MXPA01003422A - Geminivirus resistant transgenic plants - Google Patents

Abstract

Transgenic plants with increased resistance to geminivirus infection, and nucleic acid constructs useful in producing such plants, are described. The transgenic plants express a mutant AL3/C3 geminivirus protein, which increases resistance to infection by at least one geminivirus, compared to a non-transformed control plant.

Description

TRANSGENIC PLANTS RESISTANT TO GEMINIVIRUS FIELD OF THE INVENTION The present invention relates to transgenic plants with increased resistance to infection by geminivirus, and to nucleic acid constructs useful for producing said plants. Transgenic plants express a mutant AL3 / C3 protein of geminivirus which increases resistance to infection by geminivirus.

BACKGROUND OF THE INVENTION Geminiviruses are a large and diverse family of plant DNA viruses with circular single-stranded DNA (ss) genomes that replicate through double-stranded circular DNA intermediaries. See Lazarowitz, Crit. Rev. Plant Sci. 11: 327 (1992); Timmermans et al., Annu. Rev. Plant Physiol. 45:79 (1994). Viral DNA replication, which results in both single-strand and double-strand viral DNA in large quantities, involves the expression of only a small number of viral proteins that are involved in either replication or viral transcription . Geminiviruses seem to rely primarily on host machinery to copy their genomes and express their genes, including the nuclear DNA and RNA polymerases of their plant hosts. These Geminivirus properties are rare among plant viruses, most of which are RNA viruses or that replicate through RNA intermediates using the replicase encoded by the viruses. Geminiviruses infect a wide variety of plants and cause considerable harvest losses worldwide. Geminiviruses are divided based on the host type, whether monocotyledonous or dicotyledonous, genomic structure, and insect vector. The subgroup I of geminivirus (also known as Mastrevirus) is transmitted by grasshoppers and infects mainly the monocotyledons, although it is known that a subgroup I of the geminiviruses infects the dicotyledons. The subgroup II of geminivirus (also known as Curtovirus) is transmitted by grasshoppers and infects the dicotyledons. The subgroup III of geminivirus (also known as Begomovirus) is transmitted by white mosquitoes and infect dicots. Viruses of subgroups I and II have genomes that comprise a single-stranded ssDNA component; The subgroup III of geminivirus typically has a bipartite genome comprising two DNAs of similar size (usually referred to as A and B), as illustrated by African casava mosaic virus (ACMV), tomato golden mosaic virus ( TGMV) and potato yellow mosaic virus. However, there are known monopartite geminiviruses that infect dicotyledons, for example the yellow leaf curve virus of tomato (TYLCV). The monopartite genomes of subgroup II and III of geminivirus have a gene array similar to that of the genes AL1, AL2 and AL3 found in DNA A components of the bipartite subgroup III of geminivirus. Subgroup III viruses are also divided into "old world" and "new world" viruses, a division based on evolutionary divergence. For the successful infection of plants by bipartite geminiviruses, both genomic component A and B are required. Examples of subgroup II and III of geminivirus include African casava mosaic virus (ACMV) and tomato golden mosaic virus. (TGMV). TGMV, similar to ACMV, is composed of two circular DNA molecules of the same size, both of which are required for infection. Sequence analyzes of the two genomic components reveal six open reading frames (ORFs); Four of the ORFs are encoded by DNA A and two by DNA B. In both components, the ORFs diverge from an intergenic region of 230 conserved nucleotides (common region) and are bidirectionally transcribed from a double replicative DNA form. strand. The ORFs are named according to the genome component and orientation relative to the common region (ie, left versus right (UR), or sense of the virion against the complementary sense of the virion (V / C)). It is known that certain proteins are involved in the replication of viral DNA (REP genes). See, for example, Elmer et al., Nucleic Acids Res. 16: 7043 (1988); Hatta and Francki, Virology 92: 428 (1979). The genome A component contains all the viral information necessary for the replication and encapsidation of viral DNA while the Component B codes for the functions that are required for the movement of the virus through the infected plant. The DNA A component of these viruses is capable of performing autonomous replication in a plant cell in the absence of B DNA when inserted in greater length than full-length copies within the plant genome, or when a copy is introduced transiently within the plant cells. In the genomes of monopartite gemínívirus. the unique genomic component contains all the viral information necessary for the replication, encapsidation, and movement of the virus. Geminivirus causes substantial losses among economically important crops, including tomatoes, beans and cucurbits. Current strategies to control the infection of geminiviruses are directed to the insect vectors that carry the viruses. However, the use of insecticides to control or fight a geminivirus infection can be expansive and inefficient. Additionally, host insects can vary in their susceptibility to available insecticides, and over time they can develop resistance to insecticides. See Markham et al., Pestic. Sci. 42: 123 (1994). Several methods have been used in an attempt to generate plants resistant to geminiviruses, including the classical methods of intermixing and transgenesis, with limited success. Unlike plant RNA viruses, the introduction of geminivirus sequences into transgenic plants does not confer resistance and, conversely, frequently results in the production of functional viral proteins.

((Hayes and Buck, Nucleic Acids Res. 17: 10213 (1989); Hanley-Bowdoin et al., Proc. Nati, Acad. Sci. USA (1990).) Kunik et al. Reported transgenic tomatoes containing a geminivirus cover protein (Kunik et al., BioTechnology 12: 500 (1994)). The expression of antisense RNAs against DNA replication proteins in transgenic plants reduces the level of viral DNA accumulation in more 70% (Day et al., Proc. Nati, Acad. Sci. USA 88: 6721 (1991)), at a level that is still sufficient to confer wild-type viral symptoms (Hanley-Bowdoin et al., Plant Cell 1: 157 (1989)) .- Similarly, the presence of defective replicons that interfere in transformed plants can reduce the level of viral DNA accumulation by about 70% (Frischmuth and Stanley, Virology 200: 826 (1994)). Antisense RNAs and defective replicons that interfere work best against their cognate viruses (Bejarano et al., Plant Mol. Bi. Ol. 24: 241 (1994)), also limited their utilities. Aptisense RNA directed to Rep protein mRNA (encoded by the C1 gene) was used by Bendahmane and Gronenborn to produce transgenic Nicotiana benthamiana plants with altered responses to TYLCV. Plant Mol.Biol. 33: 351 (1997) Therefore, it is convenient to design new strategies to control the infection by geminivirus.

BRIEF DESCRIPTION OF THE INVENTION A first aspect of the present invention is a plant containing transformed plant cells, which contain a heterologous nucleic acid construct comprising a promoter operable in plant cells, a nucleic acid sequence encoding an AL3 / C3 mutant protein, and a termination sequence. The expression of the AL3 / C3 mutant protein increases the resistance of the plant to the infection of at least one geminivirus, compared to the control, compared to the non-transformed control. A further aspect of the present invention is a tomato plant containing transformed plant cells, which contain a heterologous nucleic acid construct comprising a promoter operable in plant cells, a nucleic acid sequence encoding an AL3 mutant protein / C3, and a termination sequence. The expression of the AL3 / C3 mutant protein increases the resistance of the plant to the infection of at least one geminivirus, compared to the non-transformed control. A further aspect of the present invention is a plant of the Solanaceae family that contains transformed plant cells, which contains a heterologous nucleic acid construct comprising a promoter operable in plant cells, a nucleic acid sequence encoding a mutant protein AL3 / C3, and a termination sequence.

The expression of the AL3 / C3 mutant protein increases the resistance of the tomato plant of at least one geminivirus, compared to the non-transformed control. A further aspect of the present invention is a method of combating infection by geminiviruses in an agricultural field, by planting the field with a crop of plants comprising transformed plant cells, wherein the transformed plant cells contain a heterologous nucleic acid construct. comprising a promoter operable in plant cells, a nucleic acid sequence encoding an AL3 / C3 mutant protein, and a terminator sequence. The expression of the AL3 / C3 mutant protein increases the resistance of the plant to the infection of at least one geminivirus, compared to the untransformed control. A further aspect of the present invention is a method for making a transgenic plant that has increased resistance to infection by geminivirus, by transforming a plant cell with a DNA construct comprising a promoter, a nucleic acid sequence encoding a AL3 / C3 mutant protein, a termination sequence. A plant is generated from the transformed plant cell and the expression of the AL3 / C3 mutant protein increases the resistance of the plant to the infection of at least one geminivirus, compared to the untransformed control.

A further aspect of the present invention is a nucleic acid construct containing a promoter operable in a plant cell, a nucleic acid sequence encoding an AL3 / C3 mutant protein, and a terminator sequence located towards the 3 'end of the nucleic acid. of the nucleic acid sequence. A further aspect of the present invention is a method for producing a nucleic acid construct useful for improving resistance to geminvirus in plants. The method includes the identification of mutants of an AL3 / C3 protein of geminivirus that improves the resistance to geminivirus in plant cells when expressed in them, and prepare a nucleic acid construct containing an operable promoter in a plant cell, a sequence of nucleic acid encoding the AL3 / C3 mutant protein, and a terminator sequence located at the 3 'end of the nucleic acid sequence.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A provides a genomic map of the representative bipartite geminivirus (African casava mosaic virus (ACMV)). The components of DNA A and DNA B are shown with the location of the coding regions in the virion sense (AR1, AR2) and in the complementary sense (AL1 m AL2, AL3, AL4). The intergenic sequence is shaded.

Figure 1B provides a genomic map of a representative monopartite geminivirus (corn line virus (MSV)). The location of the coding regions in the direction of the virion (V1, V2) and in the complementary sense (C1, C2) is shown. The LIR refers to the long intergenic region and SIR refers to the small intergenic region. Figure 2 provides a comparison of the sequences of the thirteen AL3 / C3 proteins using the predicted EMBL program, which assigns a similar value to each amino acid position and predicts the secondary structure of the protein based on all protein sequences. The thirteen AL3 / C3 proteins show a general similarity value of about 80% and include the release of predicted a-helix structure sections. Figure 3 compares the sequences of sixteen AL3 / C3 proteins of geminivirus and the consensus sequence. Figure 4 provides the sequence of thirty-one C3 mutants TYLCV. Figure 5 provides the sequence of six AL3 TGMV mutants.

DETAILED DESCRIPTION OF THE INVENTION The present invention will now be more fully described hereinafter with references to the accompanying drawings, in which preferred embodiments of the invention are shown.

The present method uses the expression of a transdominant mutant of the geminivirus accessory replication protein, AL3 / C3 (also known as Ren), to confer increased resistance to geminivirus in transgenic plants. Although not wishing to be restricted to a single theory, the present invention hypothesizes that the mutant proteins may interfere with the replication activity of the wild-type proteins produced by the infection of geminvirus, reducing the replication of infectious viruses and leading to improved resistance. . Alternatively, the mutant proteins can interact with proteins that are required at the level of the total plant, but which are not required at the level of plant cells. The AL3 / C3 proteins do not work in a virus-specific manner and thus, the mutant versions are useful for producing transgenic plants with improved resistance to multiple geminiviruses. The inventors of the present invention determined that transgenic plants expressing AL3 / C3 mutant trans-dominating geminivirus proteins have increased resistance to infection to several geminiviruses. The AL3 / C3 proteins of geminivirus that are closely related to geminiviruses (unlike AL1 / C1 proteins) are functionally interchangeable, and thus the present method results in improved resistance to several infections by geminivirus. The Geminiviridae family consists of three groups that differ with respect to the vector insect, host type and genome structure. Subgroup I includes the viruses transmitted by the jumping leaf that usually they infect monocotyledonous plants and have genomes of singular components. Subgroup III includes viruses transmitted by white mosquitoes that infect dicotyledonous plants and most commonly have bipartite genomes. The viruses of subgroup II are transmitted by skipping and have genomes of singular components similar to subgroup I, but infect dicotyledonous plants such as subgroup III. The members of the three subgroups use similar replication and transcription strategies, although there are differences. Geminiviruses have small genomes that consist of either one or two ss DNA molecules that have a size range of about 2.5 to about 3 X 103 nucleotides. The genomic DNA contains various coding sequences separated by 5 'intergenic regions. The coding capacity of the genomes varies among the different subgroups. The subgroup of viruses I specifies four open reading frames for polypeptides greater than 10 kDa, while the viruses of subgroups II and III encode six to seven open reading frames. Currently there are two nomenclatures for geminivirus genes. The first nomenclature identifies the viral genes as specified by the virion (V) or the complementary sense (C) of the DNA strands, while the second designates the genes with respect to the left (L) or right (R) ) of the 5 'ether region (see figures 1A and 1B). The designations C and L are equivalent, as well as the designations V and R.

The genomes of geminivirus subgroups III typically consist of two DNA components, designated A and B. Both components are required for efficient infection of host plants. Component A encodes all the information necessary for viral replication and encapsidation, while component B can not replicate in the absence of DNA A, but is required for the systemic movement of viruses and the production of symptoms in infected plants . Component A typically contains five open reading frames (ORFs), four of which (AL1 / C1, AL2 / C2, AL3 / C3 and AL4 / C4) are specified by sequences that overlap the complementary strand. Mutations in the AL3 gene result in severe retardation and attenuated symptoms (Morris et al., J. Gen. Virol. 72: 1205 (1991): Etessami et al., J. Gen. Virol. 72: 1005 (1991 ); Sung and Coutts, J. Gen. Virol. 75: 1773 (1995)). The proteins AL1 / C1 (Rep) and AL3 / C3 are involved in the replication of geminivirus, and the proteins AL1 / C1 (Rep) and AL2 / C2, act on the regulatory gene expression of the virus. It is known that the mutation of the open reading frame AL1 blocks viral replication, whereas an AL3 mutant results in reduced levels of DNA (Sunter et al., Virology 179: 69 (1990); Sung and Coutts, J. Gen Virol. 76: 1773 (1995)). Additionally, transgenic plants that contain the AL1 gene and that constitutively express the Rep protein in the absence of AL3 maintain DNA B replication, demonstrating that Rep is sufficient for replication in the presence of host factors (Hanley-Bowdoin et al. , Proc. Nati.

Acad. Sci. USA 87: 1446 (1990); Elmer et al., Nucleic Acids Res 16: 7043 (1988)). The AL3 / C3 protein improves the viral DNA accumulation of subgroup II and III of geminivirus through an unknown mechanism. AL3 TGMV is located in the nucleus of cells of infected plants at levels similar to that of Rep protein (Nagar et al., Plant Cell 7: 705 (1995)). Two protein interactions have been demonstrated for AL3 TGMV and BGMV: oligomerization and interaction with Rep (Settlage et al., J. Virol. 70: 6790 (1996)). None of these interactions exhibits virus specificity, consistently with the ability of proteins of different AL3 / C3 to functionally substitute in replication assays (Sunter et al., Virology 203: 203 (1994); Gladfelter et al., Wro / Oay239 : 186 (1997)). The genome of all geminiviruses employs the same general strategy for duplication and expression: a rolling circle replication system is amplified in ssDNA and produces dsDNAs that serve as cores for replication and transcription. The double strands of DNA are transcribed divergently from the 5 'intergenic region which also includes the origin of replication in the plus strand. Rolling circle replication is a two-step process; the synthesis of the leader strand and the delayed strand of DNA are separate events. The "plus" strand of a single strand is first used as a template for the synthesis of the "minus" strand, resulting in a double-stranded (RF) replicative form. The replicative form then serves as tempered for the synthesis of the plus strand to generate the free ssDNA. A specific nick site for the start of the synthesis of the DNA plus strand (a mark of the rolling circle replication systems) The synthesis of the minus strand is initiated by the RNA that most of the time is generated by a cc complex / primasa. (The plus strand corresponds to the strand of the virion that is found in both ssDNA and dsDNA) The minus strand is the complementary strand found only in dsDNAs. Thus, the replication of geminivirus requires two origins, one of the synthesis of the plus strand and another of the synthesis of the minus strand. The origin of the plus strand of the three subgroups of geminiviruses has been mapped in the 5 'intergenic region, which also contains the promoters for sense virion transcription and complementary sense. The cis elements that mediate viral replication and transcription are best characterized by subgroup III of geminivirus, TGMV. Geminiviruses fall into three groups based on their insect vectors, host type and genomic structure. Most viruses that infect dicots have two genomic components, designated A and B, and are transmitted by white flies. Most of the time, the components of the single monopartite genome, of geminiviruses that infect dicotyledons, resemble the A components of bipartite viruses. The components of the genome are similarly arranged with 5 'intergenic regions that separate divergent transcription units. The 5 'intergenic regions contain the viral origin of replication (Revington et al., Plant Cell 1: 958 (1989); Lazarowitz et al., Plant Cell 4: 799 (1992)) and the transcription signals (Eagle et al., Plant Cell 6: 1157 (1994)). The bipartite geminiviruses that infect dicotyledons encode two replication proteins, AL1 and AL3, and supply the missing replication machinery of the host plant. For monopartite geminiviruses that infect dicotyledons such as yellow tomato coil virus (TYLCV), the equivalent proteins are designated C1, and C3, respectively. The AL1 protein is the only viral protein essential for viral replication (Elmer et al., Plant Mol. Biol. 10; 225 (1988); Hayes and Buck, Nucleic Acids Res. 17: 10213 (1989); Hanley-Bowdoin et al., Proc. Nnatl. Acad Sci USA 87: 1446 (1990)). Nagar et al. reported that AL1 induces the synthesis of the host replication machinery in cells of infected plants (Nagar et al., Plant Cell 7: 705 (1995)). The AL3 protein is not required for replication, but it improves the levels of viral DNA accumulation (Etessami et al., J. Gen. Vitrol., 72: 1005 (1991); Morris et al., J. Gen Virol. 72: 1205 (1991)). No RNA has been detected that specifies only the OR3 of AL3, suggesting that the gene product of AL3 is translated from an internal ORF. The present methods utilize the expression of a partially defective trans-dominant AL3 / C3 mutant protein in the transgenic plants. The AL3 / C3 mutant protein may interfere with the function of the wild counterpart, or with the replication of the AL1 / C1 viral core protein. Alternatively, the AL3 / C3 mutant protein may interfere with the capacity of the host plant to provide the necessary replication factors. Gronenbom and colleagues reported that N. Benthamiana plants express a C1 TYLCV mutant protein less susceptible to infection by TYLCV however, the trans-dominant AL1 mutants have two disadvantages. The AL1 proteins interact with DNA in a virus-specific manner (Fontes et al., Plant Cell 4: 597 (1992), Fontes et al., J. Biol. Chem. 269: 8459 (1994)), so that Trans-dominant mutants probably act in a virus-specific manner (Fontes et al., Plant Cell 6: 405 (1994)) and exert strong selective pressure in the field for resistance-breaking viruses. Replication proteins in the host (Nagar, Plant Cell 7: 705 (1995)) through interaction with regulatory factors of the plant cell cycle (Ach et al., Mol. Cell / ./17: 5077 (1997 )), and the expression may be unstable in the transgenic plants The inventors of the present invention have determined that the AL3 / C3 protein of geminivirus is useful to produce transgenic plants with increased resistance to infection by geminivirus, using a mutant strategy tans-dominant.The inventors of this invention have determined that AL3 interacts with AL1 in a non-specific way to the virus, and interacts with itself. The present inventors use a mutant version of AL3 / C3, in which one or more of its functions are disorganized.

The methods of the present invention utilize nucleic acid constructs that encode mutant versions of naturally occurring geminivirus AL3 / C3 proteins. The term "mutated" as used herein refers to proteins or peptides and means that at least one amino acid in the wild type or naturally occurring protein or polypeptide sequence has been replaced with a different amino acid, or deletions from its sequence . Preferably at least 2 or more adjacent amino acids in the wild type sequence are replaced or deleted. The AL3 / C3 mutant proteins may contain from 2 to about 30, or more, replaced or deleted amino acids. As used herein, the term "AL3 / C3" protein refers to geminivirus proteins that are known in the art as AL3 / C3 proteins from subgroup III of geminivirus, and as C3 proteins in subgroup II of geminivirus. The subgroups II and III of geminivirus encode a protein that is identified by those skilled in the art, based on structure and / or function, such as the AL3 / C3 protein. As used herein, the term "AL3 / C3" as applied to the polypeptides includes fragments of the AL3 / C3 protein. As used herein, the term "AL3 / C3" is applied to nucleic acid sequences (including naturally occurring sequences and genes, and synthesized nucleic acid sequences) that refer to the sequences encoding an AL3 / C3 protein or naturally occurring polypeptide, or an AL3 / C3 mutated protein or peptide as described herein.

The AL3 / C3 mutated proteins and the polypeptides useful in the present methods are those which, when expressed in a plant cell, reduce the sensitivity of the cells (or a plant comprising said cells) to the infection by geminivirus. The AL3 / C3 mutated proteins and the polypeptides useful for the present methods are those which, when expressed in plant cells, increase or improve the resistance or tolerance of the cells (or a plant comprising said cells) to infection by geminivirus. As used herein, "sensitivity" of a plant to infection by geminivirus refers to the rate at which the symptoms of the infection by geminivirus develop, and the severity of the symptoms. Plants with reduced sensitivity to infection have a delayed development of symptoms and / or less severe symptoms to infection by geminivirus compared to those that occur that occur in control plants. How it is used here, "tolerance" refers to plants that are infected with and that contain geminivirus, but that do not show symptoms associated with viral infection. The tolerant corn plants are able to produce a good corn despite the infection of geminivirus. As used herein, plants "immune to infection" by gemini viruses are those in which virus replication is prevented. As used herein, plants "resistant" to infection by geminiviruses are those that show both immunity to infection and tolerance.

It will be apparent to those skilled in the art that the ability of a plant to survive and thrive when exposed to geminivirus is a continuum, from plants that are less sensitive to infection, those that are tolerant to infection, those that are resistant to geminivirus. A plant that shows improved resistance or tolerance to geminivirus infection is considered here that also shows reduced sensitivity to infection by geminivirus. In each case, the severity and / or speed of development of symptoms in plants with improved resistance (reduced sensitivity) to geminivirus is lower than that which occurs in a control plant. Sensitivity, tolerance or resistance to gemimivirus infection can be measured at the level of the plant cell or at the level of the singular plant (for example, by assessing the severity or rapidity of the development of symptoms), or at the level of the plurality of the plant (for example, when evaluating the prevalence and / or severity of the infection, on the field of the harvest). The sensitivity of transgenic plants can be evaluated by comparison with untransformed control plants of the same species. As used herein, the term "protein" and "polypeptides" are used interchangeably, and refer to a polymer of amino acids (dipeptide or greater) linked through peptide bonds. Thus, the term "polypeptide" includes proteins, oligopeptides, protein fragments, protein analogs and the like. The term "polypeptide" contemplates polypeptides as defined above that are encoded by acids nucleic acids, are produced recombinantly, isolated from an appropriate source, or synthesized. The mutated AL3 / C3 proteins useful in the methods of the present invention can be based on any AL3 / C3 protein that occurs naturally. A series of mutations directed to the site of any AL3 / C3 protein can be prepared and selected for its ability to improve its resistance to geminivirus (for example, using the tobacco protoplast complementation assays as described in the examples, below) . AL3 / C3 mutant proteins or polypeptides capable of reducing the sensitivity to infection by geminiviruses in transgenic plants according to the methods identified herein, and the nucleic acid constructs capable of encoding the mutant protein are prepared according to the methods known in the art, for use in transgenic plants produced with reduced sensitivity to (or increased resistance or tolerance to) geminivirus infection. As used herein, a "native" or "naturally occurring" nucleic acid sequence is a sequence found in non-transgenic tissues or cells. Native nucleic acid sequences are those which have not been artificially altered, such as by site-directed mutagenesis. Once the native nucleic acid sequence is determined, molecules having these sequences can be synthesized or produced using recombinant nucleic acid methods as are known in the art. As used here, a Native geminivirus nucleic acid sequence is one that can be isolated from geminivirus that occurs naturally. The AL3 / C3 mutant proteins of various geminiviruses are suitable for use in the present invention, including but not limited to: tomato golden mosaic virus (TGMV), tomato spotted virus, tomato yellow curl virus (TYLCV) tomato curl virus (TLCV), potato yellow mosaic virus (PYMV), African casava mosaic virus (ACMV), Indian cassava mosaic virus, bean golden mosaic virus (BGMV), virus in mosaic of bean dwarfism, pumpkin curl virus, cotton curl virus (CLCV), beet ripple virus (BCTV), texas pepper virus and capsicum virus huasteco. A preferred mutant protein or polypeptide is one in which one or more amino acid residues are replaced with alanine. The sequence of the AL3 / C3 protein of the virus! mosaic of the Indian cassava can be found in GenBank accession number Z24758 (Hong et al, J. Gen. Virol. 74: 2437 (1993)); for tomato golden mosaic virus in GenBank accession number K02029 (Hamilton et al, EMBO J. 3: 2197 (1984)); for the tomato mottle virus in GenBank accession number L14460 (Abouzid et al, J. Gen. Virol. 73: 3225 (1992)); for the taino tomato mottle virus in GenBank accession number AF012300 (Ramos et al, Plant Dis., in press); for potato yellow mosaic virus in GenBank number of accesses D00940 (Coutts et al, J. Gen. Virol. 72: 1515 (1991); Fontes et al, J. Biol. Chem. 269: 8459 (1994)); for the Texas capsicum virus Tamaulipas strain in GenBank accession number U57457 (Torres-Pacheco, Phytopathology, 86: 1186 (1996)); for the huasteco pepper virus in GenBank accession number X70418 (Torres-Pacheco et al, J. Gen. Virology 74: 2225 (1993)); for the bean golden mosaic virus in GenBank accession number M91604 (Gilbertson et al, Phytopathology, 81: 980 (1991)); for GenBank bean golden mosaic virus accession numbers L01635 and M88686 (Gilberstson et al., Phytopathology 81: 980 (1991); Gilberston et al., Plants Dis 75: 336 (1991); for mosaic virus of dwarfism of bean in GenBank access no m88179 (Gilbertson et al, Phytopathology 81: 980 (1991); and for pumpkin curl virus in GenBank accession number M38183 (Lazarowitz and Lazdins, Vitrology 180: 58 (1991)). The present methods are useful for producing transgenic plants with improved resistance to one or more of the geminiviruses listed above, or related.Unless otherwise mentioned, the nucleotide sequences are mentioned herein by unique strands only, in the 5 'direction 3 ', from left to right The nucleotides are represented here in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission The methods and constructions of the present invention are useful for r dicotyledonous plant species to produce plants with reduced sensitivity to infection by geminivirus. The Dicotyledons suitable for use in the practice of the present invention include plants of the families Fabaceae, Solanaceae, Brassicaceae, Rosaceae and Compositae. Examples of plant species suitable for transformation with the DNA constructs of the present invention include but are not limited to tobacco (Nicotiana tabacum), potato (Solanum tuberosum), soybean (Glycine max), tomato (Lycopersicon esculentum) , cassava (Manitol esculenta) beet, peanut (Arachis hypogaea) cotton (Gossypium hirsutum), citrus trees (Citrus spp.), corn (Zea mays), beans (for example, green beans (Phaseolus vulgaris) and lima bean ( Phaseolus limensis), peas (Lathyus spp.), Sugar beet, sunflower, carrot, celery, flax, pumpkins and other cruciferous plants, pepper, strawberry, lettuce, alfalfa, oats, wheat, rye, rice, barley, sorghum and cañola . Thus an illustrative category of plants which can be transformed with the constructions of the present invention are the members of the Solanaceae family, and a particular plant that can be transformed using the constructions of the present invention is cotton. A variety of techniques are available in the field for the introduction of DNA constructs within a host plant cell. These include, but are not limited to, Agrobacterium-mediated transfection, injection, electroporation, microparticle bombardment. In preferred embodiments, the plants are transfected using Agrobacterium-mediated transfection, on intact plants that are inoculated using microprojectiles carrying a nucleic acid construct according to the present invention. In practice, a crop comprising a plurality of plants of the invention can be planted together in an agricultural field. By "agricultural field" is meant a common place of land or a greenhouse. Thus, the present invention provides a method for providing a crop of transgenic plants. Those familiar with the recombinant DNA methods available in the art will recognize that one can employ a nucleic acid sequence encoding an AL3 / C3 mutant protein of the present invention, bound in sense orientation with operably linked regulatory regulatory sequences, to construct transgenic cells and plants. Regulatory sequences for the expression of nucleic acid sequences in the sense of orientation include any of the sequelae of translation start eukaryotes, in addition to the promoter and polyadenylation / transcription and termination sequences. The nucleic acid constructs (or "transcription cassettes") of the present invention include, 5 'to 3' in the direction of transcription, a promoter as described above. discussed above and, operatively associated with the promoter, a nucleic acid sequence encoding an AL3 / C3 mutant protein of the present invention. The construction may optionally contain a termination sequence that includes a signal of stoppage for RNA polymerase. Each of these regulatory regions should be able to operate on the tissue cells to be transformed. Any suitable transformation signal can be employed to carry out the present invention, examples of which include, but are not limited to, the nos terminators, the CaMV terminator, or active termination signals derived from the same gene as the initiation region. transcriptional or derived from different genes. The term "operatively associated", as used herein, refers to nucleic acid sequences in a single nucleic acid molecule, whose sequences are associated such that the function is affected by the others. Thus, a promoter is operatively associated with a nucleic acid sequence when it is capable of affecting the transcription of that sequence (i.e., the sequence is under the transcriptional control of the promoter). The promoter is said to be "at the 5 'end" of the sequence, which in turn is said to be "toward the 3' end" of the promoter. The various fragments comprise the various constructs, transcription cassettes, markers and the like which can be introduced consecutively by cleavage of restriction enzymes from an appropriate replication system, an insertion of the particular construct or fragment within the available site. After ligation and cloning the nucleic acid construct can be isolated from manipulations. All these techniques are widely exemplified in the literature (see, for example, J. Sambrook et al., Molecular Cloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring Harbor Laboratory)). The term "nucleic acid sequence" as used herein refers to DNA or RNA molecules, and more particularly to linear arrays of deoxyribonucleotides or ribonucleotides connected to one another by links, typically phosphodiester bonds, between the 3 'and 5-carbon carbons. 'of the adjacent pentoses. The term "promoter" refers to a region of the DNA sequence that incorporates the signals necessary for the efficient expression of a coding sequence. This may include sequences to which an RNA polymerase binds but is not limited thereto, and may include other sequences to which other regulatory proteins bind, together with regions involved in the control of protein translation. The promoters used to carry out the present invention can be promoters that are consecutively activated in the plant cell. Numerous constitutively active promoters are available that are operable in the plants. A preferred example is the 35S promoter of the fig mosaic virus (FMV), or the 35S promoter of the cauliflower mosaic virus (CaMV). Alternatively, the promoter may be a promoter that is spatially active or active only in a tissue specific to the plant (see, for example, U.S. Patent No. 4,459,252 for specific root promoters), or an implantable promoter (e.g. a promoter active in plants that is induced by specific conditions, such as wounds or infections by specific pathogens). The methods for making transgenic (or "recombinant") plants of the present invention, in general, involve first providing a plant cell capable of regeneration (the plant cell typically resides in a tissue capable of regeneration). The plant cell is then transformed with a DNA construct comprising a transcription cassette of the present invention (as described herein) and a transgenic plant of the transformed plant cell is generated. Transformation steps can be carried out by any suitable technique that is known in the art including but not limited to bombardment of the plant cell with microparticles that transport the transcription cassette, infecting the cell with an Agro acterium tumefaciens containing a plasmid carrying the transcription cassette, or any other suitable technique. Vectors which can be used to transform the plant tissue with the nucleic acid constructs of the present invention include both Agrobacterium vectors and ballistic vectors, as well as other suitable vectors known to those skilled in the art. Agrobacterium tumefaciens cells containing a nucleic acid construct of the present invention are useful in methods for making transformed plants. The plant cells are infected with Agrobacterium tumefaciens to produce a transformed plant cell, and then the The plant is regenerated from the transformed plant cell, according to methods known in the art. Numerous Agrobacterium vector systems useful for carrying out the present invention are known (see, for example, U.S. Patent No. 4,459,355, U.S.A Patent No. 4,795,855, U.S. Patent No. 4,940,838). The microparticles that carry constructions of the present invention, whose microparticles are suitable for the ballistic transformation of the plant cell, are also useful for making transformed plants of the present invention. The microparticle is directed into the plant cell to produce a transformed plant cell and the plant is regenerated from the transformed plant cell. Any suitable methodology and apparatus for the transformation of cells by ballistics can be used to practice the present invention. Exemplary apparatuses and methods are described in Sanford and Wolf, US patent. No. 4,945,050; in Christou et al., No. 5,015.58; and in Agracetus European Patent Application Publication No. 0 270 356, entitled "Plant Transformation Mediated by Pollen". The plant species can be transformed with the nucleic acid constructs of the present invention by transformation of plant protoplasts by DNA and the subsequent regeneration of the plant from the transformed protoplast according to methods known in the art. The fusion of the protoplasts of tobacco with liposomes containing DNA or via electroporation are known in the art (Shilleto et al., Methods in Enzymology, 153: 313-336 (1987)). As used herein, "transformation" refers to the introduction of exogenous nucleic acid molecules into cells to produce transgenic cells stably transformed with the exogenous nucleic acid. Transformed plant cells are induced to regenerate intact plants through the application of cell culture techniques or tissues that are known in the art. The method for plant regeneration is chosen to be compatible with the transformation method. The stable presence and orientation of the exogenous DNA in the transgenic plant can be verified by the Mendelian inheritance of the DNA sequence, as revealed by standard methods of DNA analysis applied to the progeny resulting from the controlled crosses. Any plant tissue capable of subsequent clonal propagation, either by organogenesis or embryogenesis, can be transformed with the constructions of the present invention. The term "organogenesis," as used herein, means a process by which rods and roots develop sequentially from meristematic centers. the term "embryogenesis" as used herein, means a process by which the offspring and roots develop together in a concerted manner (not sequentially), while forming somatic cells or gametes the particular tissue chosen will vary depending on the propagation system clonally available for, and most suitable for, particular species that will be transformed. Exemplary white tissues include leaf discs, pollen, embryos, cotyledons, hypocotyledons, megagametophytes, callus tissue, existing meristematic tissue (eg, apical meristems, axillary buds, and root meristems), and induced meristematic tissue (e.g. , cotyledon meristem and hypocotyledon meristem). The transgenic plants of the present invention can take a variety of forms. Plants can be chimeras of transformed cells and untransformed cells. Plants can be cloned transformants (for example, all cells are transformed to contain the transcription cassette); the plants can comprise grafts of transformed and non-transformed tissues. Transformed plants can be propagated by a variety of means known in the art, such as clonal propagation, or classical cross-linking techniques. The following examples are set forth to illustrate the present invention, and should not be considered as limiting thereof.

EXAMPLE 1 Construction of mutants TYLCV3 and TGMV AL3 Site-directed mutants AL3 TYLCV C3 and TGMV were generated containing substitutions of conserved amino acid residues. The mutants were analyzed in tobacco protoplasm complementation assays. The ALC3 / C3 proteins are highly conserved among the different geminiviruses. The subsequences of sixteen AL3 / C3 proteins (and the consensus sequence) are compared in Figure 3 using the EMBL forecast program (Rost and Sarder J. Mol. Biol 232: 585 (1993)). This program assigns a score similar to each amino acid position and predicts the secondary structure of the protein based on all the protein sequences. Figure 2 shows, for thirteen AL3 / C3 proteins, the similarity scores for each amino acid position (circle) with 100 indicating the identity of that position among the thirteen sequences. These thirteen AL3 / C3 proteins showed a general similarity of around 80% (dotted line). The charged amino acids are marked by full circles and conserved tyrosines or histidines are indicated by shaded circles. The structural prediction scores are also plotted in Figure 2 (tables) with 9 indicating a 90% probability that a given amino acid is part of a predicted structure. The reason predicted structural high probability is marked (handle, curl, helix). The thirteen AL3 / C3 proteins included long sections of predicted helix structure. This information was used to identify, group and prioritize mutations to be introduced into the C3 TYLCV coding sequence. Mutations were introduced into the coding sequences TY3V and AL3 TGMV by site-directed mutagenesis (Kunkel, Proc. Nati, Acad, Sci USA 82: 482 (1985)) and verified by DNA sequencing. The mutated coding sequences were subcloned into a plant expression cassette (pMON10018 or equivalent) containing the FMV promoter (fig mosaic virus) and the NOS terminator which are flanked by NotI restriction sites. The mutant coding sequences were cloned towards the 3 'end of the polyhedrin promoter of pMON27025, a transfer vector allowing the generation of recombinant DNA baculovirus in Escherichia coli (Luckow et al., J. Vitrol 67: 4566 (1993) ). Thirty-one C3 TYLCV mutants were produced (SEQ ID Nos: 18-48), as shown in Figure 4. Mutants were numbered in order of preparation; in some cases, the sequences of the mutants represented a combination or extension of newly created mutant sequences (e.g., mutant # 69 (SEQ ID NO: 45) incorporates the sequence changes of mutants # 21 and # 45 (SEQ ID Nos. : 21 and 33)). Six AL3 TGMV mutants were produced (SEQ ID NOS: 49-54), as shown in Figure 5.

EXAMPLE 2 Constructions TYLCV v TGMV A copy 1.5 of the TYLCV plasmid replicon with a C3 deletion of the open reading frame (pTYLC7) and an FMV / full length wild type C3 ORF / terminator promoter from a plant expression cassette were constructed and used to establish an assay of C3 complementation (Fontes et al., J. Biol. Chem. 269: 8459 (1994); Gladfelter et al., Virology 239: 186 (1997)). The TYLCV replicon with the expanded C3 ORF was replicated inefficiently when electroporated into tobacco protoplasts, and viral DNA accumulation was assayed by DNA-blot analysis. When the full length wild type C3 FMV / ORF promoter was co-introduced into protoplasts, it complemented the TYLCV C3 mutant replicon effect, resulting in high levels of viral DNA replication. An expression cassette of mutant fig mosaic virus (FMV) -C3-E9 that contained a truncated C3 TYLCV open reading frame (pTYLC77) was also constructed. Thirty-one mutants of open reading frames (FMB) -C3-E9 and the corresponding expression cassettes were constructed. All mutants were sequenced before subcloning; and the sequence is provided in Figure 4. Six mutants directed to the open reading frame site AL3 TGMV were constructed (SEQ ID Nos: 49-54); These corresponded to the mutants C3 TYLCV mC3 # 17, mC3 # 67, mC3 # 69, mC3 # 71, mC3 # 73 and mC3 # 75. See figure 5. The E35S-AL3-E9 expression cassettes corresponding to these six AL3 TGMV mutants were also constructed. Recombinant baculovirus transfer vectors for mutants of the AL3 TGMV proteins of SEQ ID NOs: 49-54 were constructed using techniques known in the art.

EXAMPLE 3 C3 TYLCV mutant proteins: complementation of the defective replicons C3 or AL3 A wild type of the TYLCV-DR clone (pTYLC2) was evaluated for its replication in tobacco protoplasts, and the clone was shown to be functional. A C1 mutant version of TYLCV failed to replicate in tobacco protoplasts, showing that TYLCV replication is C1 dependent and not an artifact. The expression cassettes containing the mutants C3 mC3 # 21, mC3 # 23, and the double mutant mC3 # 31 were compared in the replication assays based on the tobacco protoplast using the mutant replicon C3 TYLCV (pTYLCV7), to test the ability of C3 mutants to provide functional C3 in trans. No improvement in TYLCV replication was detected in the presence of mC3 # 17 or mC3 # 31, while both mC3 # 21 and mC3 # 23 showed low levels of complementation (about 50% of the wild type C3 activity based on phosphorimage analysis). (Data is not shown). These results demonstrate that the combination of mutations in mC3 # 17 and mC3 # 31 resulted in a non-functional protein. Due to the presence of the double mutation, mC3 # 31 can prove to be less subject to reversion and, thus, is more durable in the field. Plasmids containing the mutants C3 mC3 # 17, mC3 # 21, mC3 # 23, and double mutant mC3 # 31, were also compared in replication assays based on the tobacco protoplast using the AL3 TGMV mutant replicon, to evaluate their ability to complement a mutant AL3 TGMV replicon. Plant expression cassettes containing the C3 TYLCV mutant coding sequence were counteracted within the tobacco protoplast with a modified TGMV A replicon that included a deletion of 88 bp in the open reading frame of AL3, and were evaluated for replication by TGMVA by gel DNA blot. The same results were obtained both with this heterologous TGMV geminivirus system and with the TYLCV homolog system described above, where the concept of resistance strategies based on general support is supported. (Data is not shown). The twenty-two additional C3 mutant expression cassettes (nC3 # 33 to mC3 # 75) were analyzed in replication assays using the C3 (-) TYLCV replicon. The C3 mutants # 67, 69, 71 and 73 had no detectable levels of C3 activity. Mutants # 39, 45, 47, 53, 57 supported significantly less replication of TYLCV than that of wild-type expression cassette C3. Table 1. The mutants pTYLC51 to pTYLC75 were also evaluated in replication assays using the mutant replicon AL3 TGMV (heterologous geminivirus replicon). The same results were obtained with this heterologous geminivirus system as with the TYLCV homologous replication assay described above. These results indicate that the function of C3 is highly conserved.

TABLE 1 EXAMPLE 4 TGMV mutant proteins: complementation of defective replicons A plant expression cassette corresponding to AL3 TGMV was developed that complements both the defective replicons AL3 TGMV and BGMV. The TGMV AL3 mutant expression cassette was tested in the replication assay using the mutant replicon (pNSB5). The tobacco protoplasts that contained the defective replicon AL3 TGMV were transfected with either wild-type expression cassettes TGMV AL3, mAL3 # 17, mAL3 # 67, mAL3 # 69, mAL3 # 71, mAL3 # 73 or mAL3 # 75 and with the replicon pNSB5, and were analyzed for its replication by TGMV A by gel DNA blot. The AL3 mutants exhibited phenotypes similar to their C3 TYLCV counterparts. Four of the mAL3 mutant TGMV proteins (mAL3 # 67, mAL3 # 69, nAL3 # 71 and mAL3 # 73) were unable to complement the AL3 deletion in the TGMV replication assays (data not shown). Mutant mAL3 # 17 exhibited little complementation, while mAL3 # 75 was wild type.

EXAMPLE 5 Interference test with AL3 Titration of the wild-type expression cassette AL1 established that 4 μg of the wild-type cassette supported the maximal mean replication of TGMV B (10 μg) in the presence of the wild type expression cassette AL3 (20 μm). Between 6-8 μg, the AL1 expression cassette levels became saturated and thus could mask the interference. (Data is not shown). Titration of the wild-type expression cassette AL3 established that 2.5 μg of the wild-type cassette supported the maximum mean replication of the AL3 TGMV mutant replicon (10 μg) in the presence of the wild-type expression cassette AL1 TGMV (4 μg). At 5 μg, the levels of the AL3 expression cassette were saturated and could thus mask the interference, (data not shown). These transient replication assays identified conditions for testing the AL3 TGMV mutant proteins for their interference with wild-type AL3 function. Based on these results, the interference assays included 10 μg of the TGMV B replicon and 4 μg of each of the AL1 and the AL3 expression cassettes. These conditions ensure that no viral replication protein was in excess.

EXAMPLE 6 Interference tests-TGMV The interference assays were carried out using the conditions noted in example 5. Tobacco protoplasts were "transfected with 10 μg of the TGMV B replicon, 4 μg of the TGMV AL1 expression cassette, and an expression cassette for each wild type AL3. TGMV, mAL3 # 17, mAL3 # 67, mAL3 # 69, mAL3 # 71 or mAL3 # 73. Protoplasts were analyzed for TGMV replication by gel DNA blot.The wild type cassette AL3 was 2.5 μg and the AL3 mutant cassettes were 50 μg None of the AL3 TGMV mutants interfered with the wild-type in the transient assays containing a TGMV B replicon and an AL1 expression cassette (no data are shown) .The assays were carried out using 20-fold excess of the mutant against the wild type expression cassette AL3 The present experiment, in which AL1 was also provided from the expression cassette, exhibited the possibility that the lack of interference by AL3 was due to at the highest expression level of the AL1 protein of an amplified annealed. • EXAMPLE 7 Interference tests-TYLCV Titration of the wild-type expression cassette C3 established that 0.2 μg of the wild-type expression cassette C3 supported the maximum mean replication of the mutant replicon of 5 μg of C3 (-) TYLCV. TO 0. 5 μg, the levels of the C3 expression cassette are saturated and could probably prevent interference. These amounts were used in interference assays containing 10 or 200 times plus an excess of the mutant expression cassette C3. These tests were repeated in duplicate. No interference from replication was observed for any of the expression cassettes of the C3 mutant. (Data is not shown). The ability of the C3 TYLCV mutant expression cassette to interfere with the activity of wild type C3 expression cassette in the protoplast assays was evaluated. Interference was assayed using 10 μg of the C3 (-) TYLCV replicon, 0.21 μg of the wild-type expression cassette C3 TYLCV and the following quantities of the mutant expression cassette: 20 μg of the C3 TYLCV mC3 # 17 mutant, mC3 # 67, mC3 # 69, mC3 # 71 or mC3 # 73; 40 μg of the C3 TYLCV mutant mC3 # 17, mC3 # 67, mC3 # 69, mC3 # 71, mC3 # 73 or mC3 # 77. (The C3 mutants were chosen based on the above experiments showing that they do not complement the C3 mutant replicon in the protoplasts). No interference was detected with the 100 or 200 fold excess of none of the mutant expression cassettes. Based on these experiments, it was concluded that the protoplast interference assay based on TYLCV is not feasible.

EXAMPLE 8 Biochemical analysis of recombinant proteins It was estabed that mAL3 # 17, mAL3 # 67, mAL3 # 69, mAL3 # 71, mAL3 # 73 and mAL3 # 75 proteins are stably produced in insect cells (SF9 cells) using known techniques in e! field (Luckow et al., J. Virology 67: 4566 (1993); Settlage et al., J.Virology 70; 6790 (1996)) (data not shown). It was also shown that mAL3 # 17 and mAL3 # 75 interact with AL1 TGMV in insect cells (no data are shown). Thus, the lack of support from mAL3 # 17 and mAL3 # 75 to the improvement of TGMV replication in complementation trials is not due to poor protein production or inability to bind to AL1. These results support the use of mAL3 # 17 in geminivirus resistance strategies. The location of the mutant and wild-type proteins AL3 TGMV in insect cells was studied. Nuclear and cytoplasmic extracts were prepared from SF9 cells and analyzed for AL3 proteins by immunoblotting. These experiments showed that AL3 mutant proteins can be generated within the nucleus of insect cells (data are not shown).

The present experiments estabed that mAL3 proteins are stably produced in eukaryotic systems (insect cells), which indicate that their negative genotypes in replication assays are not due to lack of stable protein. These experiments also showed that the mAL3 mutant protein (mAL3 # 17, mAL3 # 67, mAL3 # 69, mAL3 # 71 and mAL3 # 73) interacts with the insect cells in AL1 TGMV. These experiments indicate that there are differences in the interaction affinities of the various mutants.

EXAMPLE 9 Negative effect of the trans-dominant enhancer-6/30798 The interaction between wild type AL3 TGMV and AL1 TGMV was detected and quantified in the yeast double hybrid assays (Fiéis and Song, Nature 340: 245 (1989)) using the GAL4 DNA binding domain and domain functions of GAL4 activation (data not shown). This assay is also useful for identifying AL3 and C3 mutants with increased affinity for AL1 or C1, respectively. The identified mutant proteins are evaluated for their enhanced trans-dominant negative effects in the transformed plants to express the mutant protein. Mutant proteins with increased affinity can be combined with a defective replication mutant C3 or AL3 and evaluated to improve the trans-dominant negative effects in plant recombination.

EXAMPLE 10 Tomato mottle virus The previous series of experiments were repeated using the genome of the tomato mottle virus (ToMoV), a bipartite geminivirus agronomically important. ALOM TOMOV mutant proteins were constructed and evaluated as described above, to identify mutants useful in the production of transgenic plants with increased resistance to geminivirus infections. The sequence of the AL3 protein of the tomato mottle virus is provided in GenBank no. L14460 (Abouzid et al., J. Gen. Vitrol. 73: 3222 (1992)) and is: MDSRTGELIT AHQAENGVYI WELENPLYFK IHRVEDPLYT RTRVYHVAIR FNHNLRKALH LHKAYLNFQV WTTSMTASGS IYLARFRYLV NMYLDQLGVI SINNWRAVR FATNRVYVNH VLENHSIKFK FY (SEQ! D NO: 17).

EXAMPLE 11 Field tests Transformation by Agrobacterium (using methods known in the art) was used to produce transformed tomato plants expressing mutant proteins C3 or AL3 as described above. The mutant constructs C3 and AL3 can chosen initially based on their inability to complement the defective geminivirus replicons (see examples 3 and 4, above). Transformed plants are planted in a field in an area that is experiencing a natural epidemic of infection by geminivirus, or are artificially exposed to infection by geminivirus in a controlled environment. For example, transformed and control plants can be planted in fields in areas of Florida that experience an epidemic of TYLCV-DR geminivirus infection. Of the transformed plants exposed to the infection by geminivirus, some will show no signs of infection while others will show delayed symptoms of infection, or a reduced severity of symptoms (as compared to non-transformed control plants). Compared to non-transformed (wild type) control plants, transformed plants will have fewer individuals showing signs of infection by geminivirus; in comparison with those transformed plants that show signs of infection by geminivirus, the symptoms will be delayed (on average) in comparison with the control plants, and / or they will be less severe (on average) in comparison with the control plants. The above examples are illustrative of the present invention, and should not be considered as limiting thereof. The invention is described by the following claims, with equivalents of the claims to be included herein.

LIST OF SEQUENCES < 110 > Hanley-Bowdoin, Linda Seattlage, Sharon < 120 > Transgenic Plants Resistant to Geminivirus < 130 > 5051-433 < 140 > < 141 > < 160 > 54 < 170 > Patentln Ver 2.0 < 210 > 1 < 211 > 137 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: C3 consensus sequence of geminivirus; Xaa indicates that there is no consensus amino acid. < 220 > < 221 > VARIANT < 222 > (26) < 220 > < 221 > VARIANT < 222 > (50) < 220 > < 221 > VARIANT < 222 > (77) < 220 > < 221 > VARIANT < 222 > (83) < 220 > < 221 > VARIANT < 222 > (87) < 220 > < 221 > VARIANT < 222 > (90) < 220 > < 221 > VARIANT < 222 > (113) < 223 > C3 consensus sequence of geminvirus: The amino acid residues that vary between geminiviruses are indicated as Xaa < 400 > 1 Met Val Met Asp Ser Arg Thr Gly Glu Leu lie Thr Ala His Gln Wing 1 5 10 15 Glu Asn Gly Val Tyr He Trp Glu He Xaa Asn Pro Leu Tyr Phe Lys 5 20 25 30 He Thr Arg Val Glu Asp Pro Pro Tyr Thr Arg Thr Arg He Tyr His 35 40 45 Thr Xaa Gl n He Arg Phe Asn His Asn Leu Arg Lys Wing Leu Gly Leu 50 55 60 His Lys Al a Phe Leu Asn Phe Gln Val Trp Thr Thr Xaa Gln Thr Wing 65 70 75 80 ? Q Ser Gly Xaa Thr Tyr Leu Xaa Arg Phe Xaa Tyr Leu Val Leu Lys Tyr 85 90 95 Leu Asp Asn Leu Gly Val He Ser He Asn Asn Val He Arg Ala Val 100 105 110 Xaa Phe Ala Thr Phe Asp Val Ser Tyr Val Thr He Asp Val Leu Glu 115 120 125 Asn His Glu He Lys Phe Lys Phe Tyr 130 135 < 210 > 2 15 < 21 1 > 131 < 212 > PRT < 213 > Isolated from the Dominican Republic TYLCV < 400 > 2 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn 1 5 10 15 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He He Be Ser 35 40 45 Gln He Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 He As As Asn Asn Val He Arg As Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 3 < 211 > 134 < 212 > PRT < 213 > Isolated Israeli TYLCV < 400 > 3 Met Asp Leu Arg Thr Gly Glu Tyr He Thr Wing His Gln Wing Thr Ser 1 5 10 15 Gly Val Tyr Thr Phe Gly He Thr Asn Pro Leu Tyr Phe Thr He Thr 20 25 30 Arg His Asn Gln Asn Pro Phe Asn Asn Lys Tyr Asn Thr Leu Thr Phe 35 40 45 Gln He Arg Phe Asn His Asn Leu Arg Lys Glu Leu Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe His He Trp Thr Thr Leu Gln Ser Pro Thr Gly 65 70 75 80 His Phe Leu Arg Val Phe Lys Tyr Gln Val Cys Lys Tyr Leu Asn Asn 85 90 95 Leu Gly Val He Ser Leu Asn Asn Val Val Arg Ala Val Asp Tyr Val 100 105 110 Leu Phe His Val Phe Glu Arg Thr He Asp Val Thr Glu Asn Hi s Glu 115 120 125 He Lys Phe Asn Phe Tyr "130 < 210 > 4 < 21 1 > 134 < 212 > PRT < 213 > Casava Mosaic Virus from India < 400 > 4 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Wing Gln Wing Met Asn 1 5 10 15 Gly Val Phe He Trp Glu Val Pro Asn Pro Leu Tyr Phe Lys He He 20 25 30 Gln His Asp Asn Arg Pro Phe Val Met Asn Gln Asp He He Thr Val 35 40 45 Gln He Arg Phe Asn His Asn Leu Arg Lys Ala Leu Gly Leu His Gln 50 55 60 Cys Trp Met Asp Phe Lys Val Trp Thr Leu Gln Pro Gln Thr Trp 65 70 75. 80 Arg Phe Leu Arg Val Phe Lys Thr Gln Val Leu Lys Tyr Leu Asp Ser 85 90 95 Leu Gly Val He Ser He Asn Thr He Val Lys Wing Val Glu His Val 100 105 110 Leu Tyr Asn Val He His Gly Thr Asp Arg Val Glu Gln Ser Asn Leu 115 120 125 He Lys Leu Asn He Tyr 130 < 210 > 5 < 211 > 134 < 212 > PRT < 213 > TYLCU < 400 > 5 Met Asp Leu Arg Thr Gly Glu Tyr He Thr Wing His Gln Wing Thr Ser 1 5 10 15 Gly Val Tyr Thr Phe Glu He Thr Asn Pro Leu Tyr Phe Thr He Thr 20 25 30 Arg His Asn Gln Gln Pro Phe Asn Ser Lys Tyr Asn Phe Leu Thr Phe 35 40 45 Gln He Arg Phe Asn His Asn Leu Arg Lys Ala Leu Gly He His Lys Cys Phe Leu Asn Phe Arg He Trp Thr Thu Leu Gln Ser Pro Thr Gly 65 70 75 80 His Phe Leu Arg Val Phe Arg Tyr Gln Val Tyr Lys Tyr Leu Asn Asn 85 90 95 He Gly Val He Ser Leu Asn Asn Val He Arg Ala Val Asp Tyr Val 100 105 110 Leu Phe Asp Val Phe Glu Asn Thr He Asp Val He Glu Gln His Glu 115 120 125 He Lys Tyr Asn Leu Tyr 130 210 > 6 < 211 > 134 < 212 > PRT < 213 > < 400 > 6 Met Asp Ser Arg Thr Gly Glu Pro He Thr Wing Arg Gln Wing Met Asn 1 5 10 15 Gly Glu.Tyr He Trp Arg Val Pro Asn Pro Leu Tyr Phe Lys He He 20 25 30 Lys His His Lys Arg Pro Phe Asn Tyr Asn His Asp He He Gln Val 35 40 45 Arg He Gln Phe Asn His Asn Leu Arg Arg Ala Leu Ala He His Lys 50 55 60 Cys Phe Leu Asp Phe Thr Val Phe Thr Arg Leu Gln Pro Ala Thr Trp 65 - 70 75 80 Arg Phe Leu Arg Val Phe Lys Thr Gln Val Met Lys Tyr Leu Asp Ser 85 90 95 Leu Gly Val He Ser He Asn Asn Val He Arg Ser Val Asp His Val 100 105 '110 Leu Tyr Asn Val Leu Asp Ser Thr Phe Asp Val He Glu Asp His Asp 115 120 125 Lys Lys Phe Asn Phe Tyr 130 < 210 > 7 < 211 > '134 < 212 > i DRT < 213 > TYLCM < 400 > '7 Met Asp Leu Arg Thr Gly Glu Tyr ne Thr Ala H-is Gln Ala Thr Ser 1 5 10 15 Gly Val Tyr Thr Phe Gly lie Thr Asn Pro Leu Tyr Phe Thr lie Thr 20 25 30 Arg His Asn Gln Asn Pro Phe Asn Asn Lys Tyr Asn Thr Leu Tnr Phe 35 40 45 Gln He Arg Phe Asn H-is Asn Leu Arg Lys Glu Leu Gly lie H-is Lys 50 55 60 Cys Phe Leu Asn Phe His lie Trp Thr Thu Leu Gln Ser Pro Thr Gly 65 70 75 80 His Phe Leu Arg Val Phe Lys Tyr Gln Val Cys Lys Tyr Leu Asn Asn 85 90 95 Leu Gly Val lie Ser Leu Asn Asn Val Val Arg Ala Val Asp Tyr Val 100 105 110 Leu Phe H-is Val Phe Glu Arg Thr lie Asp Val Thr Glu Asn His Glu 115 120 125 lie Lys Phe Asn Phe Tyr 130 < 210 > 8 < 211 > 134 < 212 > PRT < 213 > Indian casava mosaic virus < 400 > 8 Met Asp Leu Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Met Asn 1 5 10 15 Gly Val Tyr Thr Trp Glu He Asn Asn Pro Leu Tyr Phe Thr He Thr 20 25 30 Arg His Gln Gln Arg Pro Phe Leu Leu Asn Gln Asp He He Thr Val 35 40 45 Gin Val Arg Phe Asn His Asn Leu Arg Lys Glu Leu Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Leu Arg Pro Gln Thr Gly 65 70 75 80 Leu Phe Leu Arg Val Phe Arg Tyr Gln Val Leu Lys Tyr Leu Asp Asn 85 90 95 He Gly Val He Ser He Asn Asp Val He Arg Ala Wing Cys His Val 100 '105 110 Leu Phe Asn Val He Glu Lys Thr He Glu Cys Gln Leu Thr His Glu 115 120 125 He Lys Phe Asn Val Tyr 130 < 210 > 9 < 211 > 132 < 212 > PRT < 213 > Abutilon mosaic virus < 400 > 9 Met Asp Ser Arg Thr Gly Glu Phe lie Thr Val His Gln Wing Glu Asr. 1 5 10 15 Gly Val Tyr He Trp Glu He Wing Asn Pro Leu Tyr Phe Arg He Tyr 20 25 30 Lys Val Glu Asp Pro Leu Tyr Thr Arg Thr Arg He Tyr His Val Gln 35 40 45 He Arg Phe Asn His Asn Leu Arg Arg Ala Leu His Leu His Lys Wing 50 55 60 Tyr Leu Asn Phe Gln Val Trp Thr Thr Ser Met Thr Wing Ser Gly Ser 65 70 75 80 He Tyr Leu Asn Arg Phe Arg Arg Leu Val Asn Met Ty 'Leu Asp Gln 85 90 95 Leu Gly Val He Ser He Asn Asn Val He Arg Wing Val Gln Phe Wing 100 105 110 Thr Asn Arg Thr Tyr Val Asn Tyr Val Leu Glu Asn His Ser He Lys 115 120 125 Phe Lys Phe Tyr 130 < 210 > 10 < 211 > 132 < 212 > PRT < 213 > Bean dwarf mosaic virus < 400 > 10 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Leu Gln Wing Glu Asn 1 5 10 15 Gly Val Tyr He Trp Glu He Glu Asn Pro Leu Tyr Phe Lys He Tyr Arg Val Glu Glu Pro Leu Tyr Thr Asn Ser Arg Val Tyr Ser Val Gln OJ 40 45 He Arg Phe Asn His Asn Leu Arg Arg Ala Leu His Leu His Lys Wing 55 60 Phe Leu Asn Phe Gln Val Trp Thr He Ser Thr Thr Ala Ser Gly Ser 70 75 80 Thr Tyr Leu Asn Arg Phe Lys His Leu Val lie Met Tyr Leu Asp Gln 85 90 95 Leu Gly He lie Ser He Asn Asn Val He Arg Gly Val Arg Phe Wing 100 105 110 Thr Asp Arg Ser Tyr Val Thr His Val He Glu Tyr His Ser He Lys 115 1120"125 Phe Lys Leu Tyr 130 <210> 1 1 <211> 132 <212> PRT <213> Golden bean mosaic virus <400> 11 Met Asp Ser Arg Thr Gly Glu Asn He Thr Wing His Gln Wing Glu Asn 1 5 10 15 Ser Val Phe He Trp Glu Val Pro Asn Pro Leu Tyr Phe Lys He Met 20 25 30 Arg Val Gl u Asp Pro Al a Tyr Thr Arg Thr Arg He Tyr His He Gln '40 45 H e Arg Phe Asn His Asn Leu Arg Lys Ala Leu Asp Leu His Lys Wing 50 55 60 Phe Leu Asn Phe Gln Val Trp Thr Ser He Gln Wing Ser Gly Thr 65 70 75 80 Thr Tyr Leu Asn Arg Phe Arg Leu Leu Val Leu Leu Tyr Leu Hi s Arg 85 90 95 Leu Gly Val H e Gly H e Asn Asn Val H e Arg Ala Val Gln Phe Al a 100 105 110 Thr Asn Lys Ser Tyr Val Asn Thr Val Leu Glu Asn His Asp He Lys 115 120 125 Tyr Lys Phe Tyr 130 < 210 > 12 < 21 1 > 132 < 212 > PRT < 213 > Isolated Brazilian-BGMV < 400 > 12 Met Asp Ser Arg Thr Gly Glu Arg He Thr Wing Arg Gln Wing Glu Asn 1 5 10 15 Gly Val Tyr He Trp Glu He Ser Asn Pro Leu Tyr Phe Lys Met Tyr 20 25 30 Asn Val Glu Asp Leu Gln Tyr Thr Thr Arg Val Tyr His Leu Gln 35 40 45 He Arg Phe Asn His Asn Leu Arg Asn Lys Leu Gly Leu His Lys Wing 50 55 60 Phe Leu Asn Phe Gln Val Trp Thr He Ser Leu Gln Wing Ser Gly Thr 65 70 75 80 Thr Tyr Leu Asn Arg Phe Lys Tyr Leu Val Leu Leu Tyr Leu Asp Arg 85 90 95 He Gly Val He Ser Leu Asn Asn Val He Arg Ala Val Arg Phe Ala 100 105 110 Thr Asp Lys Ser Tyr Val Asn Tyr Val Leu Glu Asn His Glu He Lys 115 120 125 Tyr Lys Phe Tyr 130 < 210 > 13 < 211 > 132 < 212 > PRT < 213 > Huasteco pepper virus < 400 > 13 Met Asp Leu Arg Thr Gly Val Pro He Thr Wing Wing Gln Wing Wing Asn 1 5 1 í n0 15 Gly Val Phe He Trp Glu Leu Arg Asn Pro Leu Tyr Phe Lys He Arg 20 25 30 Leu Val Glu Thr Pro Met Tyr Thr Arg Ser Arg Val Phe His He Gln 35 40 45 Val Arg Ala Asn His Asn Met Arg Thr Ala Leu Gly Leu His Lys Ala 50 55 60 Tyr Phe Asn Phe Gln Val Trp Thr Thr Leu Thr Thr He Ser Gly Gln 65 70 • 75 80 He Tyr Leu Asn Arg Phe Lys Leu Leu Val Met Phe Tyr Leu Asp Asn 85 90 95 Leu Gly Leu He Ser Val Asn Asn Val Lie Arg Ala Val Ser Phe Ala 100 105 110 Thr Asp Lys Arg Tyr Val Asn Al a Val Leu Glu Asn Hi s Glu He He 115 120 125 Tyr Lys Leu Tyr 130 < 210 > 14 < 21 1 > 1324 < 212 > PRT < 213 > Yellow potato mosaic virus < 400 > 14 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Arg Gl n Wing Glu Asn 1 5 10 Í5 Gly Val Phe He Trp Glu He Glu Asn Pro Leu Tyr Phe Lys He Asn 20 25 30 Gl n Val Gl u Asp Met Gl n Tyr Thr Arg Thr Arg He Tyr Ser Val Gln 35 40 45 He Arg Phe Asn His Asn Leu Arg Arg Al a Leu Asp Leu Hi s Lys Ala 50 55 60 Tyr Leu Asn Phe Gl n Val Trp Thr Thr Ser Met Thr Wing Ser Gly Ser 65 70 75 80 Asn Tyr Leu Wing Arg Phe Arg Gln Leu Val Leu Leu Tyr Leu Asp Arg 85 90 95 Leu Gly Val He Ser He Asn Asn Val He Arg Ser Val Arg Phe Wing 100 105 110 Thr Asp Arg Ser Tyr Val Asn Tyr Val Leu Glu Asn His Ser He Lys 115 120 125 Tyr Lys Phe Tyr 130 < 210 > 15 < 21 1 > 134 < 212 > PRT < 213 > Virus pumpkin curved leaves < 400 > 15 Met Val Met Asp Leu Arg Thr Trp Asp Asp He Thr Val His Gln Al a 1- 5 10 5 Glu Asn Ser Val Phe He Trp Glu Val Pro Asn Pro Leu Tyr Phe Lys 20 25 30 Met Tyr Xaa Val Glu Asp Pro Leu Tyr Thr His Thr Arg He Tyr His n Arg Phe Asn His Asn Leu Arg Arg Ala Leu Asn Leu His "He Gl 60 50 55 Lys Wing Phe Leu Asn Phe Gln Val Trp Thr Glu Ser He Arg Wing Ser 65 70 G1y Thr Thr Tyr Leu Asn Arg Phe Arg His Leu Val Met Leu Tyr Leu 85 ASP Arg Leu Gly Val He Gly Leu Asn Asn Val He Arg Ala Val Ser 100 iUD Trp A Thr Asp Arg Ser Tyr Val Asn Tyr V.1 Leu Glu Asn His el. i, XD He Lys Phe Lys He Tyr 130 < 210 > 16 < 21 1 > 132 < 212 > PRT < 213 > Golden tomato mosaic virus < 400 > 16 Met Asp Ser Arg Thr Gly Glu Pro He Thr Val Pro Gln Ala Glu Asn 5 10 15 Gly Val Tyr He Trp Glu He Thr Asn Pro Leu Tyr Phe Lys He He 20 25 30 Ser Val Glu Asp Pro Leu Tyr Thr Asn Thr Arg He Tyr His Leu Gln 35 40 45 He Arg Phe Asn His Asn Leu Arg Arg Ala Leu Asp Leu His Lys Ala 50 55 5th Phe Leu Asn Phe Gln Val Trp Thr Thr Ser Thr Thr Ala Ser Gly Ara 65 70 75 80 Thr Tyr Leu Asn Arg Phe Lys Tyr Leu Val Met Leu Tyr Leu Glu Gln 85 go 95 Leu Gly Val He Cys He Asn Asn Val He Arg Wing Val Arg Phe Wing 100 105 no Thr Asp Arg Ser Tyr He Thr His Val Leu Glu Asn His Ser He Lys 115 120 125 Tyr Lys Phe Tyr 130 < 210 > 17 < 211 > 132 < 212 > PRT < 213 > Speckled tomato virus < 400 > 17 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing His G n Al a Gl u Asn 1 5 10 15 Gly Val Tyr He Trp Glu Leu Glu Asn Pro Leu Tyr Phe Lys H and His 20 25 30 Arg Val Glu Asp Pro Leu Tyr Thr Arg Thr Arg Val Tyr His Val Gln 35 40 45 lie Arg Phe Asn Hi s Asn Leu Arg Lys Ala Leu His Leu Hi s Lys Wing 50 55 60 Tyr Leu Asn Phe Gln Val Trp Thr Thr Ser Met Thr Wing Ser Gly Ser 65 70 75 80 He Tyr Leu Wing Arg Phe Arg Tyr Leu Val Asn Met Tyr Leu Asp Gln 85 90 95 Leu Gly Val He Ser He Asn Asn Val Val Arg Ala Val Arg Phe Ala 100 105 110 Thr Asn Arg Val Tyr Val Asn His Val Leu Glu Asn His Ser He Lys 115 120 125 Phe Lys Phe Tyr 130 < 210 > 18 < 21 1 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 15) < 400 > 18 Met Ala Ser Al to Thr Gly Glu Leu He Thr Al to Pro Gln Ala Glu Asn i 5 10 15 Gly Val Phe He Trp Glu H e Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He He Ser He 35 40 45 Gln He Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 He Ser As Asn Asn Val He Arg As Val Val Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 19 < 21 1 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 17) < 400 > 19 Met Asp Ser Arg Thr Gly Glu Leu He Thr Ala Pro Gln Ala Glu Asn 1 5 10 15 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Wing Wing He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He He Be He 35 40 45 Gln He Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 He As As Asn Asn Val As Arg As Val Asp His Val Leu As Asp 105 105 110 Val Leu Gl As As Thr As Asn Val Thr Glu Thr His Asp H e Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 20 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 19) < 400 > 20 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn 1 5 10 15 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp lie He Be He 35 40 45 Gln He Arg Phe Asn His Asn He Wing Wing Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 lie Ser He Asn Asn Val He Arg Ala Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 21 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 21) < 400 > 21 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn 1 5 10 15 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He He Be Ser 35 40 45 Gln He Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lvs 50 55 60 Cys Phe Leu Asn Phe Arg He Ala Wing Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 He Be He As Asn Asn Val He Arg Ala Val Asp His Val Leu Tyr Aso 100 105 no Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 22 < 21 1 > 130 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: TYCLV C3 Mutant (mC3 # 23) < 400 > 22 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn 1 5 10 15 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gl n Arg Pro Phe Leu Met Asn His Asp He He Be Ser 35 40 45 Gl n He Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Gln Thr Gly Arg Phe Leu Arg 65 70 75 80 Val Al a Al to Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val He 85 90 95 Ser He Asn Asn Val He Arg Al to Val Asp His Val Leu Tyr Asp Val 100 105 110 Leu Glu Asn Thr He Asn Val Thr Gl u Thr His Asp He Lys Tyr Lys 115 120 125 Phe Tyr 130 < 210 > 23 < 21 1 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 25) < 400 > 23 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn 1 5? O 15 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 2 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He He Be Ser 35 40 45 Glri lie Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lys 50 55. 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 go 95 He Ser He Ala Ala Val He Arg Ala Val Asp His Val Leu Tyr ASD 100 105 no Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 24 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 27) < 400 > 24 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp Hi s Ser Gl n Arg Pro Phe Leu Met Asn His Asp He He Ser Ser 35 40 45 Gln lie Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 He Ser He Asn Asn Val He Arg Ala Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Ala Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 25 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 29) < 400 > 20 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn 1 5? O 15 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He He Ser Ser 35 40 45 Gln He Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lvs 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 0 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 go 95 He Be As Asn Asn Val He Arg Al As Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr He Asn Val Thr Gl u Thr Hi s Asp H e Lys Al a < 210 > 26 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 31) < 400 > 26 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Al a Gl u Asn 1 5 10 15 Gly Val Phe le Trp Glu lie Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He lie Be He - 35 40 45 Gln He Arg Phe Asn His Asn He Arg Lys Val Met Gly He Hi s Lys 50 55 • 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 Be Ser Wing Wing Val He Arg Wing Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 27 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 33) < 400 > 27 Met Asp Ser Arg Thr Gly Ala Leu He Al to Ala Pro Ala Al to Glu Asn 1 5 10 15 Gly Val Phe lie Trp Gl u He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He Has Ser He 35 40 45 Gln He Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 lie Ser He Asn Asn Val He Arg Wing Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 28 < 21 1 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 35) < 400 > 28 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn 1 5 10 15 Gly Val Wing He Al a Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He He Ser Ser 35 40 45 Gln He Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 He Ser He As Asn Asn Val He Arg As Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp -He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 29 < 21 1 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 37) < 400 > 29 Met Asp Ser Arg Thr Gly Glu Leu ile Thr Ala Pro Gln Ala Glu Asn 1 5 10 '15 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Ala Ala Ala Leu Met Asp His Asp He Has Ser He 35 40 45 Gln He Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 8 800 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 lie Ser lie Asn Asn Val He Arg Ala Val Asp His Val Leu Tyr Asp 100 1? N05? 110 Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 30 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 39) < 400 > 30 * Asp Ser Arg Thr 67 and Glu L «euu lile? T mhrr AAliaa P Prroo 6G1lnn Wing Glu Asn 10 15 ^ V, Pte p T Glu Ile As ro Leü Tyr ptetjs ne Thr Asp His Ser Gln Arg Pro Phe Leu "• • sn H-is Asp He 3H0 35 ..... .- Met A e Ser I 40 le 45 Wing Wing Wing Asn His Asn I 50 Arg Lys Val Met Gly ne His Lys 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 He Ser He Asn Asn Val He Arg Ala Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 Lys Phe Tyr 130 < 21O > 31 < 211 > 131 '< 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 41) < 400 > 31 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn 1 5 10 15 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He He Ser Be He 35 40 45 Gln He Arg Phe Wing Wing Wing He Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 He Ser He Asn Asn Val He Arg Ala Val Asp His Val Leu Tyr Asp 100 105 -lio Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 32 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 43) < 400 > 32 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn 1 5 10 15 Gly Val Phe Lie Trp Glu He Asn Asn Pro 'Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He He Be Ser 35 40 45 Gln He Arg Phe Asn His Asn He Arg Lys Val Met Gly He Wing Wing 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 He Ser He Asn Asn Val He Arg Ala Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 33 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 45) < 400 > 33 net Asp Ser Arg Thr Gly Glu Leu lie Thr Wing Pro Gln Wing Glu Asn 1 5 10 15 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He He Ser He 35 40 45 Gl n He Arg Phe Asn His Asn He Arg Lys Val Met Gly He Hi s Lys 50 55 60 Cys Al a Leu Ala Wing Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 He Ser lie Asn Asn Val He Arg Ala Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 34 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 47) < 400 > 34 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn 1 - 5? Or 15 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He He Ser Be He 35 40 45 Gln He Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Wing Wing Leu Wing Being Leu Gly Val 85 90 95 He Be As Asn Asn Val He Arg Wing Val Asp His Val Leu Tyr Asp 100 105 no Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 35 < 21 1 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 49) < 400 > 35 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn 1 5 10 15 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gl n Arg Pro Phe Leu Met Asn His Asp He He Be Ser 35 40 45 Gln He Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 He Ser He Asn Asn Val He Arg Ala Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr He Asn Val Thr Ala Al a Ala Ala He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 36 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 51) < 400 > 36 Met Asp Ser Glu Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn 1 5 10 15 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp Hi s Ser Gl n Arg Pro Phe Leu Met Asn His Asp He Has Ser He 35 40 45 'Gln He Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 55 70 75 80 Arg Val Phe Arg Tyr Glu Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 H e Ser He Asn Asn Val He Arg Wing Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 37 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV(mC3 # 53) < 400 > 37 Met Asp Ser Arg Thr Gly Lys Leu He Thr Wing Pro Gln Wing Glu Asn 1 5 10 15 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He He Be Ser 35 40 45 Gln He Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 He Ser He Asn Asn Val He Arg Ala Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 38 < 21 1 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 55) < 400 > 38 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn 1 5 10 15 Gly Val Phe He Trp Gl u He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He He Be Ser 35 40 45 Gln He Glu Phe Asn His Asn He Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 He Ser He Asn Asn Val He Arg Ala Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 39 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 57) < 400 > 39 Met Asp Ser Arg Thr Gly Glu Leu lie Thr Ala Pro Gln Wing Glu Asn 1 5 10 15 Gly Val Phe lie Trp Glu lie Asn Asn Pro Leu Tyr Phe Lys lie Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp lie lie Ser lie 35 40 45 Gln He Arg Phe Asn His Asn lie Glu Lys Val Met Gly .lie His Lys 50 55 60 Cys Phe Leu Asn Phe Arg lie Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 lie Be lie Asn Asn Val He Arg Ala Val Asp His Val Leu Tyr Aso 100 105 no Val Leu Glu Asn Thr lie Asn Val Thr Glu Thr His Asp lie Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 40 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 59) < 400 > 40 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn '1 5 10 15 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He He Ser Ser He 35 40 45 Gln He Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Glu Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 He Ser He Asn Asn Val He Arg Ala Val Asp His Val Leu Tyr Asp 100 105 no Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lvs Tvr 115 120 125 Lys Phe Tyr 130 < 210 > 41 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 61) < 400 > 41 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn 1 5 10 15 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He He Ser He 35 40 45 Gln He Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 He Ser He Asn Asn Val lie Glu Ala Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 42 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 63 <400> 42 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asp 1 5 10 15 Gly Val Phe lie Trp Glu lie Asn Asn Pro Leu Tyr Phe Lys lie Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp lie lie Ser 35 40 45 Gln lie Arg Phe Asp His Asn lie Arg Lys Val Met Gly lie His Lys 50 55 60 Cys Phe Leu Asn Phe Arg lie Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 lie Be lie Asn Asn Val lie Arg Ala Val Asp His Val Leu Tyr Asp 100 105 lio Val Leu Glu Asn Thr lie Asn Val Thr Glu Thr His Lys lie Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 43 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV '(mC3 # 65 <400> 43 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn 1 5? o 15 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He He Be Ser 35 40 45 Gln He Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 He Ser He Asn Asn Val He Arg Ala Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Glu Tyr 115 120 125 Lys Phe! Tyr 13C I < 210 > 44 < 21 1 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 67 <400> 44 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn 1 5 10 15 Gly Val Phe He Trp Glu He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He He Ser Ser 35 40 45 Wing Wing Wing Wing Wing Wing He Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65. 70 '75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 He Ser He Asn Asn Val He Arg Ala Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr He Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 45 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 69 <400> 45 Met Asp Ser Arg Thr Gly Glu Leu He Thr Al a Pro Gln Ala Glu Asn 1 5 10 15 Gly Val Phe He Trp Gl u He Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp He He Ser He 35 40 45 Gln He Arg Phe Asn His Asn He Arg Lys Val Met Gly He His Lys 50 55 '60 Cys Ala Leu Ala Ala Arg He Ala Ala Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu ASD Ser Leu Glv Val 85 90 95 l ie Ser lie Asn Val lie Arg Al a Val Asp Hi s Val Leu Tyr Asp 100 105 110 Val Leu Gl u Asn Thr H e Asn Val Thr Gl u Thr Hi s Asp H e Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 46 < 21 1 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 7) < 400 > 46 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn L 5 10 15 Gly Val Phe lie Trp Glu lie Asp Asn Pro Leu Tyr Phe Lys lie Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp lie lie Ser lie 35 40 45 Glp lie Arg Phe Asn His Asn lie Arg Lys Val Met Gly He His Lys 50 55 60 Cys Phe Leu Asn Phe Arg lie Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 75 80 Ala Ala Ala Ala Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 lie Be lie Asn Asn Val lie Arg Ala Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr lie Asn Val Thr Glu Thr His Asp He Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 47 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 73 <400> 47 Met Asp Ser Arg Thr Gly Glu Leu lie Thr Ala Pro Glp Ala Glu Asn 1 5 10 15 Gly Val Phe Lie Trp Glu lie Asn Asn Pro Leu Tyr Phe Lys He Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asp His Asp lie lie Ser lie 35 40 45 Gln He Arg Phe Asn His Asn lie Arg Lys Val Met Gly lie His Lys 50 55 60 Cys Phe Leu Asn Phe Arg He Trp Thr Thr Gln Thr Gly Arg Phe Leu 65 70 '75 80 Arg Val Phe Arg Tyr Gly Wing Wing Wing Wing Wing Wing Leu Gly Val 85 90 95 lie Ser lie Asn Val lie Arg Ala Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr lie Asn Val Thr Glu Thr His Asp le Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 48 < 211 > 131 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 75 <400> 48 Met Asp Ser Arg Thr Gly Glu Leu He Thr Wing Pro Gln Wing Glu Asn 1 5 10 15 Gly Val Phe lie Trp Glu lie Asn Asn Pro Leu Tyr Phe Lys lie Thr 20 25 30 Asp His Ser Gln Arg Pro Phe Leu Met Asn His Asp lie lie 35 35 45 45 Gln lie Arg Phe Asn His Asn He Glu Glu Val Met Gly lie His Lys 50 55 60 Cys Phe Leu Asn Phe Arg lie Trp Thr Thr Gl n Thr Gly Arg Phe Leu 65 70 75 80 Arg Val Phe Arg Tyr Gly Val Leu Lys Tyr Leu Asp Ser Leu Gly Val 85 90 95 lie Be He Asn Asn Val lie Arg Ala Val Asp His Val Leu Tyr Asp 100 105 110 Val Leu Glu Asn Thr lie Asn Val Thr Glu Thr His Asp lie Lys Tyr 115 120 125 Lys Phe Tyr 130 < 210 > 49 < 211 > 132 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 67 <400> 49 Met Asp Ser Arg Thr Gly Glu Pro He Thr Val Pro Gln Wing Glu Asn 1 5 10 15 Gly Val Tyr He Trp Glu He Thr Asn Pro Leu Tyr Phe Lys He He 20 25 30 Ser Val Glu Asp Pro Leu Tyr Thr Asn Thr Arg He Tyr His Leu Wing 35 40 45 Wing Wing Wing Wing Wing Leu Arg Arg Wing Leu Asp Leu His Lys Wing 50 55 60 Phe Leu Asn Phe Gln Val Trp Thr Thr Ser Thr Thr Wing Ser Gly Arg 65 70 75 80 Thr Tyr Leu Asn Arg Phe Lys Tyr Leu Val Met Leu Tyr Leu Glu Gln 85 90 95 Leu Gly Val He Cys He Asn Asn Val He Arg Wing Val Arg Phe Wing 100 105 110 'Thr Asp Arg Ser Tyr He Thr His Val Leu Glu Asn His Ser He Lys 115 120 125 Tyr Lys Phe Tyr 130 < 210 > 50 < 211 > 132 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 69 <400> 50 Met Asp Ser Arg Thr Gly Glu Pro He Thr Val Pro Gln Al a Glu Asn 1 5 10 15 Gly Val Tyr He Trp Gl u He Thr Asn Pro Leu Tyr Phe Lys He H e 20 25 30 Ser Val Glu Asp Pro Leu Tyr Thr Asn Thr Arg He Tyr Hi s Leu Gl n 35 40 45 H e Arg Phe Asn His Asn Leu Arg Arg Ala Leu Asp Leu Hi s Lys Al a 50 55 60 Al a Leu Ala Ala Gl n Val Al a Al a Thr Ser Thr Thr Wing Ser Gly Arg 65 70 75 80 Thr Tyr Leu Asn Arg Phe Lys Tyr Leu Val 'Met Leu Tyr Leu Gl u Gl n 85 90 95 Leu Gly Val He Cys He Asn Asn Val He Arg Al a Val Arg Phe Wing 100 105 110 Thr Asp Arg Ser Tyr He Thr Hi s Val Leu Glu Asn His Ser He Lys 115 120 125 Tyr Lys Phe Tyr 130 < 21O > 51 < 211 > 132 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 69) < 400 > 51 Met Asp Ser Arg Thr Gly Glu Pro He Thr Val Pro Gln Wing Glu Asn 5 10 15 Gly Val Tyr lie Trp Glu He Thr Asn Pro Leu Tyr Phe Lys lie lie 20 25 30 Ser Val Glu Asp Pro Leu Tyr Thr Asn Thr Arg He Tyr His Leu Glp 35 40 45 He Arg Phe Asn His Asn Leu Arg Arg Ala Leu Asp Leu His Lys Wing 50 55 60 Phe Leu Asn Phe Gln Val Trp Thr Thr Ser Thr Thr Wing Ser Gly Arg 6 = 70 75 80 Thr Tyr Leu Ala Ala Ala Ala Tyr Leu Val Met Leu Tyr Leu Glu Glp 85 90 95 Leu Gly Val lie Cys lie Asn Asp Val lie Arg Ala Val Arg Ph < = > Ala 100 _ 105 110 Thr Asp Arg Ser Tyr lie Thr His Val Leu Glu Asn His Ser lie Lys 115 120 125 Tyr Lys Phe Tyr 130 < 210 > 52 < 211 > 132 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 73) < 400 > 52 Met Asp Ser Arg Thr Gly Glu Pro He Thr Val Pro Gln Wing Glu Asn 1 5 10 15 Gly Val Tyr He Trp Glu He Thr Asn Pro Leu Tyr Phe Lys He He 20 25 30 Ser Val Glu Asp Pro Leu Tyr Thr Asn Thr Arg He Tyr His Leu Gln 35 40 45 He Arg Phe Asn His Asn Leu Ara? M? I 3. . , fifty ?? Ar9 Arg Al a Leu Asp Leu HTs Lys Ala 5Ü 60"the L6U AS" Pte G '"* ¡^ 1 *" Be Thr Thr * ,. Be 61and Ar9 75 80 go Tyr Le "*" ^ "» L'S ^ ^ l. Ala? L, A. Ala Ala Gl "0 95 ^ ßl, V., H C, ". Asn As "v,." Arg ",", ^ ^ 105 11 (J Thr Asp jg ser t r "e Thr m. Va, LeU ßl (1 As" "ls Ser He L" Tyr Lys Phe Tyr 130 < 210 > 53 < 211 > 132 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 75) < 400 > 53 Met Asp Ser Arg Thr Gly Glu Pro He Thr Val Pro Gln Wing Glu Asn 1 5 10. fifteen Gly Val Tyr He Trp Glu He Thr Asn Pro Leu Tyr Phe Lys He He 20 25 30 Ser Val Glu Asp Pro Leu Tyr Thr Asn Thr Arg He Tyr His Leu Gln 35 40 45 He Arg Phe Asn His Asn Leu Glu Glu Ala Leu Asp Leu His Lys Wing 50 55 60 Phe Leu Asn Phe Gln Val Trp Thr Thr Ser Thr Thr Wing Ser Gly Arg 65 70 75 80 Thr Tyr Leu Asn Arg Phe Lys Tyr Leu Val Met Leu Tyr Leu Glu Gln 85 90 95 Leu Gly Val He Cys He Asn Asn Val He Arg Wing Val Arg Phe Wing 100 105 110 Thr Asp Arg Ser Tyr He Thr His Val Leu Glu Asn His Ser He Lys 115 120 125 Tyr Lys Phe Tyr 130 < 210 > 54 < 21 1 > 132 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutant C3 TYCLV (mC3 # 17) < 400 > 54 Met Asp Ser Arg Thr Gly Glu Pro He Thr Val Pro Gl n Wing Gl u Asn 1 5 10 15 Gly Val Tyr He Trp Glu He Thr Asn Pro Leu Wing Wing He H e 20 25 30 Ser Val Gl u Asp Pro Leu Tyr Thr Asn Thr Arg He Tyr His Leu Gl n 35 40 45 lie Arg Phe Asn His Asn Leu Arg Arg Ala Leu Asp Leu His Lys Wing 50 55 60 Phe Leu Asn Phe Gln Val Trp Thr Thr Ser Thr Thr Wing Ser Gly Arg 65 70 75 80 Thr Tyr Leu Asn Arg Phe Lys Tyr Leu Val Met Leu Tyr Leu Glu Gln 85 90 95 Leu Gly Val He Cys He Asn Asn Val He Arg Wing Val Arg Phe Wing 100 105 110 Thr Asp Arg Ser Tyr He Thr His Val Leu Glu Asn His Ser He Lys 115 120 25 Tyr Lys Phe Tyr 130

Claims (18)

NOVELTY OF THE INVENTION CLAIMS

1. - A plant comprising transformed plant cells, said transformed plant cells containing a heterologous nucleic acid construct comprising, in the 5 'to 3' direction: (a) a promoter operable in said plant cells, (b) an acid sequence nucleic encoding mutant protein AL3 / C3 said nucleic acid sequence is located towards the 3 'end of said promoter and is operatively associated therewith, and (c) a terminator sequence located 3' end of said acid sequence. nucleic acid and operatively associated thereto, wherein the expression of said AL3 / C3 mutant protein increases the resistance of said plant to infection by at least one geminivirus, as compared to the untransformed control.

2. A plant according to claim 1, further characterized in that said plant has increased resistance to the geminivirus selected from the group consisting of the golden mosaic virus of tomato, tomato mottle virus, tomato yellow curl virus, virus of the tomato curl, African casava mosaic virus, india casava mosaic virus, yellow potato mosaic virus, golden bean mosaic virus, dwarf mosaic virus of bean, pumpkin curl virus, capsicum virus of texas, cotton curl virus and curl virus of radish.

3. A plant according to claim 1, further characterized in that said promoter is constitutively active in said plant.

4. A plant according to claim 1, further characterized in that said plant is selected from the group consisting of tomato, cassava, potato, bean, squash and beet.

5. A tomato plant comprising transformed tomato plant cells, said transformed tomato plant cells containing a heterologous nucleic acid construct comprising, in the 5 'to 31 direction: (a) a promoter operable in said cells vegetables, (b) a nucleic acid sequence that encodes a mutant protein AL3 / C3, said nucleic acid sequence is located towards the 3 'end of said promoter and is operatively associated with the, and (c) a determination sequence located 3' end of said nucleic acid sequence and is operatively associated with the same, wherein the expression of said AL3 / C3 mutant protein increases the resistance of the tomato plant to infection by at least one geminivirus, compared to the untransformed control.

6. A tomato plant according to claim 5, further characterized in that said plant has increased resistance to the geminivirus selected from the group consisting of gold mosaic virus of tomato, tomato mottle virus, tomato yellow curl virus, and tomato curl virus.

7. A plant of the Solanaceae family, wherein the cells of said plant are transformed to contain a heterologous nucleic acid construct comprising, in the 5 'to 3' direction: (a) a promoter operable in said plant cells, (b) a nucleic acid sequence encoding an AL3 / C3 mutant protein, said nucleic acid sequence located to the 3 'end of said promoter and operatively associated therewith, and (c) a determination sequence located to the extreme 3 'of said nucleic acid sequence and operatively associated therewith, wherein the expression of said AL3 / C3 mutant protein increases the resistance of the tomato plant to infection by at least one geminivirus, as compared to the untransformed control .

8. A method to combat infection by geminiviruses in an agricultural field, which consists of planting in the field a crop of plants comprising transformed plant cells, said transformed cells contain a heterologous nucleic acid construct comprising, in the 5 'to 3' direction: (a) a promoter operable in said plant cells, (b) a nucleic acid sequence encoding an AL3 / C3 mutant protein. , said nucleic acid sequence located towards the 3 'end of said promoter and operatively associated therewith, and (c) a determination sequence located towards the 3' end of said nucleic acid sequence and operatively associated therewith, where the Expression of said AL3 / C3 mutant protein increases the resistance of said plants to infection by at least one geminivirus, as compared to the untransformed control.

9. A method to build a transgenic plant that has increased resistance to infection by geminivirus, said method consists of: providing a plant cell capable of regeneration; transforming said plant cell with a DNA construct comprising, in the 5 'to 3' direction, a promoter operable in said plant cells, a nucleic acid sequence encoding an AL3 / C3 mutant protein, and said localized nucleic acid sequence towards the 3 'end of said promoter and operatively associated with it and a determination sequence located 3' end of said nucleic acid sequence and operatively associated with it; and then regenerating the geminivirus-resistant transgenic plant from said transformed plant cell, wherein the expression of said mutant-AL3 / C3 protein increases the resistance of said plants to infection by at least one geminivirus, in comparison with the control not turned.

10. A method according to claim 9, further characterized in that said plant cell resides in a plant tissue capable of regeneration. 1.

A method according to claim 9, further characterized in that said transforming step is carried out by bombarding said plant cell with microparticles carrying said expression cassette.

12. - A method according to claim 9, further characterized in that said transformation step is carried out by infecting said cells with an Agrobacterium tumefaciens containing a Ti plasmid carrying said expression cassette.

13. A nucleic acid construct comprising an expression cassette, which comprises the construction, in the 5 'to 3' direction of: (a) a promoter operable in a plant cell, (b) a nucleic acid sequence encoding an AL3 / C3 mutant protein, said nucleic acid sequence located towards the 3 'end of said promoter and operatively associated with the, and (c) a determination sequence located 3' to said nucleic acid sequence and associated operatively with him.

14. A DNA construct according to claim 13, further characterized in that it is carried out by means of a plant transformation vector.

15. A method for producing a nucleic acid construct useful for conferring increased resistance to geminiviruses in plants, consisting of: (a) identifying mutants of the geminivirus AL2 / C3 protein that improves geminivirus resistance in plant cells when they express themselves in them; (b) preparing a nucleic acid construct comprising, in the 5 'to 3' direction, a promoter operable in a plant cell, and a nucleic acid sequence encoding said AL3 / C3 mutant protein, said nucleic acid sequence it is located towards 3 'end of said promoter and is operatively associated with the, and a terminator sequence located 3' end of said nucleic acid sequence and operatively associated with it.

16. The seed or progeny of a plant according to claim 1, further characterized in that said seed or progeny has the inherited nucleic acid sequence that codes for the AL3 / C3 mutant protein.

17. The seed or progeny of a plant according to claim 5, further characterized in that the seed or progeny has an inherited nucleic acid sequence that codes for the AL3 / C3 mutant protein.

18. The seed or progeny of a plant according to claim 7, further characterized in that said seed or progeny has the inherited nucleic acid sequence that codes for the AL3 / C3 mutant protein.