WO2004065557A2 - Aptameres peptidiques qui se lient aux proteines rep des virus ssdna - Google Patents

Aptameres peptidiques qui se lient aux proteines rep des virus ssdna Download PDF

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WO2004065557A2
WO2004065557A2 PCT/US2004/001564 US2004001564W WO2004065557A2 WO 2004065557 A2 WO2004065557 A2 WO 2004065557A2 US 2004001564 W US2004001564 W US 2004001564W WO 2004065557 A2 WO2004065557 A2 WO 2004065557A2
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seq
plant
virus
polypeptide
plant cell
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WO2004065557A3 (fr
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Linda Hanley-Bowdoin
Luisa Lopez Ochoa
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North Carolina State University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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

Definitions

  • the present invention relates to products and methods for detecting viral infections and inhibiting viral replication.
  • Single-stranded DNA (ssDNA) viruses cause severe disease problems in plants and animals (Moffat (1999) Science 286:1835). Geminiviruses and nanoviruses infect many important crops worldwide, such as cassava, bean, pepper, tomato, sugar beet, cotton and maize (Brown and Bird (1992) Plant Disease 7:220- 225; Czosnek and Laterrot (1997) Arch. Virol. 142:1391-1406; Lotrakul, et al. (1998) Plant Dis. 82:1253-1257; Zhou, et al. (1997) J. Gen. Virol. 78:2101-2111 ; ansoor, et al.
  • Circoviruses cause significant disease losses among livestock and poultry (Allan, et al. (1998) J. Vet. Diagn. Invest. 10:3-10; Bassami, et al. (1998) Virology 249:453-9; Nayar, et al. (1999) Can. Vet. J. 40:277-8).
  • a human circovirus in Hepatitis C patients has also been identified (Miyata, et al. (1999) J. Virol. 73:3582-3586 ; Mushahwar, et al. (1999) Proc. Natl. Acad. Sci. USA. 96:3177-3182). Even though these viruses have diverse host ranges and cause different diseases, they are highly related to each other.
  • Tomato Yellow Leaf Curl Virus (TYLCV) resistance genes have been introgressed from a wild relative into tomato breeding lines (Vidavsky and Czosnk
  • Transgenic resistance strategies have also met with limited success. Unlike plant RNA viruses, the introduction of geminivirus sequences into transgenic plants does not interfere with infection. There is one report of transgenic tomatoes that contain a geminivirus coat protein gene and display virus tolerance (Kunik, et al. (1994) BioTechnology 12:500-504); however, this result has not been reproduced (Azzam, et al. (1996) Annu. Rep. Bean Impr ⁇ v. Coop. 39:276-277; Sinisterra, et al.
  • RNAs similar in size to RNAi have been detected in geminivirus-infected cells and tissues (Lucioli et al., (2003) J. Virol. 77:6785-6798; Vanitharani et al., (2003) Proc. Natl. Acad. Sci USA 100:9632-9636), suggesting that geminivirus infection is not blocked by the silencing process.
  • Antisense RNA and defective-interfering replicon strategies have also been of limited success, resulting in some reduction of viral DNA and amelioration of symptoms (Stanley, et al. (1990) Proc. Natl. Acad. Sci. USA 87:6291-6295; Day, et al. (1991 ) Proc. Natl. Acad. Sci. USA 88:6721-6725; Frischmuth and Stanley (1994) Virology 200:826-830; Bendahmane and Gronenborn (1997) Plant Mol. Biol. 33:351-357; Frischmuth, et al. (1997) Mol. Breed. 3:213-217; Aragao, et al. (1998) Mol. Breed. 4:491-499).
  • Transgenic plants that inducibly express dianthin upon geminivirus infection also display resistance (Hong, et al. (1996) Virology 220:119-127), but the safety of a toxic ribosome-inactivating protein has not been established. Expression of mutant geminivirus movement proteins in transgenic plants also results in resistance, but the phenotype is variable (Pascal, et al. (1993) supra; Duan, et al. (1997) Mol. Plant Microbe Interact. 10:1065-74; Duan, et al. (1997) Mol. Plant Microbe Interact. 10:617-623; Hou, et al. (2000) Mol. Plant Microbe Interact. 13:297- 308).
  • plant ssDNA viruses occur as mixed infections.
  • geminivirus complexes are responsible for bean golden mosaic disease and geminivirus/nanovirus combinations cause cotton leaf curl and Ageratum yellow vein diseases (Mansoor, et al. (1999) Virology 259:190-199; Saunders and Stanley (1999) Virology 264:142-152).
  • This complexity is compounded by the rapid evolution of ssDNA viruses (Navas-Castillo, et al. (2000) supra; Monci, et al. (2002) supra; Padidam, et al. (1999) Virology 265:218-225).
  • Geminiviruses, nanoviruses, and circoviruses amplify their circular ssDNA genomes via a rolling circle mechanism through the combined action of a single viral protein, Rep, and the host DNA replication machinery (Laufs, et al. (1995) Biochimie 77:765-773; Mankertz, et al. (1997) J. Virol. 71:2562-2566; Katul, et al. (1998) J. Gen. Virol. 79:3101-3109; Mankertz, et al. (1998) J. Gen. Virol. 79:381-384; Hanley- Bowdoin, et al. (1999) Cht. Rev. Plant Sci. 18:71-106).
  • Rep initiates plus-strand DNA synthesis by cleaving the viral origin within a hairpin structure at an invariant sequence, acts as a DNA ligase to terminate rolling circle replication, and hydrolyzes ATP. Because of the functional conservation, Rep proteins from all three ssDNA virus families are highly homologous.
  • TGMV tomato golden mosaic virus
  • TGMV Rep is a multifunctional protein that mediates both virus-specific recognition of its cognate origin (Fontes, et al. (1994) J. Biol. Chem. 269:8459-8465) and transcriptional repression (Eagle, et al. (1994) Plant Cell 6:1157-1170; Eagle and Hanley-Bowdoin (1997) J. Virol. 1 :6947-6955).
  • Rep initiates and terminates plus-strand replication within a conserved hairpin motif (Laufs, et al.
  • Rep mutants have proven to be effective in interfering with geminivirus replication. Mutations in motif III, the ATP binding site and the oligomerization domain ( Figure 1) interfere with wild-type virus replication in transient assays (Orozco, et al. (2000) J Biol. Chem. 275:6114-6122; Hanson and Maxwell (1999) Phytopathology 89:480-486; Chatterji, et al. (2001 ) J Biol. Chem. 276:25631- 25638). However, efforts to produce mutant Rep proteins from transgenes have been less successful. Another strategy has been to express truncated versions of Rep in plants (Noris, et al.
  • Aptamers are small peptide or nucleic acid molecules that specifically recognize and bind proteins. When targeted to residues critical for function, aptamers can inactivate a protein of interest and interfere with cellular processes.
  • an aptamer that binds to the active site of the cell cycle regulator, cdk2 was isolated by screening a combinatorial peptide library in yeast dihybrid assays (Colas, et al. (1996) Nature 380:548-550). The peptide blocks cdk2/cyclin E kinase activity in vitro and, when expressed in vivo, retards cell division (Cohen, et al.
  • Single-stranded DNA (ssDNA) viruses cause severe disease problems in plants and animals. Geminiviruses and nanoviruses infect many important plant crops worldwide, whereas circoviruses cause significant disease losses among livestock and poultry. Even though these viruses have diverse host ranges and cause different diseases, their replication initiation proteins (Rep) are highly related to each other.
  • the present invention can be used to develop a broad-based strategy for interference against eukaryotic viruses, in particular, eukaryotic ssDNA viruses.
  • a first aspect of the invention provides a polypeptide comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 , SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61 , SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO
  • Additional aspects of the invention provide an isolated nucleic acid comprising a nucleotide sequence encoding the polypeptide of the invention.
  • a further aspect of the present invention provides a vector or a cell comprising an isolated nucleic acid comprising a nucleotide sequence encoding the polypeptide comprising an amino acid sequence as recited above.
  • transgenic plant comprising transformed plant cells, the transformed plant cells comprising the isolated nucleic acids of the invention.
  • transgenic plants having increased resistance to a virus infection comprising providing a plant cell capable of regeneration, transforming the plant cell with an isolated nucleic acid comprising an isolated nucleic acid as described above, and regenerating a transgenic plant from said transformed plant cell, wherein expression of the isolated nucleic acid to produce the polypeptide increases resistance of the transgenic plant to infection by a virus.
  • Additional methods of making transgenic plants having increased resistance to a virus infection comprise introducing an isolated nucleic acid, as recited above, into a cell to produce a transgenic plant, wherein expression of the isolated nucleic acid to produce the polypeptide increases resistance of the transgenic plant to infection by a virus.
  • Another aspect of the present invention provides a method of inhibiting viral replication in a plant cell comprising introducing an isolated nucleic acid, as recited above, into the plant cell in an amount effective to inhibit virus replication.
  • the present invention further provides methods of detecting a viral infection, comprising: (a) contacting a sample with a polypeptide as recited above or a fusion protein as recited above; and (b) detecting the presence or absence of binding between the polypeptide and a target, wherein the binding of the polypeptide to the target in the sample indicates the presence of a virus.
  • FIG. 1 shows the results of initial aptamer replication interference assays. Tobacco protoplasts were cotransfected with the TGMV A replicon pMON1565 (2 ⁇ g) and the indicated plant expression cassettes (40 ⁇ g). Viral replication was analyzed at three days post-transfection on DNA gel blots. Each assay was performed in triplicate.
  • RepM13 is a TGMV Rep mutant that strongly interferes with replication in protoplasts and plants.
  • Two Trx-aptamer cassettes, A42 and C1 :60 (striped bars), interfere with replication to a similar extent as RepM13.
  • the error bars represent two standard errors.
  • Figure 3 depicts proteins used as bait in peptide aptamer screens.
  • TGMV AL1 is represented by the striped rectangle with open boxes corresponding to motifs I, II and III of the DNA cleavage domain and the NTP binding motif of the helicase domain. The oval is the LexA DNA binding domain.
  • Figure 4 shows groups 1-9 (Panels A-l), respectively, of aptamer sequences aligned and classified using Vector NT. Positions showing greater than 50% conservation of closely related (uppercase) or weakly similar (lowercase) amino acids are underlined in the alignments and are indicated as the consensus sequence. The brackets denote each position within consensus sequences with the amino acids shown in order of frequency with the most frequent shown first. Numbers to the left of each sequence indicate the clone number.
  • Figure 5 shows gel blot analysis of replication interference assays with heterologous geminiviruses.
  • the double-stranded (ds) DNA product is indicated and the relative accumulation in each panel is provided at the bottom.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragment thereof, and to naturally occurring or synthetic molecules. Where "amino acid sequence” is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence,” and like terms, are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
  • a “functional fragment” of an amino acid sequence as used herein refers to portion of the amino acid sequence that retains at least one biological activity normally associated with that amino acid sequence.
  • a “functional variant” of an amino acid sequence as used herein refers to no more than one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions in the sequence of interest.
  • the functional variant retains at least one biological activity normally associated with that amino acid sequence.
  • the "functional variant” retains at least about 40%, 50%, 60%, 75%, 85%, 90%, 95% or more biological activity normally associated with the full-length amino acid sequence.
  • a “functional variant” is an amino acid sequence that is at least about 60%, 70%, 80%, 90%, 95% 97% or 98% similar to the polypeptide sequence disclosed herein (or fragments thereof).
  • Polypeptide as used herein, is used interchangeably with “protein,” and refers to a polymer of amino acids (dipeptide or greater) linked through peptide bonds.
  • polypeptide includes proteins, oligopeptides, protein fragments, protein analogs and the like.
  • polypeptide contemplates polypeptides as defined above that are encoded by nucleic acids, are recombinantly produced, are isolated from an appropriate source, or are synthesized.
  • Fusion protein refers to a protein produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different polypeptides, or fragments thereof, are fused together in the correct translational reading frame.
  • the two or more different polypeptides, or fragments thereof include those not found fused together in nature or include naturally occurring mutants.
  • isolated nucleic acid refers to a nucleic acid separated or substantially free from at least some of the other components of the naturally occurring organism or virus, such as for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the nucleic acid.
  • an "isolated" polypeptide means a polypeptide that is separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.
  • Vector refers to a viral or non-viral vector that is used to deliver a nucleic acid to a cell, protoplast, tissue or subject.
  • Transgenic refers a plant that comprises a foreign nucleic acid incorporated into the genetic makeup of the plant, such as for example, by stable integration into the nuclear genome.
  • Plant cell refers to plant cells, plant protoplasts and plant tissue cultures, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants, such as leaves, pollen, embryos, cotyledon, hypocotyl, roots, root tips, anthers, flowers and parts thereof, ovules, shoots, stems, stalks, pith, capsules, and the like.
  • “Resistance to a virus infection” refers to the reduced susceptibility of a plant or animal subject to viral infection as compared with a control susceptible plant or animal subject under conditions of infestation. “Resistance” can refer to reduced onset, severity, duration and/or spread of viral infection.
  • a polypeptide comprises, consists essentially of or consists of: (a) the amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 , SEQ ID NO:42, SEQ ID NO-43, SEQ ID NO-44, SEQ ID NO-45, SEQ ID NO-46, SEQ ID NO-47, SEQ ID NO-48, SEQ ID NO-49, SEQ ID NO:50, SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO-60, SEQ ID NO:61 , SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO-66, SEQ ID NO-68, SEQ ID NO:
  • polypeptides of the invention encompass those amino acids that have at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or higher amino acid sequence similarity with the polypeptide sequences specifically disclosed herein (or fragments thereof).
  • sequence identity and/or similarity can be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2, 482 (1981 ), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35, 351-360 (1987); the method is similar to that described by Higgins & Sharp, CABIOS 5, 151 -153 (1989).
  • BLAST is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993).
  • a particularly useful BLAST program is the WU- BLAST-2 program which was obtained from Altschul et al., Methods in Enzymology, 266, 460-480 (1996); http://blast.wustl/edu/blast/ README.html.
  • WU-BLAST-2 uses several search parameters, which are preferably set to the default values. The parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values can be adjusted to increase sensitivity.
  • a percentage amino acid sequence identity value can be determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region.
  • the "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
  • the alignment can include the introduction of gaps in the sequences to be aligned.
  • sequences which contain either more or fewer amino acids than the polypeptides specifically disclosed herein it is understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical amino acids in relation to the total number of amino acids. Thus, for example, sequence identity of sequences shorter than a sequence specifically disclosed herein, will be determined using the number of amino acids in the shorter sequence, in one embodiment. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as, insertions, deletions, substitutions, etc.
  • the present invention also encompasses functional fragments of the polypeptides disclosed herein.
  • a functional fragment of an amino acid sequence recited above retains at least one of the biological activities of the unmodified sequence, for example, binding to the Rep protein and/or inhibiting viral replication.
  • the functional fragment of the amino acid sequence retains all of the activities possessed by the unmodified sequence.
  • By “retains" biological activity it is meant that the amino acid sequence retains at least about 10%, 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the native amino acid sequence (and can even have a higher level of activity than the native amino acid sequence).
  • a "non-functional" amino acid sequence is one that exhibits essentially no detectable biological activity normally associated with the amino acid sequence (e.g., at most, only an insignificant amount, e.g., less than about 10% or even 5%).
  • the invention further provides functional variants of the polypeptides disclosed herein.
  • a functional variant of an amino acid sequence recited above has no more than one, two, three, four, five or six amino acid substitutions in the amino acid sequence of interest.
  • one, more than one, or all of the amino acid substitutions are conservative substitutions.
  • amino acid substitutions facilitate binding affinity of the polypeptide to the Rep protein and/or improve inhibitory properties.
  • a functional variant has no more than 1 , 2, 3, 4, 5 or 6 amino acid substitutions, insertions and/or deletions in the amino acid sequence of interest.
  • polypeptides containing such deletions or substitutions are a further aspect of the present invention.
  • polypeptides containing substitutions or replacements of amino acids one or more amino acids of a polypeptide sequence may be replaced by one or more other amino acids wherein such replacement does not affect the function of that sequence.
  • Ala may be replaced with Val or Ser; Val may be replaced with Ala, Leu, Met, or lie, preferably Ala or Leu; Leu may be replaced with Ala, Val or lie, preferably Val or lie; Gly may be replaced with Pro or Cys, preferably Pro; Pro may be replaced with Gly, Cys, Ser, or Met, preferably Gly, Cys, or Ser; Cys may be replaced with Gly, Pro, Ser, or Met, preferably Pro or Met; Met may be replaced with Pro or Cys, preferably Cys; His may be replaced with Phe or Gin, preferably Phe; Phe may be replaced with His, Tyr, or Trp, preferably His or Tyr; Tyr may be replaced with His, Phe orTrp, preferably Phe or Trp; Trp may be replaced with Phe or Tyr, preferably Tyr; Asn may be replaced with Gin or Ser, preferably Gin; Gin may be replaced with His, Lys, Glu, Asn, or Ser, preferably Asn or Ser; Ser may be replaced with Gin,
  • the polypeptides bind anywhere in the Rep protein. In some other embodiments, the polypeptide binds to the catalytic domain for DNA cleavage of the Rep protein. Binding of the polypeptide to the Rep protein can occur in one or more DNA cleavage motifs (Motif I, Motif II and Motif III) located in the Rep N-terminus ( Figure 1). In certain embodiments, the polypeptide binds to Motif III within the catalytic domain for DNA cleavage. In other representative embodiments, the polypeptide binds to the DNA binding domain of the Rep protein or any other conserved region of the Rep protein (e.g., the N-terminal portion). In still other embodiments, the polypeptide binds to the Rep protein and further inhibits viral replication.
  • DNA cleavage motifs Motif I, Motif II and Motif III
  • the polypeptides and fusion proteins can bind to a viral Rep protein and optionally inhibit replication and/or infection.
  • the viruses can include any single- stranded eukaryotic DNA virus employing a rolling circle replication mechanism.
  • the virus is a plant pathogen or an animal pathogen.
  • the virus is a geminivirus, a nanovirus, or a circovirus.
  • viral infection can be caused by a combination of viruses, i.e., is a mixed infection.
  • Infectious clones for a variety of geminiviruses, nanoviruses and circoviruses are available in the art. See, e.g., Table 3, which provides sequences for geminivirus, nanovirus and circovirus type members. The nucleic acid sequences of other infectious clones are available at http://www.ncbi.nlm.nih.gov/ICTVdb/lctv/fr-fst- q.htm.
  • Circovirid ⁇ e Circovirus Porcine circovirus type I (PCVl) 00.016.0.01.001 NKEYCSKEGH U49186 Gyrovirus Chicken anaemia vims (CAV) 00.016.0.02.001 NLTYVSKIGG M55918
  • BBTV B ⁇ buvirus Banana bunchy top virus
  • Motif III sequences of virus type members Rep proteins Motif III sequences of Rep proteins from geminiviruses, nanoviruses and circoviruses are shown. The invariant Y and K residues are in bold type.
  • Geminiviruses are subgrouped as begomoviruses, curtoviruses, topocuviruses or mastreviruses. See Virus Taxonomy: The Seventh Report of the International Committee on Taxonomy of Viruses M.H. van Regenmortel, CM. Fauquet, D.H.L. Bishop et al. (eds.)Academic Press, 1024 pp. (2000) San Diego, Wien New York.
  • the invention provides a fusion protein comprising, consisting essentially of or consisting of the polypeptide recited above.
  • the polypeptide conformation is constrained.
  • polypeptides wherein conformation is constrained can bind to the target with higher affinity as compared to polypeptides wherein conformation is random.
  • the fusion protein comprises thioredoxin (or a fragment thereof).
  • the fusion proteins binds to Rep and/or inhibits viral replication. In certain embodiments, the fusion protein reduces geminivirus replication.
  • Embodiments of the present invention further provide an isolated nucleic acid comprising, consisting essentially of or consisting of a nucleotide sequence encoding the polypeptides and fusion proteins of the invention.
  • the isolated nucleotide comprises a nucleotide sequence encoding the polypeptide recited above.
  • the nucleic acid can be DNA, RNA or a chimera thereof, and can further include naturally occurring bases and/or analogs and derivatives of naturally occurring bases. Further, the isolated nucleic acid can be double-stranded, single- stranded or a combination thereof.
  • the present invention further provides a vector comprising the isolated nucleic acid recited above.
  • the vector is an expression vector.
  • the vector is compatible with bacterial, yeast, animal (e.g., mammalian, insect) or plant (e.g., monocot, dicot) cells.
  • Exemplary vectors include but are not limited to plasmids (including the Ti plasmid from Agrobacteria), virus vectors, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophage and the like.
  • cells comprising the isolated nucleic acids, vectors, polypeptides and fusion proteins of the invention.
  • the cell can be any cell known in the art including plant cells and protoplasts, animal cells, bacterial cells, yeast cells, and the like. Further, in particular embodiments, the cell can be a cultured cell or a cell in an intact plant or subject in vivo. Exemplary viruses
  • the geminiviruses are single-stranded plant DNA viruses. They possess a circular, single-stranded (ss) genomic DNA encapsidated in twinned "geminate" icosahedral particles. The encapsidated ssDNAs are replicated through circular double stranded DNA intermediates in the nucleus of the host cell, presumably by a rolling circle mechanism. Viral DNA replication, which results in the simulation of both single and double stranded viral DNAs in large amounts, involves the expression of only a small number of viral proteins that are necessary either for the replication process itself or facilitates replication or viral transcription. The geminiviruses therefore appear to rely primarily on the machinery of the host for viral replication and gene expression.
  • Geminiviruses are subdivided on the basis of host range in either monocots or dicots and whether the insect vector is a leaf hopper, tree hopper or a whitefly species.
  • Monocot-infecting geminiviruses the mastreviruses, are transmitted by leaf hoppers and their genome comprises a single ss DNA component about 2.7 kb in size (monopartite geminivirus).
  • This type of genome the smallest known infectious DNA, is typified by wheat dwarf virus which is one of a number from the subgroup that have been cloned and sequenced. A few mastreviruses infect dicot species as illustrated by bean yellow dwarf virus.
  • geminiviruse begomovirus genus infect dicot hosts and are transmitted by the whitefly. Most possess a bipartite genome comprising similarly sized DNAs (usually termed A and B) as illustrated by African cassava mosaic virus (ACMV), tomato golden mosaic virus (TGMV) and potato yellow mosaic virus. For successful infection of plants, both genomic components are required. Some begomoviruses possess single component genomes, as illustrated by tomato yellow leaf curl virus (TYLCV). The curtoviruses, typified by beet curly top virus, occupy a unique intermediary position between the above two genera as they infect dicots but are transmitted by leaf hoppers. The fourth geminivirus genus, the topocuviruses, is comprised of a single virus, tomato pseudo-curly top virus, which has single component genome and is transmitted by tree hoppers.
  • a and B African cassava mosaic virus
  • TGMV tomato golden mosaic virus
  • TYLCV tomato yellow leaf curl virus
  • the curtoviruses typified
  • the bipartite geminiviruses contain only the viruses that infect dicots.
  • Exemplary is the African Cassava Mosaic Virus (ACMV) and the Tomato Golden Mosaic Virus (TGMV).
  • TGMV like ACMV, is composed of two circular DNA molecules of the same size, both of which are required for infectivity. Sequence analysis of the two genome components reveals six open reading frames (ORFs); four of the ORFs are encoded by DNA A and two by DNA B. On both components, the ORFs diverge from a conserved 230 nucleotide intergenic region (common region) and are transcribed bidirectionally from double stranded replicative form DNA.
  • the ORFs are named according to genome component and orientation relative to the common region (i.e., left versus right).
  • the AL2 gene product transactivates expression of the TGMV coat protein gene, which is also sometimes known as "AR1". Functions have not yet been attributed to some of the ORFs in the geminivirus genomes. However, it is known that certain proteins are involved in the replication of viral DNA (REP genes). See, e.g., Elmer et al., Nucleic Acids Res. 16, 7043 (1988); Hatta and Francki, Virology 92, 428 (1979).
  • the A genome component contains all viral information necessary for the replication and encapsidation of viral DNA, while the B component encodes functions required for movement of the virus through the infected plant.
  • the DNA A component of these viruses is capable of autonomous replication in plant cells in the absence of DNA B when inserted as a greater than full-length copy into the genome of plant cells, or when a copy is electroporated into plant cells.
  • the single genomic component contains all viral information necessary for replication, encapsidation, and movement of the virus.
  • the geminivirus A component carries the Rep (also known as C1 , AC1 or ALI), the AL2 (also known as C2 or TRAP), AL3 (also known as C3, AC3 or REN), and AR1 (also known as V1 or coat protein) sequences.
  • the geminivirus B component carries the BR1 (also known as BV1) and BL1 (also known as BC1) sequences. Additionally, monopartite geminiviruses encode a protein that is homologous to the Rep protein of bipartite viruses.
  • geminiviruses encompass viruses of the Genus Mastrevirus, Genus Curtovirus, Genus Topocuvirus and Genus Begomovirus.
  • Exemplary geminiviruses include, but are not limited to, Abutilon Mosaic Virus, Ageratum Yellow Vein Virus, Bhendi Yellow Vein Mosaic virus, Cassava African Mosaic Virus, Chino del Tomato Virus, Cotton Leaf Crumple Virus, Croton Yellow Vein Mosaic Virus, Dolichos Yellow Mosaic Virus, Horsegram Yellow Mosaic Virus, Jatropha Mosaic virus, Lima Bean Golden Mosaic Virus, Melon Leaf Curl Virus, Mung Bean Yellow Mosaic Virus, Okra Leaf Curl Virus, Pepper Hausteco Virus, Potato Yellow Mosaic Virus, Rhynchosia Mosaic Virus, Squash Leaf Curl Virus, Tobacco Leaf Curl Virus, Tomato Australian Leaf Curl Virus, Tomato Indian Leaf Curl Virus, Tomato
  • Nanovirus Rep proteins differ from those of members of the Geminiviruses in being smaller (about 33 kDa), having a slightly distinct dNTP-binding motif and lacking the Rb-binding motif. Moreover, the Nanoviruses are distinct from Geminivirus particle morphology, genome size, number and size of DNA components, and mode of transcription. The Nanoviruses have a conserved nona- nucleotide motif at the apex of the stem-loop sequence which is consistent with the operation of a rolling circle model for DNA replication. As used herein, Nanoviruses include but are not limited to Banana Bunchy
  • BBTV Top Virus
  • FBNYN Faba Bean Necrotic Yellows Virus
  • MVDV Milk Vetch Dwarf Virus
  • SCSV subterranean clover stunt virus
  • AAVV Ageratum yellow vein virus
  • Circoviruses infect animal species and are characterized as round, non- enveloped virions with mean diameters from 17 to 23.5 nm containing circular ssDNA.
  • the ssDNA genome of the circoviruses represent the smallest viral DNA replicons known.
  • at least six viruses have been identified as members of the family according to The Sixth Report of the International Committee for the Taxonomy of Viruses (Lukert, et al. (1995) Arch. Virol. 10 Suppl.:166-168).
  • Circoviruses include but are not limited to members of the
  • Circoviridae family including chicken anemia virus (CAV), beak and feather disease virus (BFDV), porcine circovirus type 1 (PCV1), porcine circovirus type 2 (PCV2) and pigeon circovirus and any other virus designated as a nanovirus by the ICTV.
  • CAV chicken anemia virus
  • BFDV feather disease virus
  • PCV1 porcine circovirus type 1
  • PCV2 porcine circovirus type 2
  • pigeon circovirus any other virus designated as a nanovirus by the ICTV.
  • Embodiments of the present invention further provide a transgenic plant or plant cell comprising the isolated nucleic acid recited above.
  • the plant or cell can be stably transformed with the isolated nucleic acid.
  • the isolated nucleic acid is flanked by a T-DNA border sequence, optionally by 5' and 3' T-DNA border sequences.
  • the invention provides a plant cell or plant comprising the polypeptides or fusion proteins of the present invention.
  • Plants can be transformed according to the present invention using any suitable method known in the art. Intact plants, plant tissue, explants, meristematic tissue, protoplasts, callus tissue, cultured cells, and the like may be used for transformation depending on the plant species and the method employed. In a preferred embodiment, intact plants are inoculated using microprojectiles carrying a nucleic acid to be transferred into the plant. The site of inoculation will be apparent to one skilled in the art; leaf tissue is one example of a suitable site of inoculation. In preferred embodiments, intact plant tissues or plants are inoculated, without the need for regeneration of plants (e.g., from callus). Exemplary transformation methods include biological methods using viruses and Agrobacterium, physicochernical methods such as electroporation, polyethylene glycol, ballistic bombardment, microinjection, floral dip method and the like.
  • the vector is microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA (Crossway, Mol. Gen. Genetics 202:, 179 (1985)).
  • the genetic material is transferred into the plant cell using polyethylene glycol (Krens, et al. Nature 296, 72 (1982)).
  • protoplasts are fused with minicells, cells, lysosomes, or other fusible lipid-surfaced bodies that contain the nucleotide sequence to be transferred to the plant (Fraley, et al., Proc. Natl. Acad. Sci. USA 79, 1859 (1982)).
  • DNA may also be introduced into the plant cells by electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)).
  • plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide and regenerate.
  • electroporation is that large pieces of DNA, including artificial chromosomes, can be transformed by this method.
  • Viral vectors include RNA and DNA viral vectors (e.g., geminivirus, badnavirus, nanoviruses and caulimovirus vectors).
  • RNA and DNA viral vectors e.g., geminivirus, badnavirus, nanoviruses and caulimovirus vectors.
  • Ballistic transformation typically comprises the steps of: (a) providing a plant tissue as a target; (b) propelling a microprojectile carrying the heterologous nucleotide sequence at the plant tissue at a velocity sufficient to pierce the walls of the cells within the tissue and to deposit the nucleotide sequence within a cell of the tissue to thereby provide a transformed tissue.
  • the method further includes the step of culturing the transformed tissue with a selection agent.
  • the selection step is followed by the step of regenerating transformed plants from the transformed tissue.
  • the technique may be carried out with the nucleotide sequence as a precipitate (wet or freeze-dried) alone, in place of the aqueous solution containing the nucleotide sequence.
  • Any ballistic cell transformation apparatus can be used in practicing the present invention.
  • Exemplary apparatus are disclosed by Sandford et al. (Particulate Science and Technology 5, 27 (1988)), Klein et al. (Nature 327, 70 (1987)), and in EP 0 270 356.
  • Such apparatus have been used to transform maize cells (Klein et al., Proc. Natl. Acad. Sci. USA 85, 4305 (1988)), soybean callus (Christou et al., Plant Physiol.
  • an apparatus configured as described by Klein et al. (Nature 70, 327 (1987)) may be utilized.
  • This apparatus comprises a bombardment chamber, which is divided into two separate compartments by an adjustable-height stopping plate.
  • An acceleration tube is mounted on top of the bombardment chamber.
  • a macroprojectile is propelled down the acceleration tube at the stopping plate by a gunpowder charge.
  • the stopping plate has a borehole formed therein, which is smaller in diameter than the microprojectile.
  • the macroprojectile carries the microprojectile(s), and the macroprojectile is aimed and fired at the borehole. When the macroprojectile is stopped by the stopping plate, the microprojectile(s) is propelled through the borehole.
  • the target tissue is positioned in the bombardment chamber so that a microprojectile(s) propelled through the bore hole penetrates the cell walls of the cells in the target tissue and deposit the nucleotide sequence of interest carried thereon in the cells of the target tissue.
  • the bombardment chamber is partially evacuated prior to use to prevent atmospheric drag from unduly slowing the microprojectiles.
  • the chamber is only partially evacuated so that the target tissue is not desiccated during bombardment.
  • a vacuum typically between about 400 to about 800 millimeters of mercury is suitable. In alternate embodiments, ballistic transformation is achieved without use of microprojectiles.
  • an aqueous solution containing the nucleotide sequence of interest as a precipitate may be carried by the macroprojectile (e.g., by placing the aqueous solution directly on the plate-contact end of the macroprojectile without a microprojectile, where it is held by surface tension), and the solution alone propelled at the plant tissue target (e.g., by propelling the macroprojectile down the acceleration tube in the same manner as described above).
  • Other approaches include placing the nucleic acid precipitate itself ("wet" precipitate) or a freeze-dried nucleotide precipitate directly on the plate-contact end of the macroprojectile without a microprojectile.
  • nucleotide sequence In the absence of a microprojectile, it is believed that the nucleotide sequence must either be propelled at the tissue target at a greater velocity than that needed if carried by a microprojectile, or the nucleotide sequenced caused to travel a shorter distance to the target tissue (or both).
  • the nucleotide sequence can be carried on a microprojectile.
  • the microprojectile may be formed from any material having sufficient density and cohesiveness to be propelled through the cell wall, given the particle's velocity and the distance the particle must travel.
  • materials for making microprojectiles include metal, glass, silica, ice, polyethylene, polypropylene, polycarbonate, and carbon compounds (e.g., graphite, diamond).
  • Metallic particles are currently preferred.
  • suitable metals include tungsten, gold, and iridium.
  • the particles should be of a size sufficiently small to avoid excessive disruption of the cells they contact in the target tissue, and sufficiently large to provide the inertia required to penetrate to the cell of interest in the target tissue. Particles ranging in diameter from about one-half micrometer to about three micrometers are suitable. Particles need not be spherical, as surface irregularities on the particles may enhance their DNA carrying capacity.
  • the nucleotide sequence may be immobilized on the particle by precipitation.
  • the precise precipitation parameters employed will vary depending upon factors such as the particle acceleration procedure employed, as is known in the art.
  • the carrier particles may optionally be coated with an encapsulating agents such as polylysine to improve the stability of nucleotide sequences immobilized thereon, as discussed in EP 0 270 356 (column 8).
  • plants may be transformed using Agrobacterium tumefaciens or Agrobacterium rhizogenes, preferably Agrobacterium tumefaciens.
  • Agrobacterium- mediated gene transfer exploits the natural ability of A. tumefaciens and A. rhizogenes to transfer DNA into plant chromosomes.
  • Agrobacterium is a plant pathogen that transfers a set of genes encoded in a region called T-DNA of the Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, into plant cells.
  • the typical result of transfer of the Ti plasmid is a tumorous growth called a crown gall in which the T-DNA is stably integrated into a host chromosome. Integration of the Ri plasmid into the host chromosomal DNA results in a condition known as "hairy root disease". The ability to cause disease in the host plant can be avoided by deletion of the genes in the T-DNA without loss of DNA transfer and integration.
  • the DNA to be transferred is attached to border sequences that define the end points of an integrated T-DNA.
  • Agrobacterium mediated transformation has been achieved in several monocot species, including cereal species such as rye (de la Pena et al., Nature 325, 274 (1987)), maize (Rhodes et al., Science 240, 204 (1988)), and rice (Shimamoto et al., Nature 338, 274 (1989)).
  • A. rhizogenes Transformation using A. rhizogenes has developed analogously to that of A. tumefaciens and has been successfully utilized to transform, for example, alfalfa, Solanum nigrum L., and poplar.
  • U.S. Patent No. 5, 773,693 to Burgess et al. it is preferable to use a disarmed A. tumefaciens strain (as described below), however, the wild-type A. rhizogenes may be employed.
  • An illustrative strain of A. rhizogenes is strain 15834.
  • the Agrobacterium strain is typically modified to contain the nucleotide sequences to be transferred to the plant.
  • the nucleotide sequence to be transferred is incorporated into the T-region and is typically flanked by at least one T-DNA border sequence, preferably two T-DNA border sequences.
  • a variety of Agrobacterium strains are known in the art, and can be used in the methods of the invention. See, e.g., Hooykaas, Plant Mol. Biol. 13, 327 (1989); Smith et al., Crop Science 35, 301 (1995); Chilton, Proc. Natl. Acad. Sci. USA 90, 3119 (1993); Mollony et al., Monograph Theor. Appl. Genet NY 19, 148 (1993); Ishida et al., Nature Biotechnol. 14, 745 (1996); and Komari et al., The Plant JoumaHO, 165 (1996).
  • the Ti (or Ri) plasmid contains a vir region.
  • the vir region is important for efficient transformation, and appears to be species-specific.
  • Two exemplary classes of recombinant Ti and Ri plasmid vector systems are commonly used in the art.
  • the shuttle vector containing the gene of interest is inserted by genetic recombination into a non- oncogenic Ti plasmid that contains both the cis-acting and trans-acting elements required for plant transformation as, for example, in the PMLJ1 shuttle vector of DeBlock et al., EMBO J 3, 1681 (1984), and the non-oncogenic Ti plasmid pGV2850 described by Zambryski et al., EMBO J 2, 2143 (1983).
  • the gene of interest is inserted into a shuttle vector containing the cis-acting elements required for plant transformation.
  • the other necessary functions are provided in trans by the non-oncogenic Ti plasmid as exemplified by the pBIN19 shuttle vector described by Bevan, Nucleic Acids Research 12, 8711 (1984), and the non-oncogenic Ti plasmid PAL4404 described by Hoekma, et al., Nature 303, 179 (1983).
  • Binary vector systems have been developed where the manipulated disarmed T-DNA carrying the heterologous nucleotide sequence of interest and the vir functions are present on separate plasmids.
  • a modified T-DNA region comprising foreign DNA (the nucleic acid to be transferred) is constructed in a small plasmid that replicates in E. coli.
  • This plasmid is transferred conjugatively in a tri-parental mating or via electroporation into A. tumefaciens that contains a compatible plasmid with virulence gene sequences.
  • the vir functions are supplied in trans to transfer the T-DNA into the plant genome.
  • Such binary vectors are useful in the practice of the present invention.
  • Plant cells may be transformed with Agrobacteha by any means known in the art, e.g., by co-cultivation with cultured isolated protoplasts, or transformation of intact cells or tissues.
  • the first requires an established culture system that allows for culturing protoplasts and subsequent plant regeneration from cultured protoplasts. Identification of transformed cells or plants is generally accomplished by including a selectable marker in the transforming vector, or by obtaining evidence of successful bacterial infection.
  • the nucleotide sequence of interest is incorporated into the plant genome, typically flanked by at least one T-DNA border sequence.
  • the nucleotide sequence of interest is flanked by two T-DNA border sequences.
  • transgenic plants may be produced using the well-established 'floral dip' method (See, e.g., Clough and Bent (1998) Plant Journal 16:735).
  • plants are grown in soil until the primary inflorescence is about 10 cm tall.
  • the primary inflorescence is cut to induce the emergence of multiple secondary inflorescences.
  • the inflorescences of these plants are dipped in a suspension of Agrobacterium containing the vector of interest. After the dipping process, the plants are grown to maturity and the seeds are harvested.
  • Transgenic seeds from these treated plants are selected by germination in soil under selective pressure (e.g., using the chemical bialaphos).
  • Transgenic plants containing the selectable marker survive treatment and are transplanted to individual pots for subsequent analysis. See Bechtold, N. and Pelletier, G. Methods Mol Biol 82, 259- 266 (1998); Chung, M.H. et al. Transgenic Res 9, 471-476 (2000); Clough, S.J. and Bent, A.F. Plant J 16, 735-743 (1998); Mysore, K.S. et al. Plant J 21 , 9-16 (2000); Tague, B.W. Transgenic Res 10, 259-267 (2001); Wang, W.C. et al. Plant Cell Rep 22, 274-281 (2003); Ye, G.N. et al. Plant J., 19:249-257 (1999). Plant cells, which have been transformed by any method known in the art, can also be regenerated to produce intact plants using known techniques.
  • Plant regeneration from cultured protoplasts is described in Evans et al., Handbook of Plant Cell Cultures, Vol. 1 : (MacMilan Publishing Co. New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. II, 1986). It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugar-cane, sugar beet, cotton, fruit trees, and legumes.
  • Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently root. Alternatively, somatic embryo formation can be induced in the callus tissue. These somatic embryos germinate as natural embryos to form plants.
  • the culture media will generally contain various amino acids and plant hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable. A large number of plants have been shown capable of regeneration from transformed individual cells to obtain transgenic whole plants.
  • the regenerated plants are transferred to standard soil conditions and cultivated in a conventional manner.
  • the plants are grown and harvested using conventional procedures.
  • the particular conditions for transformation, selection and regeneration may be optimized by those of skill in the art. Factors that affect the efficiency of transformation include the species of plant, the tissue infected, composition of the media for tissue culture, selectable marker genes, the length of any of the above- described step, kinds of vectors, and light/dark conditions. Therefore, these and other factors may be varied to determine what is an optimal transformation protocol for any particular plant species. It is recognized that not every species will react in the same manner to the transformation conditions and may require a slightly different modification of the protocols disclosed herein. However, by altering each of the variables, an optimum protocol can be derived for any plant species.
  • transgenic inbred or doubled-haploid lines may be used for producing transgenic inbred or doubled-haploid lines.
  • Transgenic inbred/doubled-haploid lines could then be crossed, with another (non-transformed or transformed) inbred or doubled-haploid line, in order to produce a transgenic hybrid plant.
  • a genetic trait which has been engineered into a particular line using the foregoing transformation techniques could be moved into another line using traditional backcrossing techniques that are well known in the plant breeding arts.
  • Plants that may be employed in practicing the present invention include any plant (angiosperm or gymnosperm; monocot or dicot). Exemplary plants include, but are not limited to corn (Zea mays), canola
  • Vegetables include Solanaceous species (e.g., tomatoes; Lycopersicon esculentum), lettuce (e.g., Lactuea sativa), carrots (Caucus carota), cauliflower (Brassica oleracea), celery (apium graveolens), eggplant (Solanum melongena), asparagus (Asparagus officinalis), ochra (Abelmoschus esculentus), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), members of the genus Cucurbita such as Hubbard squash (C. Hubbard), Butternut squash (C.
  • moschata Zucchini (C. pepo), Crookneck squash (C. crookneck), C. argyrosperma , C. argyrosperma ssp sororia, C. digitata, C. ecuadorensis, C. foetidissima, C. lundelliana, and C. martinezii, and members of the genus Cucumis such as cucumber (Cucumis sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
  • Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (dianthus caryophyllus), poinsettia (Euphorbia pulcherima), and chrysanthemum.
  • Conifers which may be employed in practicing the present invention, include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).
  • pines such as loblolly pine (Pinus taeda), slash pine (
  • Turfgrass include but are not limited to zoysiagrasses, bentgrasses, fescue grasses, bluegrasses, St. Augustinegrasses, bermudagrasses, bufallograsses, ryegrasses, and orchardgrasses.
  • plants that serve primarily as laboratory models, e.g., Arabidopsis.
  • the transgenic plant can be a tobacco plant, a potato plant, a soybean plant, a peanut plant, a tomato plant, a melon plant, a cassava plant, a bean plant, a squash plant, a maize plant, a cotton plant or a vegetable plant.
  • the plant is a cassava plant.
  • the present invention provides methods of providing resistance against a plant virus infection, in an agricultural field, comprising planting the field with a crop of plants as recited above.
  • Embodiments of the present invention further provide transgenic plants having increased resistance to a virus infection from viruses such as a geminivirus, a nanovirus and combinations thereof as compared to a non-transgenic control.
  • Resistance may be evaluated by any suitable method known in the art, e.g., measuring inhibition of viral replication, detecting specific mutations within the genome of the viral agent, detecting and quantifying viral load and measuring surrogate markers of viral replication.
  • the term "resistant/resistance” is not intended to indicate that the subject is absolutely immune from viral infection.
  • the degree of resistance may be assessed with respect to a population of subjects or an entire field of plants. A subject may be considered “resistant” to viral infection if the overall incidence of infection is reduced, even if particular subjects may be susceptible to disease.
  • Particular embodiments of the present invention provide methods of making transgenic plants having increased resistance to a virus, wherein the method comprises providing a plant cell capable of regeneration; transforming the plant cell with an isolated nucleic acid comprising an isolated nucleic acid recited above; and regenerating a transgenic plant from that transformed plant cell, wherein expression of the isolated nucleic acid to produce the polypeptide increases resistance of the transgenic plant to infection by a virus.
  • the methods of making transgenic plants having increased resistance to a virus comprises introducing an isolated nucleic acid recited above into a plant cell to produce a transgenic plant, wherein expression of the isolated nucleic acid to produce the polypeptide increases resistance of the transgenic plant to infection by a virus.
  • the plant cell can be a tobacco plant cell, a potato plant cell, a soybean plant cell, a peanut plant cell, a tomato plant cell, a melon plant cell, a cassava plant cell, a bean plant cell, a squash plant cell, a maize plant cell, a cotton plant cell and a vegetable plant cell.
  • the plant cell is stably transformed with the isolated nucleic acid.
  • the plant cell is transformed by an Agrobacterium-medlated transformation method.
  • the plant cell is transformed by a biolistic transformation method.
  • the present invention provides methods of inhibiting viral replication in a plant cell (e.g., a cultured plant cell or protoplast or a plant cell in vivo) comprising introducing an isolated nucleic acid recited above into the plant cell in an amount effective to inhibit virus replication as compared to non- transgenic control.
  • virus replication is inhibited by at least about 10%, 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more.
  • the virus can be a geminivirus, a nanovirus and combinations thereof.
  • the invention provides methods of inhibiting viral replication in a plant cell by introducing a polypeptide or fusion protein of the invention into a plant cell. In still other embodiments, the invention provides a method of providing increased resistance to a viral infection comprising introducing a polypeptide or fusion protein of the invention into a plant.
  • a further embodiment of the present invention is a method of detecting a viral infection.
  • the method can involve contacting a sample with at least one polypeptide of the present invention or a fusion protein thereof and detecting the presence or absence of binding between the polypeptide and a target, wherein the binding of the polypeptide to the target in the sample indicates the presence of a virus.
  • a sample is intended to include biological or environmental material which may be suspected of containing a virus of the family Geminiviridae, Nanoviridae or Circoviridae.
  • a sample suspected of containing a virus is one which may have come in contact with a virus or may be at risk of having or acquiring a virus.
  • a sample may be blood, plasma, serum, cell products, cell line cultures, cell extracts, cerebrospinal fluid (CSF), tissue homogenates, urine, organs for transplantation, or semen isolated preferably from a bird, such as chicken or pigeon, or a pig.
  • CSF cerebrospinal fluid
  • the sample may be a plant tissue culture, fruit, leaf, root, stem, or seed.
  • a sample of environmental origin may include, but not be limited to, soil, water, and food samples including canned goods, meats, and animal fodder. It is contemplated that the method of the invention may be useful in detecting viral contaminants in an environmental sample, viral presence in an organ being used in a transplantation, or viral infection of plant seeds or tissue cultures.
  • polypeptide of the present invention can be labeled, preferably with a fluorescent or bioluminescent tag. Fluorochromes such as
  • Phycocyanine, Allophycocyanine, Tricolor, AMCA, Eosin, Erythrosin, Fluorescein, Fluorescein Isothiocyanate Hydroxycoumarin, Rhodamine, Texas Red, Lucifer Yellow, and the like may be attached directly to a polypeptide of the invention through standard groups such as sulfhydryl or primary amine groups.
  • Methods of imaging and analyzing any of the above-mentioned labels are well-known in the art and the method employed will vary with the type of analysis being conducted, i.e. individual samples or multiple sample analyses in high-throughput screens. Measurement of the label can be accomplished using flow cytometry, laser confocal microscopy, spectrofluorometer, fluorescence microscopy, fluorescence scanners and the like.
  • a polypeptide of the present invention may be biotinylated and detection of a biotinylated polypeptide may be performed using any of the well- known avidin or streptavidin reagents.
  • Detection of biotin-avidin or biotin-streptavidin complexes typically involves conjugated forms of avidin or streptavidin including, but are not limited to, enzyme-conjugates (e.g., alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, horseradish peroxidase) or fluorescent-conjugates (e.g., 7-amino- 4-methylcoumarin-3-acetic (AMCA), fluorescein, phycoerythrin, rhodamine, TEXAS RED®, OREGON GREEN®) or antibodies which specifically bind to avidin or streptavidin.
  • enzyme-conjugates e.g., alkaline phosphatase,
  • Antibodies which specifically interact with a polypeptide of the present invention can also be used in the detection of binding between said polypeptide and its target.
  • a bound polypeptide-target complex is contacted with an antibody specific for said polypeptide and standard methods for detecting antibodies are employed for detecting binding of the antibody to the polypeptide-target complex, e.g., spectrofluorometer, fluorescence microscopy, immunocytochemistry, western blotting, ELISA, fluorescence scanners, and the like.
  • Other methods for detecting antibodies are well-known to those of skill in the art (see, e.g., "Methods in Immunodiagnosis", 2nd Edition, Rose and Bigazzi, eds.
  • the presence or absence of a bound polypeptide-target complex is then correlated with the presence or absence of a virus from which the target was derived.
  • the detection method of the invention may be used to detect one or more specific viruses, genera, or family of viruses depending on the specificity of the polypeptide being used.
  • the products of the present invention can be used for the preparation of a medicament or agricultural product.
  • the present invention is applicable to animal, avian and plant subjects, where appropriate, for medicinal, diagnostic, veterinary, or agricultural purposes.
  • geminiviruses and nanoviruses affect plants
  • circoviruses affect livestock and poultry
  • a human circovirus has been identified in patients with Hepatitis C.
  • Animal subjects include, but are not limited to, humans, primates, canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g. rats and mice), lagomorphs, and the like, and mammals in utero.
  • Avian subjects include, but are not limited to, chickens, ducks, turkeys, geese, quail, pheasant, ratites (e.g., ostrich) and domesticated birds (e.g., parrots and canaries), and birds in ovo. Plant subjects are described above.
  • Methods of the present invention can be carried out in a manner suitable for administration or application to the suitable subject. Administration to a plant or a plant cell is described above.
  • An isolated nucleic acid vector, polypeptide or fusion protein of the present invention can be used to formulate pharmaceutical compositions comprising a vector of the invention in a pharmaceutically acceptable carrier and/or other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc.
  • the carrier will typically be a liquid.
  • the carrier may be either solid or liquid.
  • the carrier will be respirable, and will preferably be in solid or liquid particulate form.
  • physiologically acceptable carrier is one that is not toxic or unduly detrimental to cells.
  • physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline, physiologically acceptable carriers include pharmaceutically acceptable carriers.
  • pharmaceutically acceptable it is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects.
  • a pharmaceutical composition may be used, for example, in transfection of a cell ex vivo or in administering a viral particle or cell directly to a subject.
  • a “therapeutically effective” amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
  • Dosages will depend upon the mode of administration, the disease or condition to be treated, the individual subject's condition, and can be determined in a routine manner. See e.g., Remington, The Science And Practice of Pharmacy (20 tn Ed. 2000).
  • more than one administration e.g., two, three, four or more administrations
  • more than one administration e.g., two, three, four or more administrations
  • Exemplary modes of administration include oral, rectal, transmucosal, topical, transdermal, in utero (or in ovo), inhalation, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, and intraarticular) administration, and the like, as well as direct tissue or organ injection, alternatively, intrathecal, direct intramuscular, intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • administration may be by local rather than systemic manner, for example, in a depot or sustained-release formulation.
  • Geminivirus Rep proteins have been studied extensively, and many of the assays for studying this protein are well-established in the art. Consequently, geminiviruses were used to test the capacity of aptamers to interfere with ssDNA virus replication and infection in eukaryotes.
  • the well-characterized virus, tomato golden mosaic virus (TGMV) was primarily studied.
  • TGMV is a bipartite geminivirus that infects solanaceous species and encodes a typical Rep protein.
  • Other viruses such as CbLCV, ACMV, EACMV, MSV and TYLCV are also contemplated as being useful in the analysis of selected aptamers.
  • CbLCV has a bipartite genome and is a severe pathogen in brassica.
  • CbLCV is representative of a small group of dicot- infecting geminiviruses that encode an atypical Rep protein.
  • TYLCV is a monopartite geminivirus that causes significant losses in tomato crops through out the world. Genomic clones as well as replication and infectivity assays are well-established for these viruses. Together, these viruses are used to establish the efficacy and breadth of the aptamer resistance strategy for ssDNA viruses.
  • Rep contains several conserved sequences, including the cleavage motifs, the -helices and the NTP binding site ( Figure 1); however, sequences that met all of the criteria were within the cleavage motifs.
  • the selected sequences listed in Table 1 were involved in initiation of rolling circle replication, highly conserved and unique to rolling circle initiators.
  • Geminiviruses are subgroup ed as Begomoviruses, Curtoviruses, Topocuviruses or Mastreviruses
  • the Begomoviruses are further grouped according to whether they are of New or Old World lineage.
  • Motif III of Rep ( Figure 1) was focused on because of its strong conservation across all ssDNA virus Rep proteins (Table 1), its catalytic role in rolling circle replication and its involvement in Rep/DNA binding. Motif III contains invariant Tyr and Lys residues that appear essential for viral replication (Orozco and Hanley- Bowdoin (1998) supra; Hoogstraten, et al. (1996) Mol. Plant Microbe Interact. 9:594- 599). The Tyr residue mediates DNA cleavage and is covalently crosslinked to the 5'-phosphorus of the cleaved DNA (Laufs, et al. (1995) FEBS Lett. 377:258-262), whereas the role of the Lys residue is unknown.
  • Trx E. coli thioredoxin
  • the Trx-peptide fusions were part of a coding sequence that also included the SV40 nuclear localization signal, the B42 activation domain and a hemagglutinin epitope tag.
  • Expression of the fusion gene was under the control of the yeast GAL1 promoter and the ADH1 terminator.
  • a yeast dihybrid screen of 10 7 Trx-aptamers identified 40 fusions that bind to TGMV Rep. Eleven of these contained intact Trx- aptamer sequences whereas the remainder were either truncated or out of frame. Two of the 40 fusions contained the same aptamer sequence.
  • Trx-aptamers were subcloned into a plant expression cassette containing the CaMV 35S promoter and rbcS E9 3'- end and analyzed in an in vivo replication interference assay (Example 7).
  • Two of the Trx-aptamers (A42 and C1 :60 reduced TGMV A replication to a level similar to a strong Rep trans- dominant mutant (RepM13).
  • RepM13, A41 and C1 :60 each reduced replication to about 40% of wild-type levels.
  • RepM13 typically reduces replication to >5% (Orozco, et al. (2000) supra), indicating that the interference assays may not have been optimal.
  • EXAMPLE 4 Aptamer Library Screening After the initial screening, a subsequent aptamer library screen was performed to identify aptamers sequences that specifically bind to the N-terminal domain of Rep that contains the three cleavage motifs, including motif III.
  • Two plasmids were generated for screening the Trx-aptamer library in yeast dihybrid assays ( Figure 3).
  • pNSB1118 encodes full-length TGMV (Rep) fused to a LexA DNA binding domain (DBD-AL1 ), whereas pNSB1162 specifies a fusion with the first 130 amino acids of TGMV Rep (DBD-AL1 ⁇ - 13 o).
  • Both plasmids were constructed using MATCHMAKER LEXA dihybrid system (CLONTECH, Palo Alto, CA), which incorporates four separate screens, e.g. ⁇ -galactosidase activity and leucine auxotrophy, in the presence of galactose and in the absence of glucose, to reduce the frequency of false positives. Authenticity of the interactions was confirmed by recovering candidate clones in E. coli, retransforming them into yeast, and confirming that individually they did not show activity. Peptides that bind to the DBD- AL1 bait may interact with all the functional domains of TGMV AL1 , while interactions with DBD- A I 1 -. 130 are confined the to N-terminus.
  • the N-terminus was focused on because it contains the DNA cleavage/ligation and origin binding domains, both of which are essential for geminivirus replication and contain highly conserved motifs.
  • yeast dihybrid assay Karlonin, et al. (2000) Methods Enzymol.
  • pNSB1118 - A total of 8x10 6 cells were plated onto galactose containing plates minus histidine, tryptophan, leucine and uracil (+Gal, -HTLU). Seven hundred colonies positive for AL1 interaction were selected. These colonies were picked and frozen at -80°C for subsequent analysis.
  • pNSB1162 - A total of 1.5x10 7 cells were plated on +Gal, -HTLU plates. Candidates (597 colonies) were selected and AL1 interactions were confirmed for 287 candidates using LacZ and leucine auxotrophy assays.
  • Positive clones may also be tested for loss of interaction using bait plasmids containing alanine substitutions at the invariant Tyr and Lys residues in motif III, which have been shown to knockout Rep function (Orozco et al. (1998) J. Biol. Chem., 273:24448-24456). Because alanine substitutions are structurally neutral changes, the mutants retain conformation and, thus, enable one to focus on aptamers that specifically interact with motif III. A similar approach was taken in the isolation and characterization of a Trx-peptide that binds to wild-type human cdk2 but not to variants with point mutations in the kinase active site (Cohen, et al. (1998) supra). Because of the strong conservation across motif III, any interacting peptides identified using TGMV Rep may also bind other ssDNA virus Rep proteins such as those of CbLCV and TYLCV.
  • Peptides in Panels B to I of Figure 4 contain recognizable motifs with consensus sequences enriched for hydrophobic amino acids that are characteristic of protein interaction surfaces and may mediate binding to Rep. Sequences in Panels B, C, D, F and H of Figure 4 had conserved residues which were tightly clustered and may constitute a well-defined primary sequence that binds to Rep. Sequences in Panels E, G and I of Figure 4 had conserved residues which were more scattered but may come together in the folded peptide to interact with Rep. There were also numerous stretches of amino acids that were conserved between 2-3 peptides that were not included in the groups. These may constitute Rep binding motifs that are not well represented in the current peptide population.
  • pNSB1226 contains the CaMV 35S promoter, nos terminator and kanamycin selection in E. coli.
  • pNSBI 226 was constructed for these experiments to facilitate recovery of the plant expression cassettes in the presence of the yeast library clones, which confer ampicillin resistance to E. coli.
  • Replication interference assays were developed using a semi-quantitative PCR protocol that facilitated high throughput screening for replication interference. An excess amount of Trx-aptamer expression cassette DNA was cotransfected into tobacco By-2 protoplasts with TGMV A replicon DNA. Total DNA was isolated at 36 hours post transfection and analyzed for replication interference after Dpnl digestion to eliminate input DNA. Replicated DNA was detected using divergent primers corresponding to TGMV A. This assay was initially developed using a plasmid that expresses a TGMV AL1 trans-dom ' mant interfering mutant, and a cassette that expresses an AL1 -binding Trx-peptide.
  • Trx-peptides that bind to the N-terminus of TGMV AL1 are tested.
  • the experimental design is based on comparative sequence analysis ( Figure 4), which indicates that the 81 unique peptides constitute 9 groups. Members of each group are first tested together for replication interference. The strongest interfering Trx-peptides are then compared across groups. It may also be determined whether interfering properties of the different Trx- peptides are additive, indicating that they bind to different regions of the AL1 N- terminus. A limited set of Trx-peptides are identified and it is determined if they bind to the replication proteins of other geminiviruses, including ACMV and EACMV, and interfere with their replication in transient assays.
  • in vivo replication interference assays are performed. Plant expression vectors are constructed which contain the 35S promoter driving expression of the Trx-peptide fusions. A nuclear localization signal is also included in the plant cassettes because Rep is found exclusively in nuclei of infected plant cells (Nagar, et al. (1995) Plant Cell 7:705- 719). The SV40 nuclear localization signal has been used to target T7 RNA polymerase to tobacco nuclei (Lassner, et al. (1991) Plant Mol. Biol. 17:229-234).
  • a pET16 vector with a SV40 nuclear localization signal sequence immediately downstream of the histidine tag is generated and used to make the E. coli Trx-peptide expression cassettes described.
  • This approach allows direct transfer of the Trx-peptide coding sequences from the E. coli vectors to the plant vectors.
  • Expression of the Trx-peptide constructs is not expected to be toxic to plant cells because the thioredoxin active site is disrupted by the peptide insertions. However, if toxic, fusions of the peptides occurs, the green fluorescent protein or another nontoxic protein may be used (Baulcombe, et al. (1995) Plant J. 7:1045-1053).
  • a TGMV A replicon encoding wild-type Rep is transfected into tobacco protoplasts either alone or in the presence of increasing amounts of the 35S-Trx- peptide cassettes. Total DNA is isolated after a 36-hour culture period, and TGMV A replication is quantified by DNA gel blot and phosphorimage analyses (Orozco, et al. (2000) supra). Control cells transfected with TGMV A and a 35S cassette corresponding to Trx alone serve as the 100% replication control. Alternatively, a 20-mer peptide derived from GST (Trx-GST) that does not bind to Rep or interfere with its function is cloned into the Trx active site and used as a control.
  • Trx-GST 20-mer peptide derived from GST
  • the amount of each 35S-Trx-peptide cassette required to inhibit replication by 50% (IC50) is determined and compared for the different aptamers.
  • the aptamers are also scored as to whether they partially or fully abolish TGMV A replication.
  • Transient replication assays are used to determine if an aptamer confers broad-based interference.
  • TYLCV, ACMV, EACMV and CbLCV A replicons are used in place of TGMV A. Similar strategies have been used to show that Trx-peptides produced from expression cassettes can interfere with transcription and cell division in animal systems (Cohen, et al. (1998) supra; Fabbrizio, et al. (1999) supra).
  • the in vivo replication interference assay may be used with a variety of viruses (Figure 5).
  • Tobacco protoplasts were transfected with a replicon corresponding to the viral DNA indicated above each panel in Figure 5.
  • the replicon was cotransfected with an empty plant expression cassette.
  • the replicon was cotransfected with a plant expression cassette corresponding to a frar/s-dominant mutant of TGMV Rep protein.
  • Total DNA was isolated 48 hours post- transfection and analyzed for viral DNA replication on DNA gel blots hybridized with virus-specific probes labeled with 32P.
  • Trx-peptides that are most effective in in vivo assays are tested for interference with replication functions in vitro.
  • Peptides corresponding to the aptamer clones are tested for interference with Rep DNA binding and cleavage activities in vitro.
  • Trx-peptide fusions are used for these assays.
  • E. coli expression cassettes corresponding to each Trx-peptide insert are constructed using PCR-generated fragments and pET16b (NOVAGEN®, Madison, WI), which contains the T7 promoter and encodes an N-terminal histidine tag.
  • Recombinant histidine-tagged Trx-peptides are produced in BL21 cells under ITPG induction and purified by nickel affinity chromatography. This approach has been used to produce a number of purified proteins (Settlage, et al. (1996) supra; Kong, et al. (2000) supra; Pedersen and Hanley-Bowdoin (1994) Virology 202:1070- 1075).
  • TGMV Rep are expressed as a glutathione sulphonyl transferase (GST) fusion in insect cells.
  • GST-Rep purified by glutathione affinity chromatography is functional for DNA binding and cleavage (Orozco and Hanley-Bowdoin (1996) supra; Orozco, et al. (1997) supra).
  • Trx-peptides For the DNA binding assays, increasing amounts of purified Trx-peptide are incubated with GST-Rep prior to the addition of a radiolabeled double-stranded DNA containing the Rep recognition sequence. Bound complexes are resolved on agarose gels and quantified by phosphorimage analysis (Orozco, et al. (1998) Virology 242:346-356). The concentration of Trx-peptide required to inhibit binding by 50% (IC50) is determined for each aptamer. Comparison of the IC50 values for different aptamers provides a measure of their relative interfering activities. Trx- peptides have been used successfully to interfere with E2F/DNA binding activity (Fabbrizio, et al. (1999) Oncogene 18:4357-4363).
  • Trx-peptides The abilities of the different Trx-peptides to interfere with DNA cleavage is also evaluated.
  • increasing amounts of purified Trx-peptide and GST-Rep are preincubated, and then a radiolabeled single-stranded oligonucleotide containing the cleavage recognition sequence is added to the reaction.
  • the DNA reaction products are isolated, resolved on denaturing polyacrylamide gels and quantified. Again the amount of Trx-peptide required to inhibit cleavage by 50% (IC50) is determined and compared for the different aptamers.
  • An analogous approach has been used to assess the kinase inhibitory activities of cdk2-specific Trx-peptides (Cohen, et al. (1998) supra).
  • Nonspecific effects due to Trx or trace E. coli contaminants in the DNA binding and cleavage assays are analyzed through the expression and purification of a histidine-tagged version of Trx containing a peptide sequence that does not bind to Rep or interfere with its function in replication assays , which serves as a negative control in the above experiments.
  • Nicotiana benthamiana is a permissive host of TGMV, TYLCV, ACMV, EACMV and CbLCV. Thus, the ability of these viruses to target the same species is used to establish the efficacy of using aptamers that bind Rep cleavage motifs to confer broad-based disease resistance.
  • Transgenic N. benthamiana that express the Trx-peptide fusions are generated using Agrobacterium transformation technology. Plant transformation vectors with expression cassettes flanked by matrix attachment regions to reduce the risk of transgene silencing may preferably be used (Allen, et al. (2000) Plant Mol Biol. 43:361-376). Plant expression cassettes containing the FMV 34S promoter are preferably used to generate the transformation vectors.
  • the FMV promoter has several advantages over the related CaMV 35S promoter for these experiments because of its strong activity and uniform expression pattern in transgenic plants (Sanger, et al. (1990) Plant Mol. Biol. 14:433-443).
  • the only known FMV host is Scrophularia californica, thereby reducing the risk of silencing of the 34S promoter by pathogen infection of a crop species (Al-Kaff, et al. (1998) Science 279:2113-2115; Al-Kaff, et al. (2000) Nat. Biotechnol. 18:995-999).
  • transgenic plants Three types are generated; two that express the most effective Trx-aptamers as determined by the interference assays and a control line that expresses a Trx-aptamer fusion that does not bind to or interfere with Rep activity.
  • the transgenic plants are generated using an Agrobacterium transfer vector with a kanamycin selectable marker and a unique Not ⁇ site for direct cloning of the 34S-Trx-aptamer cassettes. Immunoblotting techniques are used in combination with an anti-HA antibody to assess Trx-aptamer expression in a minimum of 40 primary transformants for each construct. Seed from expressing lines is tested for 3:1 segregation on kanamycin and insert copy number on DNA gel blots. The identification of lines with single copy inserts is necessary to minimize any effects due to gene silencing in later experiments.
  • T1 plants of N. benthamiana lines that test positive for Trx- aptamer expression are co-inoculated with Agrobacterium carrying T-DNAs with partial tandem copies of wild-type TGMV A or B DNA (Elmer, et al. (1988) Plant Mol. Biol. 10:225-234).
  • Agroinoculation results in TGMV infection frequencies of greater than 95%.
  • a minimum of 10 kanamycin-resistant plants for each line are inoculated to allow statistical analysis of the data. Plants are monitored for the appearance and severity of symptoms to determine if any of the lines expressing the Trx-aptamers are asymptomatic or show delayed or attenuated symptoms.
  • TGMV DNA levels are monitored in each plant on squash blots to determine if there is a reduction in viral DNA accumulation (Orozco and Hanley-Bowdoin (1996) supra). Those lines that show resistance or tolerance at the T1 generation are analyzed in subsequent generations to assess the stability of the trait. Transgenic lines that are resistant to TGMV infection are also challenged with ACMV, EACMV and TYLCV to assess the breadth of the trait. These viruses are inoculated by Agrobacterium or biolistics. Infectious clones of CbLCV (Hill, et al. (1998) Plant Cell 1 :1057-1067; Kong and Hanley-Bowdoin (2002) supra), ACMV and EACMV (Sangare, et al.

Abstract

L'invention concerne des polypeptides et des protéines de fusion qui se lient aux virus eucaryotes, notamment aux virus d'ADN à brin unique eucaryotes (ssDNA). Ces polypeptides et protéines de fusion se lient aux protéines d'initiation de réplication (Rep) des virus ssDNA et, facultativement, inhibent la réplication virale et/ou l'infection virale. Ce virus peut être un agent pathogène des plantes ou un agent pathogène des animaux.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007019532A2 (fr) * 2005-08-04 2007-02-15 North Carolina State University Aptamères peptidiques se liant aux protéines rep de virus ssdna
US9371564B2 (en) 2008-08-08 2016-06-21 Bayer Bioscience N.V. Methods for plant fiber characterization and identification

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ACH R.A. ET AL: 'RRB1 and RRB2 Encode Maize Retinoblastome-Related Proteins That Interact with a Plant D-Type Cyclin and Geminvirus Replication Protein' MOL BEL BIOL vol. 17, no. 9, September 1997, pages 5077 - 5086, XP000973560 *
GRAFI ET AL: 'A Maize cDNA Encoding a Member of the Retinoblastoma Protein Family: Involvement in Endoreduplication' PROC NATL ACAD SCI USA vol. 93, August 1996, pages 8962 - 8967, XP002042542 *
KONG ET AL: 'A Geminivirus Replication Protein Interacts with a Protein Kinase and a Motor Protein that Display Expression Patterns During Plant Development and Infection' PLANT CELL vol. 14, August 2002, pages 1817 - 1832, XP002986033 *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007019532A2 (fr) * 2005-08-04 2007-02-15 North Carolina State University Aptamères peptidiques se liant aux protéines rep de virus ssdna
WO2007019532A3 (fr) * 2005-08-04 2007-11-22 Univ North Carolina State Aptamères peptidiques se liant aux protéines rep de virus ssdna
US8168748B2 (en) 2005-08-04 2012-05-01 North Carolina State University Peptide aptamers that bind to the rep proteins of ssDNA viruses
US9102705B2 (en) 2005-08-04 2015-08-11 North Carolina State University Peptide aptamers that bind to the rep proteins of ssDNA viruses
US9371564B2 (en) 2008-08-08 2016-06-21 Bayer Bioscience N.V. Methods for plant fiber characterization and identification

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