower mosaic virus could be used for this pu ⁇ ose.
  • this promoter is duplicated in a vector known in the art as pKYLX 71:35S 2 (Morgan et ai, 1990).
  • pKYLX 71:35S 2 Morgan et ai, 1990.
  • other plant expression vectors could be used for this pu ⁇ ose.
  • the gene is transformed into a bacterium or other vector which is able to introduce the gene into a plant cell.
  • the gene may be introduced into plant cells by a biolistic method (Carrer, 1995).
  • competent Agrobacterium cells are used for this piupose, and plant sections are exposed to the Agrobacterium harboring the BC 1 gene. Regeneration of the plant cells in a selective medium to ensure the efficient uptake ofthe gene is preferred, followmg which the regenerated plants are grown under optimized conditions for survival.
  • the mutant gene of this invention has also been deposited prior to filing the instant patent application with the American Type Culture Collection (ATCC), 12301 Parkiawn Drive, Rockville, Maryland 20852 USA.
  • ATCC American Type Culture Collection
  • the mutant gene was cloned in a bacterial vector (pGEM-T) and the construct is named TMBC lm.
  • the deposit has been assigned accession number ATCC No. 97244 by the repository.
  • the subject deposit will be stored and made available to the public in accord with the provisions ofthe Budapest Treaty for the Deposit of Microorganisms, i.e., it will be stored with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample ofthe deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the culture.
  • the depositor acknowledges the duty to replace the deposit should the depository be unable to furnish a sample when requested, due to the condition ofthe deposit. All restrictions on the availability to the pubhc ofthe subject culture deposit will be irrevocably removed upon the granting ofa patent disclosing it.
  • the DNA is solubilized in an appropriate transformation buffer for the cell type into which the gene is to be transformed.
  • competent cells are prepared and transformed according to methods well known in the art (see Maniatis et al. , 1982), and transformed cells selected in an ampicillin growth medium.
  • the plasmid is then isolated from the E. coli and excised from the pGEM-T vector using, for example, HindHI restriction enzyme.
  • the excised gene fragment has a size of about 1100 bp.
  • the Hindlll fragment is then cloned into the Hindlll site of an appropriate expression vector as described below.
  • Figure 1 provides the sequence of the mutant gene of this invention, except for a stretch of 25 nucleotides corresponding to positions 1742-1766, which were not identified in initial sequencing efforts.
  • Figure 2 provides the complementary strand of the mutant polynucleotide and shows the HindHI termini.
  • Figure 3 provides the deduced amino acid sequence ofthe mutated gene product, except for amino acids 151- 159 which were not identified in the initial sequencing. The differences in amino acid sequence between the wild-type BCl product and mutant BCl products are shown in Figure 9B.
  • Figure 4 shows a comparison between the wild-type (upper sequence) and mutant protein (lower sequence) based on initial sequencing efforts.
  • BCl gene into an expression vector.
  • the BCl gene (nucleotides between 1278 and 2311 of the B component of tomato mottle geminivirus; Abouzid et al. , 1992) was amplified from the extracts of tomato mottle geminivirus infected tomato plants by polymerase chain reaction (PCR) technology.
  • PCR polymerase chain reaction
  • the amplified BCl segment was cloned into a pGEM-T vector and then digested with Hind HI.
  • the excised BCl segment was ligated into the unique Hind IH site ofthe binary pKYLX 71:35 S 2 vector.
  • Competent cells oi Agrobacterium tumefaciences LBA 4404 were prepared as described by An, et al, (1985).
  • the BCl gene in the pKYLX 71:35 S 2 vector was directly transferred into the Agrobacterium.
  • the clone was kept in a -80 ° C freezer for further use.
  • the Agrobacterium carrying the BCl gene in the pKYLX 71:35 S 2 vector was used to transform the leaf discs of Nicotiana tobacum cv. Xanthi.
  • the Agrobacterium cells were cultured in YEP broth containing 50 ⁇ g/ml kanamycin and 10 ⁇ g/ml tetracycline and 25 ⁇ g/ml streptomycin for 24-30 hours.
  • Agrobacterium cells were collected and resuspended in YEP broth.
  • Leaf discs cut from expanded young sterile seedlings were dipped into the Agrobacterium suspension and then placed on a selective medium containing 200 ⁇ g/ml Mefoxin and 100 ⁇ g/ml kanamycin. Regeneration and selection were carried out with the media, and took 6-8 weeks.
  • the kanamycin resistant plants were individually grown in soil under sterile condition for a week, and then transplanted to pots in a growth room and/or greenhouse.
  • Transformation of the tobacco plants was confirmed by PCR analysis for BCl gene in chromosomal DNA extracts, by Southern blotting with a BCl probe, and by ELISA analysis for NPT II (Neomycin Phosphotransferase II). Twenty-three plants were transgenic for BC 1.
  • Infected leaves of tomato plants were powdered after freezing in hquid nitrogen and extensively ground with a mortar and pestle in two volumes of ice-cold grinding buffer (GB: 100 mM Tris-HCl, pH 8.0, 10 mM EDTA and 5 ml dithiothreitol)(Deom, et al, 1990).
  • Membrane and cell-wall fractions were prepared as described by Pascal, et al., ( ⁇ 993).
  • the blotting procedure was conducted essentially as described by Towbin, et al, (1979) using a Bio-Rad Mini-Protein Electrophoresis Cell and Bio-Rad Trans-Blot Electrophoretic Transfer Cell.
  • the separator gel for small proteins was prepared with 12.5% polyacrylamide in gel buffer (Laemmli, 1970). The protein gels were transferred to nitrocellulose membrane (Bio-Rad Trans-Blot, 0.4 ⁇ m).
  • the detection of expressed BCl protein in transgenic tobacco plants was conducted with Western- Light- Chemiluminescent Detection System (TROPIX, Inc.). The BCl protein was detected at a relatively high level and extracts from about 50% ofthe plants showed a smaller (truncated) BCl protein (28k Da) than the wild-type (33k Da).
  • the BC 1 gene has been implicated as a symptom inducing element of a bipartite geminivirus during infection. Eleven transgenic tobacco plants which expressed the full length BCl protein showed disease symptoms. Twelve plants expressing the truncated BCl protein did not show disease symptoms.
  • Example 5 Resistance to tomato mottle geminivirus and tobacco mosaic tobamovirus
  • Transformed tobacco plants (R, generation) expressing BCl were tested for susceptibility to tomato mottle geminivirus infection by natural transmission with the whitefly vector and by mechanical inoculation with extracts from infected plants.
  • the inoculated plants were evaluated for resistance to tomato mottle gemimvirus by symptom development, and by enzyme linked immuno ⁇ assays (ELISA) using antiserum reactive to tomato mottle geminivirus coat protein.
  • the transgenic plants expressing the truncated BCl protein were free of symptoms and had very low ELISA readings.
  • Transgenic tobacco plants subjected to mechanical inoculation with tobacco mosaic tobamovirus showed reduced disease symptoms compared to inoculated non-transgenic plants.
  • the BCl gene from the tobacco plants expressing the truncated BCl protein was PCR- amplified and sequenced. This data indicates that the BCl gene has undergone spontaneous mutation(s) in about 50% ofthe transgenic BCl tobacco plants. During the tissue culture phase, plant cells containing the mutated BCl gene may have a selective advantage over the wild-type BCl expressing cells.
  • the mutated BCl gene in the pKYLX 71:35 S 2 vector is suitable for the production of tomatoes transgenic for the gene via Agrobacterium transformation as described above for tobacco.
  • the mutated BCl gene provides similar resistance to tomato mottle geminivirus in tomato as seen in transgenic tobacco.
  • the introduction of this mutated BCl gene into the chromosome of desirable tomato lines leads to tomato mottle geininivirus resistance in commercially acceptable tomato cultivars hybrids. In addition, it is predictable that this resistance is active against other geminivirus infections.
  • Resistance to tobacco mosaic virus was also detected in the transgenic tobacco expressing the mutated BCl gene, indicating that resistance to RNA viruses also is possible with the expression of this mutated gene from a DNA plant virus.
  • the mutated gene in tomato offers resistance to tomato mosaic tobamovirus, a virus related to tobacco mosaic tobamovirus.
  • Example 8 Production of BC 1 gene fragments useful for conferring virus resistance to plants.
  • Fragments ofthe mutant BC 1 gene which are useful for conferring virus resistance to plants can be produced by use of BAL31 exonuclease for time-controlled limited digestion ofthe mutant
  • BC 1 gene Methods of using BAL31 exonuclease for this pu ⁇ ose are well known in the art, and have been widely, used for over a decade (Wei et al. , 1983).
  • BAL31 exonuclease By using BAL31 exonuclease, one can easily remove nucleotides from either or both ends ofthe mutant BCl gene to systematically and certainly generate a wide spectrum of DNA fragments which have controlled lengths and are from controlled locations along the entire length of the mutant BCl gene. Hundreds of such fragments from various points along the entire mutant BCl gene DNA sequence can be systematically generated in one afternoon.
  • These gene fragments are then cloned into appropriate vectors and ultimately transferred into plant cells according to the methods disclosed above. Plant cells transformed with these fragments are routinely cultured and regenerated into plants, which are then tested for resistance to viruses. In this manner, fragments of the mutant BCl gene which are sufficient to confer viral resistance are routinely and predictably identified.
  • Example 9 Production of additional mutants conferring virus resistance to plants.
  • Tobacco was transformed with the movement protein (pathogenicity) gene (BCl) from tomato mottle geminivirus (TMoV) using Agrobacterium-mediated transformation.
  • BCl movement protein
  • ToV tomato mottle geminivirus
  • Different transgenic tobacco lines expressing the BCl protein had phenotypes ranging from plants with severe stunting and leaf mottling to plants with no visible symptoms.
  • the sequence data for the BCl transgene for the different phenotypes indicated unexpected mutation(s).
  • a mutated BC 1 transgene suppressed the phenotypic expression ofthe symptomatic BCl gene in tobacco lines containing both copies of the BCl gene.
  • the present invention shows spontaneous mutations in the transgene to be common Agrob ⁇ cterium-msdiated transformations, and this phenomenon can be utilized in the creation and selection of pathogen-resistant plants using pathogenicity genes during transformation.
  • TMV tomato mottle gemini virus
  • BCl movement protein gene
  • the three phenotypes were observed in the R, generation derived from a R Q plant which did not show any apparent stunting (Fig. 5).
  • the three observed phenotypes were: 1) Severe stunting and mottling, more severe than the typical symptoms associated with TMoV infections in tobacco; 2) Mottling with no stunting of growth; and 3) No visible symptoms, plants indistinguishable from nontransformed plants.
  • the transgenic plant showing slight mottling with no stunting had two copies ofthe BC 1 gene.
  • BCl proteins full-length or tmncated form
  • BCl proteins from the non-symptomatic, transgenic plants were not detected in older tissue, unlike that seen for the transgenic plants expressing the severe symptom type BCl protein. This indicated that certain mutations in the BC 1 protein may affect its stability in planta.
  • the transcript level for the plants expressing the truncated BCl protein was high and therefore the low level of truncated BCl protein detected in Western blots (Fig. 7) is not due to transcript activity.
  • the larger than expected transcript is the result of a readthrough of BC 1 termination signals into the vector rbcS termination sequences ofthe pKYLX vector.
  • the BCl gene from the transgenic tobacco plants showing the different phenotypes was amplified by polymerase chain reaction (PCR) and sequenced.
  • the sequence data revealed mutations (amino acid residue 215 G-S, 219 S-L, and 247 E-G) near the carboxyl terminus ofthe BC 1 protein (Fig. 9) for the severe stunting phenotype (Fig. 5A).
  • the non-symptomatic, transgenic R ⁇ tobacco plants revealed segregation in the R, generation as indicated by the appearance of several symptomatic plants in this generation. Some lines with symptom attenuation (Fig. 5B) continued to segregate in the R 2 generation but the non- symptomatic plants did not.
  • Southern blot analysis (Fig. 6) indicated multiple copies ofthe BCl gene in the RQ tobacco. Apparently some of the R Q tobacco lines contained copies of both the symptomatic and non-symptomatic forms of BCl. This was confirmed by Southern blot and Western blot analyses of selected R, tobacco plants which were associated with the different phenotypes (Fig. 5). The mottling phenotype with no stunting described above (Fig.
  • the subject invention also concerns the polynucleotide molecules shown in Figure 9A and the polypeptides encoded thereby shown in Figure 9B, as well as other mutated polynucleotides conferring viral resistance that can be produced using the teachings ofthe present invention.
  • the spontaneous mutations that can be produced in viral movement genes using the methods and materials of the present invention during Agrobacterium-mediated transformation provide a simple way to develop pathogen-resistant plants.
  • the introduction of the pathogenicity gene (BC 1 for the bipartite geminiviruses, AC4 for the monopartite like tomato yellow leaf curl virus) into plant cells by Agrobacterium-mediated transformation will result in selection since transformed cells which express the non-mutated pathogenicity genes will not grow as well as those cells which express the mutated pathogenicity gene.

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Abstract

Cette invention concerne un gène mutant de virus végétal qui confère au tabac et aux tomates une résistance au virus de la mosaïque du tabac et aux infections dues au géminivirus de la marbrure de la tomate, ainsi qu'aux infections dues à d'autres géminivirus apparentés. On a d'abord isolé un gène du gène BC1 connu, entre les nucléotides 1278 et 2311 du composant B du géminivirus de la marbrure de la tomate. Lors du sous-clonage de ce fragment d'ADN en un vecteur d'expression approprié et de la transformation du gène dans le tabac, on obtient un produit génique tronqué qui confère à la plante de recombinaison qui est exprimée une résistance aux infections virales.
PCT/US1996/013097 1995-08-11 1996-08-12 Procedes et materiaux pour la production de plantes resistant a des agents pathogenes WO1997007217A1 (fr)

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EP96927402A EP0843728A1 (fr) 1995-08-11 1996-08-12 Procedes et materiaux pour la production de plantes resistant a des agents pathogenes
NZ315512A NZ315512A (en) 1995-08-11 1996-08-12 Methods and materials for producing pathogen-resistant plants
IL12306296A IL123062A0 (en) 1995-08-11 1996-08-12 Methods and materials for producing pathogen-resistant plants
AU67233/96A AU726197B2 (en) 1995-08-11 1996-08-12 Methods and materials for producing pathogen-resistant plants

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US6237849B1 (en) 1995-07-31 2001-05-29 Keycorp Limited Remote smartcard terminal link
WO2000043520A2 (fr) * 1999-01-25 2000-07-27 University Of Florida Materiaux et methodes permettant de produire des plantes resistantes aux geminivirus
WO2000043520A3 (fr) * 1999-01-25 2000-11-30 Univ Florida Materiaux et methodes permettant de produire des plantes resistantes aux geminivirus
US7294758B2 (en) 1999-01-25 2007-11-13 University Of Florida Research Foundation, Inc. Materials and methods for producing geminivirus resistant plants
WO2000060088A2 (fr) * 1999-04-07 2000-10-12 E.I. Du Pont De Nemours And Company Genes de proteines de mouvement virales vegetales
WO2000060088A3 (fr) * 1999-04-07 2001-04-26 Du Pont Genes de proteines de mouvement virales vegetales
US7186885B1 (en) 1999-04-07 2007-03-06 E.I. Du Pont De Nemours And Company Plant viral movement protein genes
US7572951B2 (en) 1999-04-07 2009-08-11 E. I. Du Pont De Nemours And Company Plant viral movement protein genes

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