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WO1999063054A2 - Formes mutantes du gene al2 des geminivirus - Google Patents

Formes mutantes du gene al2 des geminivirus Download PDF

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WO1999063054A2
WO1999063054A2 PCT/US1999/012680 US9912680W WO9963054A2 WO 1999063054 A2 WO1999063054 A2 WO 1999063054A2 US 9912680 W US9912680 W US 9912680W WO 9963054 A2 WO9963054 A2 WO 9963054A2
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gene
recombinant
amino acid
transcription activator
activator protein
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WO1999063054A3 (fr
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David Bisaro
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Ohio State University Research Foundation
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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

  • the Geminiviridae is a family of single-stranded DNA plant viruses.
  • the geminiviruses in the genus Begomovirus (formerly known as subgroup III) are transmitted to plants by whiteflies.
  • the geminiviruses in the genus Begomovirus infect most dicots including, for example, vegetable crops such as beans, squash, pepper, tomato, cassava, and potato; as well as tobacco, and fiber crops such as cotton and kenaf. Such infection can result in total loss of the infected crop.
  • geminivirus epidemics in susceptible plants has increased in recent years, most likely due to an increase in whitefly populations which are resistant to insecticides. Accordingly, it is desirable to have techniques to combat geminiviruses.
  • the present invention provides isolated double domain mutant AL2 genes of a geminivirus.
  • the double domain mutant AL2 gene is useful for producing transgenic plants that are more resistant to infection by geminiviruses, particularly Begomoviruses.
  • the double domain AL2 mutant gene comprises at least one mutation in the 3 ' region of the AL2 gene, i.e., the region which encodes about 46 amino acids extending from about amino acid 83 to about amino acid 129 of the wild-type AL2 gene product and at least one mutation in the central region of the AL2 gene , i.e., the region which encodes about 20 amino acids extending from about amino acid 23 to about amino acid 43 of the wild-type AL2 gene product
  • the present invention also provides isolated single domain mutant AL2 genes of a geminivirus.
  • the single domain mutant AL2 genes are useful for producing transgenic plants that are more susceptible to infection by geminiviruses, particularly Curtoviruses (formerly known as subgroup II) and Begomoviruses.
  • the single domain mutant AL2 gene comprises at least one mutation in the 3' region of the AL2 gene, the region which encodes about 46 amino acids extending from about amino acid 83 to about amino acid 129 of the wild-type AL2 gene product.
  • the present invention also relates to vectors comprising a double domain mutant AL2 gene and to vectors comprising a single domain mutant AL2 gene.
  • the present invention also relates to transgenic plants comprising a double domain mutant AL2 gene and to transgenic plants comprising a single domain mutant AL2 gene.
  • Fig. 1 shows the amino acid sequence, SEQ ID NO: l, of the AL2 gene product, hereinafter referred to as the "transcriptional activator protein" or "TrAP", of tomato golden mosaic virus (TGMV)(see arrow).
  • the amino acid sequence of TMGV TrAP is aligned with the amino acid sequences of the TrAP proteins of Abutilon mosaic virus (AbMV), bean dwarf mosaic virus (BDMV), Bean golden mosaic virus (BGMV), cabbage leaf curl virus (CabLCV), potato yellow mosaic virus (PYMV), sida golden mosaic virus (SGMV), squash leaf curl virus (SqLCV), tomato mottle virus (ToMV), and Texas pepper virus (TPV).
  • Fig. 2 shows the 5' primers and 3' primers used to construct substitution mutants of the 3' region of the TGMV AL2 gene.
  • Fig. 3 is a graph depicting the extent of transcriptional activation by fusion proteins comprising a GAL 4 binding domain fused to a full-length TGMV TrAP protein or fragments thereof.
  • Fig. 4 is a graph depicting the extent of transcriptional activation by fusion proteins comprising a GAL 4 binding domain fused to fragments of TGMV TrAP, which fragments do and do not contain amino acid substitutions in the wild-type sequence.
  • TrAP is a transcriptional activator that is required for expression of the genes involved in infectivity, systemic spread, and insect transmission of such geminiviruses. Specifically, TrAP is a transcriptional activator that controls expression of the late genes that encode the capsid protein and a movement protein, CP and BRI, respectively. TrAP is a small protein of approximately 15 kDa. Begomovirus TrAP sequences are highly conserved. The amino acid sequence of the TrAP protein of TGMV is shown in Fig. 1.
  • TrAP has a modular structure which comprises an amino terminal basic domain, a central region, and a carboxy terminal acidic domain.
  • the central region of TrAP is characterized by a series of strictly conserved cysteine and histidine residues. These conserved residues are arranged as in TrAP from all Begomoviruses except squash leaf curl virus (SqLCV), which has a slightly different spacing: C-X ⁇ -C-X 4 -H-X 2 -C-X 8 -H-X - HC, wherein the X subscript indicates the number of amino acids separating the cysteine and histidine residues.
  • SqLCV squash leaf curl virus
  • mutant forms of the AL2 gene of a geminivirus are provided.
  • the nucleotide sequences of the wild-type, i.e., non-mutant forms of the AL2 genes of the geminiviruses are readily available in GenBank.
  • the accession numbers for geminiviruses whose genomes contain an AL2 gene are as follows:
  • One mutant form of the AL2 gene is a double domain mutant which comprises mutations in at least two regions of the AL2 gene.
  • the double domain mutant comprises at least one mutation in the 3' region of the AL2 gene , i.e. the region that encodes the C terminus of TrAP which extends from about amino acid 83 to about amino acid 129 of TrAP and at least one mutation in the central region of the AL2 gene, i.e., the region which encodes from about amino acid 23 to amino acid 43 of TrAP.
  • the double domain mutant AL2 gene encodes a TrAP having at least one mutation in the acidic activation domain and at least one mutation in the cysteine-histidine domain.
  • the 3' region mutation is: a deletion of from 3 to 15 base pairs; or an addition of from 3 to 15 base pairs; or a substitution of one or more base pairs in the region that encodes the C-terminus of TrAP; or combinations thereof.
  • the first mutation comprises a substitution of base pairs so that mutant gene encodes a mutant TrAP in which one or more amino acids in the region extending from amino acid 100-129 of the wild-type protein are substituted.
  • the 3' region mutation is a substitution which results in replacement of one or more of the hydrophobic amino acid residues, or one or more acidic acid residues, or combinations thereof in the C-terminus of the wild-type protein with an amino acid that does not have an acidic, hydrophobic, or aromatic side chain.
  • the substituted amino acids be replaced with serine, threonine, proline, cysteine, methionine, lysine, histidine, arginine, asparagine, glutamine, glycine or alanine.
  • the 3' region mutation results in replacement of the conserved isoleucine, phenylalanine, tryptophan or acidic residues within amino acids 115-129 of the wild-type protein with a small, non- charged amino acid, such as, for example, alanine or glycine.
  • Good results in have been achieved using a mutant TGMV AL2 gene which encodes a protein having an alanine at positions 119, 123, 124, and 128 in place of the hydrophobic residues that are found at these positions in the wild-type TGMV TrAP (i.e.
  • the central region mutation is a deletion of 3 to 60 base pairs so that the TrAP encoded by the mutant gene comprises from one to 20 fewer amino acids in the central region, an addition of from 3 to 60 base pairs so that the TrAP encoded by the double domain mutant gene comprises 1 to 20 more amino acids in the central region thereof, or a substitution of one or more base pairs in the central region so that the amino acid sequence of the TrAP encoded by the double domain mutant has a sequence which is different from the sequence of the wild-type TrAP.
  • the second mutation comprises a deletion of from one to 10 codons which encode amino acids 33 through 43 of TrAP or, alternatively, a substitution of base pairs so that the mutant gene encodes a TrAP in which one or, preferably, more amino acids in the central region of the wild type-protein are substituted.
  • the double dominant mutant gene encodes a TrAP in which a plurality of cysteine residues or a combination of histidine and cysteine residues in the central region of the wild-type protein are replaced with an amino acid other than a cysteine, histidine, or methionine. It is highly preferred that the cysteine and histidine residues be replaced with glycine or alanine.
  • the mutant gene encodes a TrAP in which the histidine at position 40 is replaced with an alanine or glycine.
  • Double domain mutant AL2 genes are used to make defective TrAP proteins that are unable to activate transcription of CP and BR1 movement protein genes and that are also unable to interact with SNF-1 kinase. Such genes can be used as research tools to study transcriptional activation in plants, .to analyze transcription factor interactions, to study viral pathogenesis, or to study plant defense responses to pathogens.
  • the present invention also provides mutant single domain mutant AL2 genes which have at least one mutation in the 3' region of the AL2 gene.
  • Single domain mutant AL2 genes may also have one or more mutations in the region encoding the amino terminus of TrAP, i.e. amino acids 1 to about 22 in TrAP.
  • single domain mutant AL2 genes may also have one or more mutations in the region encoding the central region of TrAP, as long as such mutation or mutations are silent or involve replacement with conservative amino acids.
  • the single domain mutant AL2 gene does not encompass the double domain mutant AL2 gene.
  • the mutation or mutations in the 3' region of the single domain mutant AL2 gene is a deletion of from 3 to 36 base pairs, an addition of from 3 to 36 base pairs, a substitution of one or more base pairs, or combinations thereof.
  • the mutation comprises a substitution of base pairs so that mutant gene encodes a TrAP in which one or more amino acids in the C-terminus of the wild-type protein are substituted with a small, non- charged amino acid, such as for example alanine.
  • the mutant gene encodes a TrAP in which one or more of the hydrophobic amino acid residues, one or more acidic residues, or combinations thereof in the C-terminus of the wild-type protein are replaced with an alanine residue.
  • the substitution mutation encodes a TrAP where the conserved isoleucine, phenylalanine, tryptophan, aspartate and glutamate residues within amino acids 115-129 of the wild-type sequence are substituted with a small, non-charged amino acid, such as for example alanine.
  • Single domain mutant AL2 genes are used to make defective TrAP proteins which are unable to activate transcription of the CP and BR1 movement protein genes. Such proteins, however, are able to interact with SNF-1 kinase and block pathways that employ such kinase. . Such genes can be used as research tools to study transcriptional activation in plants, .to analyze transcription factor interactions, to study viral pathogenesis, or to study plant defense responses to pathogens.
  • Mutations are introduced into the AL2 gene by conventional techniques such as, for example, by a combination of polymerase chain reaction (PCR) and site-directed mutagenesis.
  • PCR-based site-directed mutagenesis technique preferably employs a circular plasmid or linear DNA comprising the AL2 gene from any Begomovirus, more preferably a TGMV AL2 gene.
  • the PCR-based technique also employs oligonucleotide primers which encode the desired mutation, hereinafter referred to as the "mutagenic primer”, and oligonucleotide primers that are specific for the AL2 gene and flanking sequences such that a full-length AL2 gene, or a truncated version thereof, containing the desired mutation is synthesized.
  • the mutagenic primer comprises a first sequence of nucleotides that bind to the encoding strand of the AL2 gene immediately upstream of the point of mutation and a second sequence of nucleotides that bind to the encoding strand of the AL2 gene immediately downstream of the point of mutation.
  • the present invention also provides vectors comprising a double domain mutant AL2 gene and vectors comprising a single domain mutant AL2 gene.
  • Vectors comprising mutant AL2 genes are useful for transfecting, preferably transforming plants.
  • the vector DNA is any DNA molecule, such as a plasmid or a bacteriaphage genome which comprises the mutant AL2 gene.
  • the preferred vector-containing organism is a non- tumor inducing bacteria or strain of bacteria, preferably of the family Rizobiacea more preferably Agrobacterium, most preferably Agrobacterium tumefaciens or Agrobacterium rhizogenes. Methods for transforming plants using Agrobacterium tumefaciens are described in Hosrsch et al (1985) A simple and general method for transferring genes into plants. Science 227: 1229-1231; and Rogers et al. (1986) Production of transformed plants using Ti plasmid vectors. Methods in Enzymology 118: 627-640.
  • the mutant AL2 gene flanked by promoter and polyadenylation signals, is contained in a plasmid which is located within the bacterium.
  • the plasmid also contains a drug or antibiotic resistance gene which allows for positive selection of transformants in medium containing the respective drug or antibiotic.
  • Such plasmid is introduced into the bacterium using conventional methods such as for example electroporation or cocultivation.
  • the mutant AL2 gene be inserted into a binary plasmid which is then inserted into the Agrobacterium to provide an Agrobacterium transformant.
  • the present invention also relates to a method of making plants that are more resistant to infection with geminiviruses, particularly Begomoviruses.
  • the method comprises the steps of transforming a plant sample using conventional techniques with a vector comprising a double domain mutant AL2 gene, and generating a transformed plant from the transformed plant sample.
  • the plant sample is obtained from a plant that is a host for Begomovirus.
  • Plants that are susceptible to infection with a Begomovirus include, but are not limited to, beans, squash, peppers, potatoes, cassava, cotton, tobacco, kenaf, tomato, pumpkin, Arabidopsis, and cabbage.
  • the present invention also relates to methods of making a plant that is more susceptible to infection by Begomoviruses and Curtoviruses.
  • the method comprises the steps of transforming a plant sample with a vector comprising a single domain mutant AL2 gene, and generating a transformed plant from the transformed plant sample. Any dicot can be made more susceptible to such infection using such method.
  • Conventional techniques are used for preparing transgenic plants which express the double domain mutant AL2 gene or the single domain mutant AL2 gene.
  • the desired mutant AL2 gene is introduced into the plant sample using an Agrobacterium transformant.
  • the Agrobacterium transformant is cocultivated with plant cells or plant tissues. The Agrobacterium binds to the plant cell walls and transfers the plasmid or a portion thereof into the plant cell.
  • transformation results from the transfer of a specific portion of the plasmid, referred to hereinafter as "T-DNA", into the genome of plant cells.
  • T-DNA is transferred and integrated into the plant genome as a discrete unit.
  • the T-DNA contains a double domain mutant AL2 gene or a single domain mutant AL2 gene which, preferably, is flanked by a promoter and polyadenylation signals.
  • the T-DNA also contains a selectable marker resistance gene, such as a neomycin resistance gene.
  • the transformed plant cells are selected by growth in medium containing the respective drug. Thereafter, transformed plants are regenerated from the cells using conventional techniques and analyzed to ensure that the transformed plant is expressing the mutant AL2 gene.
  • the present invention provides transgenic plants comprising a double domain mutant AL2 gene.
  • Such plants are resistant to infection with geminiviruses, particularly Begomoviruses
  • the term "resistant” means a significant reduction in geminivirus replication in the transgenic plant as compared to a non-transformed plant of the same species when such plants are inoculated with a Begomovirus. Such reduction in replication is accompanied by a significant reduction in disease symptoms in the transgenic plants. Since TrAP function is not virus specific within the Begomoviruses, a transgenic plant comprising a mutant AL2 gene of one Begomovirus is expected to be resistant to infection with all Begomoviruses.
  • the present invention also provides transgenic plants that are more susceptible to infection by a Begomovirus or a Curtovirus.
  • Such plants comprise a single domain mutant AL2 gene.
  • Examples of such plants include non-desirable plants that are employed in field studies. Following completion of the field study the transformed plants are infected with a geminiviurus from the genus Curtovirus or the genus Begomovirus. Such infection kills the plant, thereby ensuring non-propagation of the non-desirable plant.
  • Such plants also include plants that may be used as temporary ground cover or that may be used as a source of additional soil nitrogen. Such plants may also be employed as research tools for the genetic analysis of defense pathways in plants.
  • transgenic plants that comprise a single domain mutant AL2 are used as model systems for identifying micro-organisms, such as for example bacteria and other viruses, whose infectivity is controlled by the SNF-1 kinase defense pathway of the plant.
  • the present invention also provides an isolated nucleic acid encoding the activation domain of TrAP, i.e. from about amino acid 100 to amino acid 129. Such nucleic acid is a useful research tool for analysis of transcriptional activation mechanisms in eukaryotic cells.
  • a single domain mutant of the AL2 gene of TGMV encoding amino acids 1-83 of wild-type TGMV TrAP was made by PCR site-directed mutagenesis using pTGA6 as a template.
  • pTGA6 is made by cloning the TGMV DNA A containing the AL2 gene into the Ec ⁇ Rl site of plasmid pG ⁇ M4, which was obtained from Proem.
  • the wild type TGMV AL2 gene and the mutant AL2 gene fragment were ligated into an two separate expression vectors which encode a GAL4 DNA binding domain (GAL4).
  • GAL4 GAL4 DNA binding domain
  • One vector encodes a fusion protein GAL4: TrAP 1-129 which comprises the wild type TrAP protein fused the GAL4 DNA binding domain.
  • the other vector encodes a fusion protein GAL4:TrAPi-83 which comprises residues 1-83 of TGMV TrAP fused to the GAL4 DNA binding domain.
  • Example 4 Fragment of the 3' Region of an AL2 gene
  • Example 5 Fragment of the 3' Region of an AL2 gene
  • Example 6 Fragment of the 3' Region of an AL2 gene
  • Example 7 Fragment of the 3' Region of an AL2 gene
  • NIH3T3 cell assay system The ability of the fusion proteins encoded by the mutant genes of Examples 1-7 to activate transcription was assessed using an NIH3T3 cell assay system.
  • the expression plasmid comprising the mutant form of the AL2 gene fused in frame to the GAL4 DNA binding domain is cotransfected into mouse fibroblasts (NIH3T3 cells) with an indicator plasmid consisting of the CAT reporter gene driven by a minimal promoter containing multiple copies of the GAL4 upstream activating sequence (UAS), which is specifically recognized by the GAL4 DNA binding domain.
  • UAS GAL4 upstream activating sequence
  • fusion proteins bind the minimal promoter, and their ability to activate transcription from this location is measured as a function of the amount of CAT present in cell extracts.
  • CAT protein levels are quantified by ELISA using an antibody specific to CAT (Boehringer Mannheim Biochemica).
  • fusion protein GAL4:TrAP ⁇ _i29 which comprises full length TrAP fused to the GAL4 DNA binding domain, activated transcription.
  • Fusion proteins lacking the C-terminal acidic domain of TrAP GAL4:TrAPi-83 and GAL4:TrAPi- 100
  • fusion proteins containing portions of the C-terminal acidic domain of TrAP GAL4:TrAP83-114.
  • GAL4:TrAP83-125, GAL4:TrAP83-120, GAL4:TrAP120-129) were unable to activate transcription from the minimal promoter.
  • TrAP has an acidic activation domain which encompasses the C-terminal region of the protein.
  • the sequence of this region is EESIGSPQGISQLPSMDDIDDSFWENLFK. SEQ ID NO: 14.
  • SEQ ID NO: 14 These results also indicate that the carboxy terminal 15 amino acids (115-129) of TrAP are sufficient to activate CAT expression from the indicator plasmid. Fusion proteins containing the C-terminal acidic region of TrAP but lacking amino acids 1-41 thereof were at least four times more active than fusion proteins containing full length TrAP.
  • DNA molecules encoding the C-terminal region of TGMV TrAP i.e. either amino acids 81-129 of TGMV TrAP or amino acids 117-129, were used to determine the effect of substitutions in the C terminal region of TrAP on the ability of this domain to activate transcription.
  • Substitutions in the nucleic acid fragment encoding the C-terminal region of TrAP were made by PCR site-directed mutagenesis using pTGA6 as a template and the primers shown in Fig. 2
  • One substituted fragment which has an alanine rather than an aspartic acid at position 117 was ligated into an expression plasmid containing a sequence encoding the GAL4 DNA binding domain (GAL4) to produce fusion protein, GAL4:D117-A.
  • GAL4 GAL4 DNA binding domain
  • a DNA molecule which encodes fusion protein GAL4:D118-A was made as described as above in example 8.
  • the fusion protein encoded by this molecule comprises a C terminal region of TGMV TrAP in which the aspartic acid at position 118 is substituted with an alanine.
  • Example 10 Substituted C Terminal Region of TGMV TrAP
  • a DNA molecule which encodes fusion protein GAL4:I119-A was made as described as above in example 8.
  • the fusion protein encoded by this molecule comprises a C terminal region of TGMV TrAP in which the isoleucine at position 1 lp is substituted with an alanine.
  • Example 11 Substituted C Terminal Region of TGMV TrAP
  • a DNA molecule which encodes fusion protein GAL4:D120-A was made as described as above in example 8.
  • the fusion protein encoded by this molecule comprises a C terminal region of TGMV TrAP in which the aspartic acid at position 120 is substituted with an alanine.
  • Example 12 Substituted C Terminal Region of TGMV TrAP A DNA molecule which encodes fusion protein GAL4:D121-A was made as described as above in example 8. The fusion protein encoded by this molecule comprises a C terminal region of TGMV TrAP in which the aspartic acid at position 121 is substituted with an alanine.
  • Example 13 Substituted C Terminal Region of TGMV TrAP A DNA molecule which encodes fusion protein GAL4:S122-Awas made as described as above in example 8. The fusion protein encoded by this molecule comprises a C terminal region of TGMV TrAP in which the serine at position 122 is substituted with an alanine.
  • Example 14 Substituted C Terminal Region of TGMV TrAP
  • a DNA molecule which encodes fusion protein GAL4:F123-A was made as described as above in example 9.
  • the fusion protein encoded by this molecule comprises a C terminal region of TGMV TrAP in which the phenylalanine at position 123 is substituted with an alanine.
  • Example 15 Substituted C Terminal Region of TGMV TrAP
  • a DNA molecule which encodes fusion protein GAL4:W124-A was made as described as above in example 8.
  • the fusion protein encoded by this molecule comprises a C terminal region of TGMV TrAP in which the tryptophan at position 124 is substituted with an alanine.
  • Example 16 Substituted C Terminal Region of TGMV TrAP
  • a DNA molecule which encodes fusion protein GAL4:E125-A was made as described as above in example 8.
  • the fusion protein encoded by this molecule comprises a C terminal region of TGMV TrAP in which the glutamic acid at position 125 is substituted with an alanine.
  • Example 17 Substituted C Terminal Region of TGMV TrAP
  • a DNA molecule which encodes fusion protein GAL4:F128-A was made as described as above in example 8.
  • the fusion protein encoded by this molecule comprises a C terminal region of TGMV TrAP in which the phenylalanine at position 128 is substituted with an alanine.
  • Example 18 Substituted C Terminal Region of TGMV TrAP
  • a DNA molecule which encodes fusion protein GAL4:D117,118-A was made as described as above in example 8.
  • the fusion protein encoded by this molecule comprises a C terminal region of TGMV TrAP in which the aspartates at positions 117 and 118 are each substituted with an alanine.
  • Example 19 Substituted C Terminal Region of TGMV TrAP
  • a DNA molecule which encodes fusion protein GAL4:D120,121-A was made as described as above in example 8.
  • the fusion protein encoded by this molecule comprises a C terminal region of TGMV TrAP in which the aspartates at position 120 and 121 are each substituted with an alanine.
  • the yeast two-hybrid system was used to identify proteins that bind to the amino terminus and central region of TrAP. Specifically, the system was used to screen an Aribidopsis cDNA library for such proteins. A truncated form of TrAP, TrAP 1-83 was used as bait because full-length TrAP activates transcription by itself. From this screen, a cDNA encoding a homologue of yeast SNF-1 kinase was isolated. Two-hybrid analysis showed that TrAP specifically interacts with the Arabidopsis SNF-1 kinase and yeast SNF-1 kinase, but not with other TGMV proteins or other cellular negative control proteins.
  • TrAP SNF-1 interaction domain of TrAP was located to amino acids 23-43. This region contains the first four conserved cysteine and histidine residues (C-X1-C-X4-H-X2-C) of the central region.
  • L2 protein of the Curtovirus BCTV also interacts with SNF-1 kinase.
  • TRAP and L2 protein are homologous in the CCHC motif indicated above.
  • the cysteine and histidine residues comprising this motif are found at positions 33, 35, 40, and 43 in TrAP.
  • PCR site-directed mutagenesis was used to replace the cysteine and histidine residues at these positions with alanine.
  • the mutagenic primers used were: C33: 5'-gacctgaacgctggctgttcc-3'; SEQ ID NO: 15 C35: 5'-gaactgtggcgcttccatatac-3'; SEQ ID NO: 16 H40: 5'-ccatatacattgccatcgactgc-3'; SEQ ID NO: 17 C43: 5'-cacatcgacgccagaaacaatgg-3', SEQ ID NO: 18.
  • Mutations were made using these primers and the Muta-gene in vitro Mutagenesis Kit from Bio-Rad.
  • Template DNA used in the mutagenesis consisted of the TGMV AL2 open reading frame cloned into Ml 3m.pl 8RF DNA. Ml 3 phage containing the desired mutations were confirmed by sequencing. Single-stranded DNA was then used as template for PCR reactions which amplified the mutant AL2 ORF. Primers used in these reactions were as follows:
  • 35S promoter for subsequent expression in plant cells.
  • mutant AL2 genes were amplified by PCR using as 5' primer: 5' gcgggcgccatgcgaaattcgtcttcc-3', SEQ ID NO: 21 and as 3' primer: 5' cgcgagctcctatttaaataagttctccca-3', SEQ ID NO: 22.
  • the resulting PCR products were then digested with BamHl and Ehel and cloned into pAS2 and pACT2 for testing in the yeast two hybrid system.
  • TrAP H40A which comprises an alanine at position 40 rather than a histidine lost the ability to interact with SNF-1 kinase.
  • TrAP C33A, and C43A were still able to interact with SNF-1 kinase.
  • Example 21 Transgenic Plants Comprising a Single Domain Mutant AL2 Gene
  • Transgenic N. benthamiana plants comprising single domain mutant AL2 genes were made using an A. tumefaciens transformant that was prepared using a binary system.
  • the transgenic plants contained a single domain mutant AL2 gene which encoded one of the following TrAP proteins: TrAP F123A, TrAP W124A, TrAP F128A, TrAP 1-115, TrAP 1- 120, TrAP- 1-125 and, TrAPl-83.
  • Transgenes were constructed by PCR.
  • the 5' primer employed in all cases was 5'GCGAGATCTATGCGAAATTCGTCTTCC-3', SEQ ID NO: 23. which is complementary to a sequence at the 5' end of the TGMV AL2 gene.
  • Template DNA employed in the PCR reaction was pTGA6.
  • the binary system consists of two components: 1) a disarmed A. tumefaciens Ti plasmid, which provides functions required for excision of the T-DNA from the Ti plasmid, and for its transfer and integration into the plant genome and 2) a second, smaller binary plasmid vector of about 6 - 10 kb that contains the T-DNA to be transferred, i.e. the mutant AL2 gene and a drug resistance gene. Cultures of E. coli harboring the binary vectors were grown in LB broth containing
  • the binary vectors contained the mutant AL2 genes shown in Table III below and a selectable marker. Most of the binary vectors also included a streptomycin/spectinomycin resistance gene (outside the T-DNA) for selection in E. coli and A. tumefaciens.
  • the binary vectors used were proprietary vectors obtained from Agritope, Inc.
  • pMON521 contains the 35 S promoter. The three different Agritope vectors used are designed to express the transgene at low, moderate, and high levels.
  • Non-transformed A. tumefaciens containing the resident, disarmed Ti plasmid was obtained from Monsanto. Cultures of the non-transformed A. tumefaciens were grown overnight in LB broth containing 25 ⁇ g/ml chloramphenicol and 50 ⁇ g/ml kanamycin. Cultures of E. coli containing the mobilization plasmid (e.g. pRK2013) was grown overnight in LB broth containing 50 ⁇ g/ml kanamycin. Cultures of E. coli containing the binary plasmid were grown overnight in LB broth containing 50 ⁇ g/ml spectinomycin.
  • the plates were incubate at 28°C until colonies were detected on the plates. Several individual colonies were recovered from each plate and inoculated into LB broth containing the same antibiotics as the plate and incubate at 28°C with shaking.
  • A. tumefaciens transformants were also used to transform leaf discs from Nicotiana benthamiana plants. For petunia and tobacco leaf discs are also used. For tomato, cotyledon pieces are used. For Arabidopsis thaliana, sterile root pieces are used. Prior to transformation, the plant tissues were sterilized in a solution of 20% chlorox, 0.5% Tween 20 for 15 min.
  • the leaf discs were placed upside down on MS 104 plates and preincubated for 48 hours at room temperature in continuous light to increase the transformation efficiency
  • the sterilized leaf discs were soaked in a liquid culture of an A. tumefaciens transformant. This is done by placing 10-20 discs in a sterilin tube (with a loose cap) and adding 1 ml of an overnight culture. The discs were then removed, blotted dry with sterile filter paper, and placed upside down on an MS 104 plate with no selection. After 48 hr on the non-selective MS 104 media at room temperature, the discs were transferred to MS 104 plates containing selection medium (750 ⁇ g/ml carbenicillin to kill the Agrobacterium and 300 ⁇ g/ml kanamycin to select for the desired T-DNA marker). After about 1-2 weeks, the discs form callus around the edges of the disc. This is followed by the appearance of shoots.
  • selection medium 750 ⁇ g/ml carbenicillin to kill the Agrobacterium and 300 ⁇ g/ml kanamycin to select for the desired T-DNA marker.
  • the presence of an integrated AL2 gene in the genome of the plant is examined by restriction endonuclease digestion followed by Southern blot analysis, or by PCR using primers designed to recognize T-DNA border sequences
  • Expression of RNA encoding the mutant AL2 gene is examined by Northern blot analysis or by RT-PCR.
  • Expression of mutant TrAP is examined by Western blotting.
  • Transgenic lines are established by selfing transformed plants to homozygosity using conventional techniques.
  • C Susceptibility of the transgenic plants to infection with Begomoviruses
  • Transgenic plants made as described above and comprising the mutant AL2 genes shown in Table III were tested by challenge inoculation with TGMV and BCTV.
  • viruses were delivered to plants by the agroinoculation procedure described in Elmer et al. (1988) Agrobacterium-mediated inoculation of plants with tomato golden mosaic virus DNAS. Plant Mol. Biol. 10:225-234.
  • the virus was mechanically inoculated. Specifically, pUC based plasmid DNAs containing tandem repeats of TGMV DNA A and TGMV DNA B were mixed and mechanically inoculated into plant leaves as a DNA solution with the aid of celite abrasive.
  • TrAPl-83 a truncated form of TrAP, i.e., TrAPl-83, which was under control of the strong and constitutive 35S promoter from cauliflower mosaic virus.
  • Transgenic plants comprising this mutant gene exhibited a normal phenotype until inoculated with TGMV and BCTV. Following inoculation these transgenic plants showed an enhanced susceptibility phenotype, characterized by a much reduced latent period and by infection at much lower inoculum doses that non-transformed plants of the same species. Plants transfected with a full-length wild-type AL2 gene died prior to inoculation. It is believed that the enhanced susceptibility of the transgenic plants is due to interaction of the modified TrAP with SNF1 kinase.
  • Another mutant gene used for transformation encode a full-length TrAP protein in which the tryptophan at position 124 in the wild-type sequence is replaced with an alanine.
  • the transgenic plant comprising this single domain mutant AL2 gene also exhibited enhanced susceptibility to infection.
  • Example 22 A double domain mutant AL2 gene encoding a mutant TrAP having an amino acid substitution in the central Region (H40A) and an amino acid substitution in the C terminal Region (F 123 A) .
  • the double domain mutant was made in two steps.
  • a single domain mutant AL2 gene comprising a variant sequence which encodes a mutant TrAP having an alanine at position 40 rather than the histidine which is found at position 40 in the wild-trap TrAP was made.
  • the single domain mutant was made using the H40 mutagenic primer, SEQ ID NO 17, of example 21 and the Muta-Gene in vitro mutagenesis kit from Bio-Rad. Template DNA used in the mutagenesis consisted of the TGMV AL2 open reading frame cloned into M13mpl8RF DNA.
  • the AL2 gene cloned in the vector was first transformed into a dut-/ung- E.
  • coli strain and single-stranded phage DNA containing uracil was isolated.
  • the phage DNA was annealed with the mutagenic primer (in this case H40A, SEQ ID NO: 17), and the second strand was synthesized with T7 DNA polymerase and T7 DNA ligase.
  • the double-stranded DNA was transformed into an ung+ E. coli strain (which degrades uracil-containing DNA); consequently the parental strand was not replicated and the strand synthesized using the mutagenic primer was preferentially amplified.
  • Ml 3 phage containing the desired H40A mutation were selected and confirmed by sequencing.
  • a second mutation in the mutant H40A mutant AL2 gene was carried out by PCR amplification of the mutant H40 AL2 gene, using a wild type 5' primer (618A-5'; Figure 2, SEQ ID NO: 26 ) and a 3' primer (620-3'; Figure 2, SEQ ID NO: 31) designed to create a full-length AL2 gene containing the F123A substitution.
  • the double mutant AL2 gene encodes a full-length mutant TrAP having an alanine at position 40 in the amino acid sequence rather than a histidine and an alanine at position 123 rather than a phenylalanine
  • Example 23 A double domain mutant AL2 gene encoding a mutant TrAP having a deletion within the central (delta 33-43) region and a deletion within the C terminal region (delta 115- 129).
  • the double domain mutant AL2 gene comprising a variant sequence which encodes a TrAP protein lacking amino acids 33 through 43 of the central region, and lacking amino acids 115 through 129 of the C terminal region were made by bidirectional PCR.
  • a wt 5' primer (618 A; Figure 2, SEQ ID NO: 26) was used in combination with 3' primer (CCHC-1 ; GTTCAGGTCAATTCGTCGCCT).
  • This primer combination permits amplification of the AL2 sequence encoding amino acids 1 through 32.
  • CCHC-2 AGAAACAATGGATTCACGCAC
  • SEQ ID NO: 37 Figure 2 was used as 3' primer.
  • This primer combination permits amplification of the AL2 sequence encoding amino acids 44 through 114.
  • the products of the two reactions were ligated together with a plasmid cloning vector (pUC) and transformed into E. coli. Plasmids containing the desired double domain mutant AL2 gene, encoding amino acids 1-32 and 44-114, were selected.
  • the double domain mutant AL2 gene encodes a mutant TrAP which lacks amino acids 33 through 43 and amino acids 115 through 129 of the wild-type TrAP.
  • Example 24 Transgenic Plants Comprising the Double Domain Mutant AL2 Genes of Example 22 and 23
  • the mutant AL2 genes of Examples 22 and 23 are incorporated into an Agrobacterium vector as described in Example 21 and used to prepare transgenic plants as described in Example 21.

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Abstract

L'invention concerne des gènes AL2 mutants, à double domaine, isolés d'un geminivirus. Le gène AL2 mutant à double domaine présente au moins une mutation dans la région 3' du gène AL2, soit une région qui code environ 30 acides aminés allant environ de l'acide aminé 100 à l'acide aminé 129 environ du produit génique AL2 de type sauvage, et au moins une mutation dans la région centrale du gène AL2, soit dans la région qui code environ 20 acides aminés allant environ de l'acide aminé 23 à l'acide aminé 43 environ du produit génique AL2 de type sauvage. L'invention concerne également des gènes mutants AL2 à un seul domaine d'un geminivirus. Ces gènes AL2 mutants à un seul domaine présentent au moins une mutation dans la région 3' du gène AL2, une région qui code environ 30 acides aminés compris entre l'acide aminé 100 et l'acide aminé 129 environ du produit génique AL2 de type sauvage. L'invention concerne en outre des vecteurs comprenant un gène AL2 mutant à double domaine et sur des vecteurs comprenant un gène AL2 mutant à un seul domaine. L'invention concerne également des plantes transgéniques comprenant un gène AL2 mutant à double domaine et des plantes transgéniques comprenant un gène AL2 mutant à un seul domaine.
PCT/US1999/012680 1998-06-05 1999-06-04 Formes mutantes du gene al2 des geminivirus Ceased WO1999063054A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2806095A1 (fr) * 2000-03-10 2001-09-14 Gentech Sequences polynucleotidiques purifiees de plantes et de levure codant pour des proteines qui interagissent avec les produits du genome des geminivirus
CN102640642A (zh) * 2012-04-25 2012-08-22 浙江大学 用含双生病毒侵染性克隆的固体菌落扎刺接种植物的方法及其应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GILLETTE ET AL.: 'Genetic Determinants of Host-Specificity in Bipartite Geminivirus DNA A Components' VIROLOGY vol. 251, 1998, pages 361 - 369, XP002927219 *
HARTITZ ET AL.: 'The Tomato Golden Mosaic Virus Transactivator (TrAP) is a Single-Stranded DNA and Zinc-Binding Phosphoprotein with an Acidic Activation Domain' VIROLOGY vol. 263, 1999, pages 1 - 14, XP002927222 *
SUNG ET AL.: 'Mutational Analysis of Potato Yellow Mosaic Geminivirus' J. GEN. VIROL. vol. 76, 1995, pages 1773 - 1780, XP002927220 *
SUNTER ET AL.: 'Regulation of a Geminivirus Coat Protein Promoter by AL2 Protein (TraP): Evidence for Activation and Derepression Mechanisms' VIROLOGY vol. 232, 1997, pages 269 - 280, XP002927221 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2806095A1 (fr) * 2000-03-10 2001-09-14 Gentech Sequences polynucleotidiques purifiees de plantes et de levure codant pour des proteines qui interagissent avec les produits du genome des geminivirus
WO2001068863A3 (fr) * 2000-03-10 2002-06-20 Gentech S A R L Sequences polynucleotidiques purifiees de plantes et de levure codant pour des proteines qui interagissent avec les produits du genome des geminivirus
CN102640642A (zh) * 2012-04-25 2012-08-22 浙江大学 用含双生病毒侵染性克隆的固体菌落扎刺接种植物的方法及其应用

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