WO2000043494A9

WO2000043494A9 - Control of virus infection using replication associated proteins, compositions and methods of use

Info

Publication number: WO2000043494A9
Application number: PCT/US2000/001849
Authority: WO
Inventors: Claude Fauquet; Anju Chatterji
Original assignee: Scripps Research Inst; Claude Fauquet; Anju Chatterji
Priority date: 1999-01-26
Filing date: 2000-01-27
Publication date: 2001-08-09
Also published as: EP1147177A4; WO2000043494A3; EP1147177A2; MXPA01007582A; WO2000043494A2; AU2736600A

Abstract

The invention is generally directed to methods and compositions for controlling infection of plants by geminiviruses using replication associated proteins. In particular, the invention relates to iteron and Rep DNA and polypeptide sequences as well as transgenic plants expressing such sequences.

Description

Control of Virus Infection

Using Replication Associated Proteins,

Compositions and Methods of Use

Background of the Invention

Field of the Invention

The invention relates to methods and compositions for controlling virus infection using replication associated proteins.

Related Art

Viruses can infect both animals and plants. Plant viruses damage plants following infection, and are the cause of substantial agricultural losses. Geminiviruses in particular are extremely devastating plant viruses found all over the world. Natural resistance genes are rare and usually not in the plant species needed and modern biotechnology (including genetic engineering) has not yet provided extremely effective measures to control the geminiviruses, as well as other viruses whose replication involves binding of a replication associated protein (Rep) to an iteron.

Geminiviruses

Geminiviruses belong to a family of plant viruses that cause economically important diseases in a wide range of cereal, vegetables and fiber crops (Brown,

J. K., FAO Plant Prot. Bull. 41:3-31 (1994)). They are characterized by twinned icosahedral particle morphology and covaiently closed circular, single stranded DNA as their genome (Stanley, J., Virol. 1: 139-150 (1991); Lazarowitz, S., Crit. Rev. Plant. Sci. 11:19-349 (1992)). Geminiviruses have a single stranded DNA genome that replicates in the infected nuclei by a rolling circle mechanism (Stenger, D.C., et al, Proc. Natl. Acad. Sci. USA 55:8029-8033 (1991); Saunders, K., et al, Nucleic. Acids. Res.19:1315-1330 (1991)).

The geminiviruses are transmitted by leafhoppers or whitefly vectors and have monopartite or bipartite genomes. Geminiviruses with a bipartite genome have their essential viral functions divided on two DNA components referred to as DNA-A and DNA-B. The DNA-A encodes the replication associated protein (Rep), the replication enhancer (REn), the transcriptional activator protein

(TrAP), and the coat protein (CP), while the movement functions are located on DNA-B. Genetic studies have shown that both the movement protein (MP) and the nuclear shuttle protein (NSP) encoded by DNA-B are necessary for systemic infection (Brough, C.L., et al, J. Gen. Virol. 69:503-514 (1988); Etessami, E., et al, J. Gen. Virol. 72: 1005-1012 (1991)). In DNA-A and DNA-B the open reading frames (ORFs) are arranged in two divergent clusters separated by an intergenic region (LR) of about 200 nucleotides. The IR contains sequences that are conserved between the two DNA components and are referred to as the common region (CR). The CR contains the origin of replication (ori) sequences that are crucial to initiate replication and consists of a conserved hairpin structure and a binding site for the Rep protein located upstream of the hairpin (Fontes, E.P.B., etal, J. Biol. Chem. 269:8459-8465 (1994); Fontes, E.P.B., etal, Plant Cell 6:405-416 (1994)). The ori for Squash leaf curl virus (SqCLV) has been mapped to a 90 nucleotide DNA segment within the CR (Lazarowitz, S.G., et al, Plant Cell 4:199-809 (1992)) and within a 100 bp fragment in tomato golden mosaic virus (TGMV, Fontes, E.P.B., et al, Plant Cell 4:591-608 (1992)).

Replication of the viral DNA occurs in the nuclei of infected cells via double stranded DNA intermediates (Saunders, K., et al, Nucleic Acids Res. 79:2325-2330 (1991); Stenger, D.C., et al, Proc. Natl. Acad. Sci. USA 55:8029-8033 (1991)). The IR contains a GC rich inverted repeat which is conserved in all geminiviruses and has the potential to form a stem-loop structure. These inverted repeats flank an AT rich sequence of 11-16 bases that contains the conserved nonamer motif, T AAT ATT AC (SEQ LD NO 187)

Replication Associated Protein (Rep)

Of the different gene products encoded by the geminivirus, only AC1 or the replication-associated protein (Rep) is essential for viral DNA replication This protein is encoded by the ORF AC 1 and initiates rolling circle replication by a site specific cleavage within the loop of the conserved structure (Laufs, J , et al , FEBS Lett. 377 258-262 (1995a), Laufs, J , et al, Proc. Natl. Acad. Sci. USA 91 3879-3883 (1995b)) Rep is a multifunctional protein and is involved in both viral replication and transcriptional regulation (Fontes, E P B , et al, J. Biol.

Chem. 169 8459-8465 (1994a), Eagle, P A , et al, Plant Cell 6 1157-1170 (1994), Eagle, P A , and Hanley-Bowdoin L , J. Virol 71 6947-6955 (1997)) The amino terminal region of the Rep protein has been shown to be important for DNA recognition and binding (Choi, I R , and Stenger D C , Virology 106 904-912 (1995), Choi, I R , and Stenger D C , Virology 116 22-126 (1996),

Jupin, I , et al, FEBSLett. 161 116-120 (1995)) and in cleavage and ligation of the viral origin of replication The carboxy terminal region of the Rep protein has a nucleoside triphosphate binding domain (Laufs, J , et al, Proc. Natl. Acad. Sci. USA 91 3879-3883 (1995b)) Rep protein possesses a nicking-closing activity and initiates rolling circle replication by a site specific cleavage within the loop of the conserved nonamer sequence, TAATATTAC (Laufs, J S , et al, FEBS Lett. 377 258-262 (1995), Laufs, J , et al, Proc. Natl Acad. Sci. USA 91 3879-3883 (1995), Heyraud- Nitschke, F , et al, Nucleic Acid Res. 13 910-916 (1995), Orozco, B M , and Hanley-Bowdoin, L , J Virol 170 148- 158 ( 1996)) The Rep protein binding site is located between the TATA box and the transcription start site for the Rep gene and acts as the origin recognition sequence and as a negatively regulatory element for Rep gene transcription (Fontes, E P B , et al, Plant Cell 6405-416 (1994), Eagle, P A , et al, Plant Cell 61 1157-1170 (1994), Eagle, P A , and Hanley- Bowdoin, L , J. Virol. 71 6947-6955 (1997)) Recently, discrete functional domains that are responsible for protein binding, cleavage and oligomerization have been identified in the N-terminus of Rep protein of tomato golden mosaic virus, (TGMV, Orozco, B.M., etal, J. Biol. Chem. 272:9840-9846 (1997); Orozco, B. M., et al, Virology 141:346-356 (1998); Gladfelter, H.J., et al, Virology 139: 186-197 (1997)).

The high affinity binding sites for the Rep protein of TGMV, bean golden mosaic virus, BGMV (Eagle, P.A., etal, Plant Cell 6: 1157-1170 (1994); Fontes, E.P.B., et al, Plant Cell 4:591 -608 (1992); Fontes, E.P.B., etal, J. Biol. Chem. 269:8459-8465 ( 1994a); Fontes, E.P.B., etal, Plant Cell 6:405-416 (1994b)) and tomato leaf curl virus, TLCV (Behjatania, S.A., et al, Nucleic Acids Res.

26:925-931(1998)) have been mapped in the viral origin close to the TATA box and the conserved hairpin structure.

Rep interacts with at least two different DNA elements in the geminivirus origin of replication, a conserved nonanucleotide sequence containing a specific nick site for the enzyme (Heyraud-Nitschke, F., et al, Nucleic Acids Res.

23:910-916 (1995); Laufs, J., et al, FEBSLett. 377:158-161 (1995a) Laufs, J., etal, Proc. Natl. Acad. Sci. USA 92:3879-3883 (1995b)) and a directly repeated sequence motif located between the TATA box in the promoter of the AC1 gene and the transcription start site (Fontes, E.P.B., et al, Plant Cell 4:591-608 (1992); Fontes, E.P.B., et al, J. Biol. Chem. 269:8459-8465 (1994a); Fontes,

E.P.B., et al, Plant Cell 6:405-416 (1994b); Choi, I.R., and Stenger DC, Virology 116:11-116 (1996)).

The Rep proteins encoded by different geminiviruses show specificity for the replication of their cognate genomes (Lazarowitz, S.G., et al, Plant Cell 4:199-809 (1992); Fontes, E.P.B., etal, Plant Cell 6:405-416 (1994b); Jupin, I., et al, FEBS Lett. 262: 116-120 (1995); Choi, I.R., and Stenger D.C., Virology 116:11-116 (1996)). This specificity of origin recognition is determined in part by the high affinity binding site of the Rep (Choi, I.R., and Stenger D.C., Virology 106:904-911 (1995); Choi, I.R, and Stenger D.C., Virology 116:11-116 (1996)) and the N-terminal domain of the Rep protein. In the case of tomato yellow leaf curl virus (TYLCV), the N-terminal domain comprises the first 116 amino acid residues of the Rep protein (Jupin, I., et al, FEBSLett. 161: 116-120 (1995)).

Attempts have been made to modify the viral genes involved in viral replication to obtain a virus-resistant plant. In one instance, there has been a disclosure of the use of a modified gene encoding the ALl structural protein, i.e. a replicase, and its effect on viral replication. U.S. Patent No. 5,850,023.

Iterons

Based on a phylogenetic and structural analysis of the IR from 30 different dicot infecting geminiviruses (Arguello-Astorga, G.R., etal, Virology 103:90-100 (1994)) identified a series of sequence elements 6 to 12 nucleotides in length which are repeated 3 to 6 times within the origin of replication. Their research indicated that the nucleotide sequence of the iterated elements (iterons) is generally virus specific and proposed that these iterons may represent specific sites for binding of Rep protein. Further, they showed that the orientation, sequence and arrangement of the iterons was highly conserved between different subgroups of the family geminiviridae. In many viruses, both those that infect plants and animals there is an organization of replication that is similar to the geminiviruses. These other viruses include Nanoviruses and Ciroviridae.

The first step in the replication process of geminiviruses involves recognition of the iterons in the common region of the virus by the Rep protein.

These iterons mostly occur as direct repeat motifs of 6-12 bp within the common region of the viral genome between the TATA box and the start site for the transcription of the AC1 gene. The iterons have been proposed to serve as the high affinity binding sites of the Rep protein and therefore function as origin recognition sequence. Specific regions on the N-terminus of the AC1 gene involved in DNA binding have been identified for tomato golden mosaic virus TGMV (Fontes, E.P.B., et al, Plant Cell 6:405-416 (1994a); Fontes, E.P.B., et al, J. Biol. Chem. 269:8459-8465 (1994b)) and ACMV (Hong, Y., and Stanley, J., J.Gen. Virol 76:2415-2422 (1995)). The list of iteron sequences and the corresponding N-terminal sequences of the Rep protein from different viruses reiterates that variability at the iteron levels in geminiviruses is limited (See Figure 1 A- 1 C) . Even though the viruses may originate in different geographical regions or have diverse host range they share similar iteron sequences. Similarly, the sequence of the amino acid residues that might be involved in origin recognition of binding show limited variability and have certain amino acids in the N-terminal sequence of the Rep protein that give them a common base. For instance, all geminiviruses with the iteron GGTAC (SEQ ID NO. 188) in the origin, have the amino acids FQIN (SEQ ID NO: 148) at [psotopms 7- 10 on the N-terminus of the Rep protein common between them, other than the motif 1 , FLTY (SEQ LD NO : 189 ), which is conserved throughout the geminiviridae. In other cases, the homology is not so remarkable, nevertheless, the 4th residue downstream of the conserved phe (F), is almost always identical between different viruses having the same iteron sequence. Recent reports involving the formation of pseudorecombinants between closely related viruses provide evidence in favor of the observations that viruses sharing same iteron sequence can form pseudorecombinants as demonstrated in case of CaLCV and SqLCV (Hill et al, Virology, 150: 283-292 (1998)) between BDMV and ToMoV (Hou et al, J Virol, 70:5430-5436 (1997)) and SiGMV/Ho and SiGMV/CR (Frishmuth et al, J Gen. Virol, 78: 2675-2682. (1997)). These data strengthen the view that the iteron and the N-terminal sequences in the Rep are two key components that influence origin recognition and replication of the virus, therefore, a control strategy based upon blocking this fundamental step offers a broad spectrum of resistance effective against all geminiviruses having the same iterons. Further, other viruses such as for example the Nanovirus or

Circoviruses that have similar replication mechanisms may also be inhibited by blocking a similar step in the process.

Nanoviruses

Nanovirus is a genus of virus that includes plant infecting viruses with a genome consisting of a multiple (at least 6) circular ssDNA molecules each of approximately 1 kb in size and encapsidated in an icosahedral (non-geminate) virion about 20 nm in diameter. It includes species such as the Subterranean clover virus (SCSV).

The virions are 17 to 20 nm in diameter and exhibit icosahedral symmetry. They are not enveloped. Capsomeres may be evident, producing an angular or hexagonal outline. They have buoyant density of 1.24 to 1.30 g/cm³ in Cs₂SO₄, and 1.34 g/cm³ in CsCl. Instability in CsCl has been reported for SCSV. An S_20w of 46S has been reported for Banana bunchy top virus (BBTV). Particle morphology is not affected by freezing of tissue before virion extraction. The nanovirus genome is composed of several species of circular ssDNA ranging in size from 985 to l l l l nts. All of them seem to be structurally similar in being positive sense, transcribed in one direction, and containing a conserved stem-loop structure (and other conserved domains) in the non-coding region. Six to 10 DNA components, each of which appears to be encapsidated in a separate particle, have been isolated from virion preparations of different species and their isolates. The number and types of ssDNA components constituting the integral genome parts have not been determined yet for any of the nanoviruses. The virions have a single capsid protein with a Mr of 19 - 20 x 10³.

The genomic information of the nanovirus is distributed over at least 6 molecules of circular ssDNA. Since the the nanovirus DNAs are structurally similar to those of the geminiviruses and at least one of the DNA components of each species codes for a replication-associated protein (Rep), nanovirus DNAs are proposed to be replicated from transcriptionally and replicationally active dsDNA forms via a rolling circle type of replication mechanism. Nicking and joining activity of the BBTV Rep protein has been demonstrated in vitro. Complementary strand synthesis of BBTV genomic ssDNA is attributed to a population of endogenous primers derived from BBTV-DNA 5, which appears to encode a protein that is potentially involved into cell cycle regulation.

All ssDNAs found associated with the assigned species contain a major virion sense ORF and appear to be transcribed unidirectionally. Each coding region is preceded by a promoter sequence with a TATA box and followed by a poly(A) addition signal. At least one of the genome components codes for a Rep protein (Rep; Mr 32.4 - 33.6 x 10³). For some isolates of the four assigned species, two to four Rep components have been described, however, some of the additional Rep components may actually be satellite components. A second virion-sense ORF, completely within the Rep ORF and encoding a putative 5 x 10³ protein of unknown function, was also identified for the BBTV Rep component

(DNA-1), which is present in all BBTV isolates studied. Available information suggests that the genome of each virus has 5 to 6 components coding for protein other than Rep proteins, which are referred to as non-Rep proteins. The genus includes species with multiple genomic DNAs that are unidirectionally transcribed, Coconut foliar decay virus (CFDV), a tentative species within the genus, has similar morphology but differs from the assigned members by containing a single circular ssDNA of 1291 nts which is proposed to be transcribed bidirectionally, by having a capsid protein of Mr ca. 24 x 10³ and by being transmitted by a plant hopper.

There are a number of species of Nanoviruses including, banana bunchy top virus (BBTV) (S56276, U18077 to U18079, L32166 & L32167, U02312, L32166 & L32167, U02312), Faba bean necrotic yellows virus (FBNY) (X80879, Y11405 to Yl 1409, (FBNYV) AJ005964 to AJ005968, Milk vetch dwarf virus (MDV) (AB000920 to 000927, 0009046, 0009047), Subterranean clover stunt virus (SCSV) (U16730 to U16736).

Comparison of the 15 potential Rep proteins identified from assigned and tentative members of the genus revealed amino acid identities ranging from about 28% to 90%. In addition, there is variability among Rep proteins, not only from different nanovirus species but also from a given virus. All Rep proteins of the assigned Nanovirus species have most of the amino acid domains characteristic of Rep proteins. The nanovirus Rep proteins differ form those of members of the family Geminiviridae in being smaller (about 33 kDa) having a slightly distinct dNTP-binding motif (GP/SXGG/NEGKS/T), lacking the Rb-binding motif, and in sharing amino acid sequence identities of only 17 to 22% with them. Moreover, the assigned species are clearly distinct from the geminiviruses in particle morphology, genome size, number and size of DNA components, mode of transcription, as well as in vector species The putative Rep protein of porcine circovirus (family Circoviridae) has only insignificant identity (18 to 23%) with nanovirus Rep proteins, and has a bidirectionally transcribed ssDNA genome resembling that of CFDV All of these viruses have a conserved nonanucleotide motif at the apex of the stem-loop sequence which is consistent with operation of a rolling circle model for DNA replication Additional information concerning Nanoviruses is found in Boevink, P, J. Virol 107354-361 (1995), Katul, L et al. , J. Gen. Virol 79 3101-3109 (1998), Harding, R etal., J. Gen. Virol 78 479-486 (1993), Katul et al, Virology 133 247-259 (1997)

Circoviridae

The Circovirus virions exhibit icosahedral symmetry and do not possess an envelope. Ranges of reported virion sizes for Chicken anemia virus (CAV), Porcine circovirus (PCV) and Beak and feather disease virus (BFDV) are 19 1 - 26 5 nm, 17 - 20 7 nm and 12 - 20 7 nm respectively Diameters of virus particles are approximately 20% greater when negatively stained using uranyl acetate as opposed to the more commonly used phosphotungstate A capsid structure consisting of 32 hollow morphological subunits arranged in a T = 3 icosahedron has been proposed for CAV The buoyant density of virions in CsCl is 1 33 D 1 37g/cm³ CAV and PCV possess sedimentation coefficients of 91 S and 57S respectively CAV and PCV are resistant to inactivation by treatment at pH 3 and both viruses can withstand incubation at 70 |C for 15 min CAV is resistant to treatment with chloroform and ether, PCV is resistant to treatment with chloroform Both CAV and PCV are at least partially resistant to sodium dodecyl sulfate The effects of pH, solvents and detergents on the infectivity of BFDV are not known due to the lack of an in vitro culture system

The virions contain circular ssDNA The genomes of CAV and PCV contain 2,298 and 1,759 bases respectively The BFDV genome is about 2 0 kb in size The CAV genome is of negative sense Information concerning the sense of the PCV and BFDV genomes has not been reported The PCV genome contains a nonanucleotide sequence motif (TAGTATTAC), which is found at the apex of a potential stem loop and which is identical or highly similar to those found in bacterial and plant viruses with circular, ssDNA genomes (Microviridae, Nanovirus and Geminiviridae). CAV and PCV virions contain one protein, Mr = 50 and 36 x 10³ respectively.

BFDV is reported to contain three proteins, Mr = 26.3, 23.7, and 15.9 x 10³. CAV has two non-structural proteins, Mr = 24 x 10³ (VP2) and 13.6 x 10³ (VP3), the smaller of which causes apoptosis in vitro. The non-structural proteins of PCV have not been characterized. The N-terminal of the CAV CP shares homologies with histone proteins consistent with it having a DNA-binding role within the virion. The protein (Mr = 35.7 x 10³) encoded by the largest PCV ORF has amino acid sequence homology with the replication-associated proteins of plant viruses with circular, ssDNA genomes.

CAV and PCV DNA replicate using circular ds replicative form (RF) DNAs. Nucleic acid and protein homologies shared with plant geminiviruses are consistent with PCV DNA replicating by a rolling circle mechanism. The origin of replication of PCV DNA has been mapped. Only one strand of the CAV RF is transcribed to produce a polycistronic messenger RNA ( ~ 2.1 kb) which contains

3 partially overlapping ORFs encoding proteins of Mr = 52 x 10³ (VPl, CP), 24 x 10³ (VP2) and 13.6 x 10³ (VP3). All three proteins are detected in electron dense bodies within the nuclei of virus-infected cell cultures. CAV particles have not been directly observed within infected cells. The PCV RF sequence has 6

ORFs larger than 200 nts, which occur in both positive and negative sense orientations. CAV causes transient anemia and immunosuppression in baby chicks. BFDV causes chronic and ultimately fatal disease in large psittacine birds. PCV-like viruses have been associated with a recently described condition of pigs known as post-weaning multisystemic wasting syndrome. Cells of the hematopoietic system are infected by CAV and BFDV At present all assigned members of the family have been classified within a single genus. However differences in virion size, genome size and genome organization may provide the basis for definition of more than one genus in the future.

Circoviruses include Beak and feather disease virus (BFDV), Chicken anemia virus (M55918, M81223)(CAV) and Porcine circovirus (U49186, Y09921) (PCV). PCV is similar to members of the families, Geminiviridae and

Microviridae, in that it exhibits nucleic acid and protein homologies related to rolling circle DNA replication. The animal circoviruses are similar to plant nanoviruses such as Banana bunchy top virus, Coconut foliar decay virus and Subterranean clover stunt virus, which possess non-enveloped, icosahedral capsids (18 - 20 nm in diameter) and circular, ssDNA genomes (0.85 - 1.3 kb in size). These plant viruses, formerly regarded as Ounassigned viruses in the family CircoviridaeO, are now classified in an unassigned genus Nanovirus. Further information cocnerning circoviruses can be found in Mankertz, A et al, J. Virol 71:1561-1566 (1997), Tischer, etal, Nature 195:64-66 (1982), Todd, D. et al, Arch. Virol. 117:119-135 (1991).

There is a need for technology aimed at controlling viruses such as geminiviruses or any virus that can be inhibited by a Rep-iteron antagonist. Basically, there are two approaches. A first approach is altering the movement of the virus in the infected plant or animal and the second approach is shutting down the virus replication. In addition there are added complications concerning such viruses. These are that the viruses, are difficult to distinguish from each other with a complicated taxonomy, often infect plants in mixtures and recombine frequently (Padidam et al, Virol. 265:218-225, (1999) and the time frame for these recombinations is unknown. Therefore, there are several issues associated with virus infection: the precise and proper molecular identification of field isolate, the development of one or more methods to control by biotechnology infection on any and all crops by geminiviruses where needed, and the development of a method that will either be general (or non-specific) or alternatively, be very specific but exploitable in such way that it would become non-specific. The invention meets many of these needs. Summaty of the Invention

The invention is directed to a method for producing resistance in a plant to a geminivirus comprising introducing a geminivirus replication associated protein (Rep)-iteron antagonist into a plant, plant cell or propagule, wherein said antagonist is selected from the group consisting of a nucleotide sequence of a geminivirus iteron capable of binding to a Rep protein and a defective Rep, wherein said defective Rep comprises a conserved geminivirus iteron binding site Preferably, the invention is directed to the use of sequences found in Figs (SEQ LD NOS 1-8, 10-35, 37-41, 43-49, 54-57, 59-62, 64-98) Embodiments of the invention are drawn to use of a Rep protein that may form a dimer with a wild- type geminivirus Rep protein or where the Rep protein comprises from two to thirty different or the same conserved iteron binding sites

Another embodiment of the invention is directed to use in the above method of a defective replication associated protein (Rep) selected from the group consisting of truncated geminivirus Rep protein, a modified Rep protein capable of binding a geminivirus iteron sequence, or a Rep protein fragment capable of binding a geminivirus iteron sequence

The invention is further directed to a vector containing a nucleotide sequence that encodes a defective geminivirus replication associated protein, wherein said encoded protein comprises a polypeptide having an amino acid sequence of a conserved geminivirus iteron binding site or a mutant thereof Preferably the vector is expressed in plants The vector also preferably encodes a polypeptide comprising a sequence as shown in Figs 1 A-1C (SEQ LD NO 1-8, 10-35, 37-41, 43-49, 54-57, 59-62, 64-98) An embodiment of the invention is directed to a polypeptide that forms a dimer with a wild-type geminivirus Rep protein or one that comprises from two to thirty different conserved iteron binding sites Another embodiment of the invention comprises a nucleotide sequence encoding at least two different Rep proteins

The invention is also directed to a nucleic acid molecule containing a nucleotide sequence comprising an isolated conserved geminivirus iteron A preferable embodiment comprises an isolated DNA sequence comprising GGTGTCTGGAGTC (SEQ LD NO: 111).

The invention is further directed to compositions for producing resistance to a geminivirus in plants comprising the vectors or nucleic acids of any of the embodiments of the invention.

Another aspect of the invention is directed to transgenic plants, cells, propagules or seeds that comprise any of the vectors or nucleic acids of the invention. A preferred embodiment comprises a nucleic acid molecule having a nucleotide sequence comprising a conserved geminivirus iteron. The invention is further directed to an isolated nucleic acid molecule comprising a nucleotide sequence for a conserved geminivirus iteron. A preferable embodiment of the invention may be directed to a nucleotide sequence comprising at least two geminivirus iterons. Another embodiment of the invention is directed to a nucleotide sequence that comprises from two to thirty different classes of geminivirus iteron shown in Figs. lA-lC.

The invention is also directed to a truncated Rep protein. Preferably the truncated Rep protein may be an isolated polypeptide selected from the group consisting of AC1_1-21, ACl^, ACl^, ACl^ AC1_M14; ACl^ and ACl _8a or a nucleic acid encoding said polypeptides. In another preferable embodiment the truncated protein comprises at least ACl^,,.

A further aspect of the invention is directed to a method for inhibiting geminivirus replication in a plant comprising introducing a geminivirus replication associated protein (Rep)-iteron antagonist into said plant, said antagonist selected from the group consisting of a nucleotide sequence defining a geminivirus iteron capable of binding to a Rep protein and a defective Rep protein, wherein said defective Rep comprises a conserved geminivirus iteron binding site.

The invention is further drawn to a method for providing resistance to infection by geminiviruses in a susceptible plant comprising: a)trans forming susceptible plant cells with a DNA molecule that comprises operatively linked in sequence in the 5' to 3' direction i) a promoter region that functions in plant cells to cause the production of an RNA sequence; and ii) a gene encoding a defective Rep protein, wherein said defective Rep protein comprises a conserved geminivirus iteron binding site; said method further comprising b) selecting said plant cells that have been transformed; c) regenerating said plant cells to provide a differentiated plant; and d) selecting a transformed plant that expresses said defective Rep gene at a level sufficient to render the plant at least partially resistant to infection by the geminivirus.

The invention is further directed to an at least partially virus-resistant transformed plant normally susceptible to infection by a geminivirus having inserted into its genome a DNA molecule that comprises operatively linked in sequence in the 5' to 3' direction; i) a promoter region that functions in plant cells to cause the production of an RNA sequence; and ii) a gene encoding a defective Rep protein, wherein said defective Rep comprises a conserved geminivirus iteron binding site.

The invention is directed to a method for producing at least partial resistance to a virus or a method for reducing replication of a virus in a plant, plant cell, propagule, animal or animal cell comprising introducing a replication associated protein (Rep)-iteron antagonist into a plant, plant cell, propagule, animal or animal cell, wherein said antagonist is selected from the group consisting of a nucleotide sequence of an iteron capable of binding to a Rep protein and a defective Rep, wherein said defective Rep comprises a conserved iteron binding site, and wherein said Rep-iteron antagonist renders the infected plant, plant cell, propagule, animal or animal cell at least partially resistant to the infection.

Preferably, the invention is directed to the use of sequences at least 50% identical to those found in Figs 1 A-1C (SEQ LD NOS: 1-8, 10-35, 37-41, 43-49, 54-57, 59- 62, 64-107). More preferably the sequences are at least 60%, 70%, 80%, 90%

95% or 99% identical. Another preferable embodiment is directed to producing resistance to infection from a Nanovirus or Circoviridae. Additional embodiments of the invention are drawn to use of a Rep protein that may form a dimer with a wild-type geminivirus Rep protein or where the Rep protein comprises from two to thirty different or the same conserved iteron binding sites.

Another embodiment of the invention is drawn to reducing infection or reducing DNA replication of any virus that replicates in a manner similar to the geminivirus, l e dependent on the binding of a Rep protein to an iteron Embodiments of the invention are also drawn to use of a Rep protein that may form a dimer with a wild-type geminivirus Rep protein or where the Rep protein comprises from two to thirty different or the same conserved iteron binding sites

The invention is further directed to a composition for producing at least partial resistance to a virus or for reducing replication of a virus in a plant, plant cell, propagule, animal or animal cell wherein said composition is used in any of the above methods The compositions may also include solutions that are physiologically compatible with the organism of interest

The invention is also directed to a Rep-iteron antagonist comprising a nucleic acid sequence encoding a Rep protein or fragment thereof that binds to an iteron wherein viral infection or DNA replication of the virus causing the infection is reduced following said antagonist binding to an iteron The invention is further directed to a Rep-iteron antagonist comprising a nucleic acid sequence that competes for binding of a Rep protein with the iteron of the virus causing the infection, wherein viral infection or DNA replication of the virus causing the infection is reduced following said antagonist binding to the Rep protein Another embodiment of the invention is directed to a Rep-iteron antagonist comprising a polypeptide or the nucleic acid sequence encoding a polypeptide comprising the sequence FLTY or KAYTDK

The invention is further directed to a Rep-iteron antagonist comprising a polypeptide or the nucleic acid sequence encoding a polypeptide selected from the group consisting of FLTYPqC wherein q is a basic or a polar amino acid, HTHxUUQ wherein U is a bulky hyrophobic residue and xxYxxK wherein x may be any amino acid

The invention is also directed to a vector comprising a nucleic acid sequence encoding any of the Rep-iteron antagonists of the invention Brief Description of the Figures

Figs. lA-lC. Sequences of Iterons and Rep N-terminal Sequences.

Figure 1A - IB. Begomoviruses (SEQ ID NOs: l-8 10-41, 76-98, 99-100, 108-110) Figure IC - Mastreviruses (SEQ ID NO:43-49, 54-69, 101-105),

Curtoviruses (SEQ ID NO:70-74,106-107) and Topcuvirus (SEQ LD NOS: 75, 107).

Fig. 2A-2C. Immuno-precipitation of Rep protein from crude lysates of Sf9 cells using anti AC1 antibody. Fig. 2 A.. The Rep proteins from the severe strain (Al, lane 1) and the mild strain (A2, lane 2) of ToLC-NdeV were expressed from the polyhedrin promoter of AcNPV in Sf9 cells and detected using anti AC1 polyclonal antiserum.

Fig. 2B. Coomassie blue stained gel showing the purified Rep proteins from the severe (lane 2) and mild (lane 5) strains of ToLC-NdeV. Lane 1 represents the marker and lane 4 shows the crude lysate from the pellet fraction.

Fig. 2C. Western blot using a polyclonal anti- AC 1 antiserum.

Stepwise eluates of the purified protein were collected from the Ni ² affinity column in 20mM Tris, 500mM NaCl a 500mM imidazole (pH 7.9) and detected using the anti AC1 antiserum. Lanes 1-4 represent stepwise aliquots of the purified protein of the severe strain and the lanes 5-7 show similar fractions of the protein purified from the mild strain of ToLC-NdeV.

Fig. 3. Electrophoretic mobility shift assays showing the interaction of the Rep protein of severe (Al) and the mild (A2) strains of ToLC-NdeV with different common region fragments. CR-s and CR-m refer to the 52 bp common region fragment derived from the intergenic region of the viral DNA. bs-s and bs-m denote the 13-bp repeat motifs in the common region of the severe and the mild strain respectively. ³²P labeled DNA fragments (CR or bs) were incubated in the presence (+) or absence (-) of competitor DNA to test the specificity of binding. All reactions contained 200 ng of poly dl.dC and were analyzed on 4% polyacrylamide gels. The reactions in lanes 3 and 8 contained 5 Ox molar excess of appropriate, unlabelled 13-bp DNA as the specific competitor and lanes 4 and 9 show the complex formation in the presence of 1000X molar excess of non-specific competitor (pUC 18) DNA. The Rep proteins of the two strains did not bind to heterologous binding site sequences as seen in lanes 5 and 10.

Figs.4A-4B. DNA sequence requirements for binding by the Rep protein.

Fig. 4A. Labeled synthetic oligonucleotides with variations in the sequence and arrangement of iterons were used as probes to analyze their effect on binding by the Rep protein of severe (lanes 1-6) and mild (lanes 7 to 10) strains of ToLC-NdeV. The key to the sequence of iterons used as probes is as follows: IT 1/2 (5' severe, 3' mild, lanes 1 and 7); I T3/4 (5'unrelated, 3' severe, lanes 2 and 8); IT 5/6 (5' unrelated, 3 'mild, lanes 5 and 9); IT 7/8 (5 'mild, 3'unrelated, lanes 6 and 10); IT 9/10 (5' severe repeated, lane 3); IT 11/12 (3' severe repeated, lane 4).

Fig. 4B. Labeled synthetic oligonucleotides with variations in the spacing and number of iterons were used as probes to analyze their effect on binding by the Rep protein. The key to the sequence of iterons used as probes is as follows: IT 13/14 (spacing within the iterons is increased to 6 nucleotides, lanes 1 and 3); IT 15/16 (no spacing between the iterons, lanes 2 and 4); IT 17/18 (5 'monomer iteron, lanes 5 and 7); IT 19/20 (5 'monomer repeated 4 times, lanes 6 and 8). Lane 9 shows the free probe.

Fig. 5. Replication of Rep protein binding site mutants. Plasmids (2μg) containing the viral replicons mutated at their binding site sequence in the origin were electrop orated into tobacco protoplasts. Total DNA was isolated 48h after transfection, resolved on agarose gels and analyzed by Southern hybridization using ³²P -labelled ACl DNA fragment (nts 2113 to 2695) as a probe. The single stranded (ss) and the supercoiled (sc) forms of the viral DNA are indicated. The virus mutants were given identical names as the oligonucleotides used to alter their iteron sequence for the sake of convenience. The key to the mutants is as follows: Lanes 2 and 13 (IT 1/2); lanes 3 and 14

(IT 3/4); lanes 4 and 15 (IT 5/6); lanes 5 and 16 (IT 7/8); lane 6 (IT 9/10); lane 7 (IT 11/12); lanes 8 and 17 (IT 13/14); lanes 9 and 18 (IT 15/16); lanes 10 and 19 (IT 17/18); lanes 1 1 and 20 (IT 19/20). Lanes 1 and 12 represent the wild type controls for the severe and the mild strain of ToLC-NdeV respectively.

Fig. 6A-6B. Accumulation of Viral DNA in BY-2 Protoplasts. The protoplasts were transfected with truncated and full length Rep proteins.

Fig. 7. Replication of Truncated Rep protein in N. benthamiana Plants.

Figs. 8A-8C. Genome organization of tomato leaf curl virus from New

Delhi, (ToLCV-Nde). This figure shows mutations made in Rep gene and in the common region. (Fig. 8A) Genome maps of DNA-A and DNA-B of severe strain. The genes encoding conserved proteins in geminiviruses are shown as solid arrows. Rep, TrAP, REn and CP on DNA-A represent the replicase associated protein (ACl), the transcriptional activator protein (AC2), the replication enhancer (AC3) and the coat protein (AVI) respectively. The MP and NSP on DNA-B are the movement protein (BC1) and the nuclear shuttle protein (BV1) respectively. The genome organization of DNA-A of both severe and the mild strain are identical. Relevant restriction sites used for mutagenesis are indicated. (Fig.8B) Schematic representation of mutants made in Rep gene of mild and severe strain DNA-A. Fragments were exchanged at the N-(Nco I to Xba I) or C- (Cla 1 to Cla I) terminal of Rep gene between the strains. The ToLCV severe strain is indicated in white hatched lines whereas the mild strain is shown by black lines. (Fig. 8C) A schematic showing organization of origin of replication in geminiviruses (not to scale). The mutations made in the putative binding site of

Rep protein and the N-terminal region of Rep gene are shown. The hairpin,

TATA box and the major ORFs in virus sense and complementary sense are indicated. The repeat sequence forming the binding site is shown as two solid arrows near the TATA box. The putative binding sites identified for the severe strain DNA-A and DNA-B and the mild strain DNA-A are indicated. Substitution mutations made in the N-terminal of Rep gene of mild and the severe strain together with point mutations made in the Rep protein binding sites are presented. (SEQ ID NO: 112-120) The panel on the left shows the sequence of first ten amino acids on the Rep protein of Al and A2 starting with the initiation codon, methionine (M), while the middle panel indicates the putative binding site sequence on the corresponding mutants (indicated on the right). (SEQ LD NOS:

121-129)

Fig. 9. Southern blot analysis of viral DNA in N. tabacum protoplasts inoculated with different mutants of ToLCV. Total DNA was extracted from protoplasts 48h after transfection and electrophoresed through 1% agarose gel without ethidium bromide, transferred to nylon membrane and hybridized with ³²p labelled DNA-A and DNA-B specific probes. Panel A shows replication ability of mutants made in severe strain DNA-A and probed with A-component (lanes 1 -

9) and B-component (lanes 10-18) specific probes. Panel B shows replication efficiency of mutants made in the mild strain DNA-A probed with A-component (lanes 1-9) and B-component (lanes 10-18) specific probes. The positions of single stranded (ss) and supercoiled (sc) viral DNA are indicated. Each lane contains 4 μg of DNA obtained from protoplasts in a single transfection.

Fig. 10. Southern blot analysis of viral DNA in N. benthamiana plants inoculated with ToLCV mutants. Total DNA was extracted from newly emerging leaves three weeks after bombardment and electrophoresed in 1% agarose gels without ethidium bromide, transferred to nylon membrane and hybridised with ³²p labeled DNA-A and DNA-B specific probes. Panel A shows replication competence of severe strain mutants probed with A-component (lanes 1-7) and B-component (lanes 8-14) specific probes. Panel B shows replication efficiency of mutants made in the mild strain DNA-A and probed with A-component (lanes 1-9) and B-component (lanes 10- 18) specific probes. The position of single stranded (ss) and supercoiled (sc) viral DNA are indicated.

Detailed Description of the Preferred Embodiments

It has been discovered that the geminivirus replication associated protein (hereafter called Rep protein or "Rep") specifically recognizes and binds to short stretches of geminivirus DNA sequences called binding sites or iterons, and this binding marks the first step in the replication of the virus in plants. These results are elaborated in the Examples of the specification that provide detailed descriptions of iterons and interactions with Rep proteins.

Definitions In order to provide a clearer understanding of the specification and claims, the following definitions are provided.

Amino Acid Sequences - The amino acid sequences herein use either the single letter or three letter designations for the amino acids. These designations are well known to one of skill in the art and can be found in numerous readily available references, such as for example in Cooper, G.M., The Cell 1997, ASM

Press, Washington, D.C. or Ausubel et al., Current Protocols in Molecular

Biology, 1994 (Also see 37 C.F.R. § 1.821).

Cloning vector: A plasmid or phage DNA or other DNA sequence which is able to replicate autonomously in a host cell, and which is characterized by one or a small number of restriction endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion, and into which a DNA fragment may be spliced in order to bring about its replication and cloning. The cloning vector may further contain a marker suitable for use in the identification of cells transformed with the cloning vector. Markers, for example, provide tetracycline resistance or ampicillin resistance.

DNA construct . As used herein, "DNA construct" should be understood to refer to a recombinant, man-made DNA, either linear or circular. Derivative or Functional Derivative: The term "derivative" or "functional derivative" is intended to include "variants," the "derivatives," or "chemical derivatives" of the Rep molecule. A "variant" of a molecule or derivative thereof is meant to refer to a molecule substantially similar to either the entire molecule, or a fragment thereof. An "analog" of a molecule or derivative thereof is meant to refer to a non-natural molecule substantially similar to either the Rep molecules or fragments thereof. Chemical and functional derivatives of the Rep protein are considered embodiments of the application.

Rep derivatives contain changes in the polypeptide relative to the native Rep polypeptide of the same size. A molecule is said to be "substantially similar" to another molecule if the sequence of amino acids in both molecules is substantially the same, and if both molecules possess a similar biological activity. Thus, two molecules that possess a similar activity, may be considered variants, derivatives, or analogs as that term is used herein even if one of the molecules contains additional amino acid residues not found in the other, or if the sequence of amino acid residues is not identical. Rep derivatives, however, need not have substantially similar biological activity to the native molecule.

As used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, strength, specificity, affinity, biological half-life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. and will be apparent to those of ordinary skill in the art. "Functional derivatives" include those polypeptides that bind iteron sequences and those nucleic acid sequences that bind Rep proteins. Expression vector. As used herein, an "expression vector" is a DNA construct that contains a structural gene operably linked to an expression control sequence so that the structural gene can be expressed when the expression vector is transformed into an appropriate host cell. Two DNA sequences (such as a promoter region sequence and a sequence encoding a Rep derivative) are said to be "operably linked" if the nature of the linkage between the two DNA sequences does not ( 1 ) result in the introduction of a frame- shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the desired sequence, or (3) interfere with the ability of the desired sequence to be transcribed by the promoter region sequence. Thus, a promoter region would be operably linked to a desired DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.

Fragment: A "fragment" of a molecule is meant to refer to any polypeptide subset of these molecules. A truncated Rep may considered to be a fragment of the whole molecule

Fusion protein: By the term "fusion protein" is intended a fused protein comprising a protein or polypeptide either with or without a "selective cleavage site" linked at its N-terminus, which is in turn linked to an additional amino acid leader polypeptide sequence.

% Identity: Whether any two polypeptides or polynucleotides are for example, at least 90% "identical" can be determined using known computer algorithms such as the "FAST A" program, using for example, the default parameters as in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:1444 (1988). Alternatively the BLAST function of the National Center for Biotechnology Information database may be used to determine identity

The terms homology and identity are often used interchangeably. In this regard, percent homology or identity may be determined by methods known to those of skill in the art. For example, by comparing sequence information using a GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (/. Mol. Biol. 48:443 (1970), as revised by Smith and Waterman (Adv. Appl. Math. 1:481 (1981). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e. , nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences.

In general, sequences are aligned so that the highest order match is obtained "Identity" per se has an art-recognized meaning and can be calculated using published techniques (See, e.g. Computational Molecular Biology, Lesk,

A M , ed , Oxford University Press, New York, 1988, Biocomputing: Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Griffin, A M , and Griffin, H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Heinje, G , Academic Press, 1987, and Sequence Analysis Primer, Gribskov,

M and Devereux, J , eds , M Stockton Press, New York, 1991) While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo, H & Lipton, D , SIAM J Applied Math 48 1073 (1988)) Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J Bishop, ed , Academic Press, San Diego, 1994, and Carillo, H & Lipton, D , SIAM J Applied Math 48 1073 (1988) Methods to determine identity and similarity are codified in computer programs Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J , et al, Nucleic Acids Research 11(1) 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S F , et al, JMolec Biol 115 403 (1990))

Therefore, as used herein, the term "identity" represents a comparison between a test and a reference polypeptide or polynucleotide More specifically, a test polypeptide may be defined as any polypeptide that is 90% or more identical to a reference polypeptide As used herein, the term at least "90% identical to" refers to percent identities from 90 to 99 99 relative to the reference polypeptides Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polynucleotide length of 100 amino acids, that no more than 10% (i e , 10 out of 100) amino acids in the test polypeptides differ from that of the reference polypeptides. Such differences may be represented as point mutations randomly distributed over the entire length of the amino acid sequence of the invention or they may be clustered in one or more locations of varying length up to the maximum allowable, e.g. 1/14 amino acid difference (approximately 90% identity). Differences are defined as amino acid substitutions, or deletions. Embodiments of the claimed invention may include those polypeptides or nucleic acid sequences that are at least 90% identical to specifically claimed sequences.

Isolated: A term meaning altered from the natural state. For example, a polynucleotide or a polypeptide naturally present in a living animal is not

"isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated," as the term is employed herein. Thus, a polypeptide or polynucleotide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention. Also intended as an "isolated polypeptide" or an "isolated polynucleotide" are polypeptides or polynucleotides that have been purified, partially or substantially, from a recombinant host cell or from a native source. For example, a recombinantly produced version of a compound or derivatives thereof can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). The terms isolated and purified are sometimes used interchangeably.

By "isolated DNA" is included DNA free of the coding sequences of those genes that, in the naturally-occurring genome of the organism (if any) from which the DNA of the invention is derived, immediately flank the gene encoding the DNA of the invention. The isolated DNA may be single-stranded or double-stranded, and may be genomic DNA, cDNA, recombinant hybrid DNA, or synthetic DNA. It may be identical to a native DNA sequence, or may differ from such sequence by the deletion, addition, or substitution of one or more nucleotides. Isolated single-stranded DNAs of the invention may be detectably labeled for use as hybridization probes, and may be antisense. Isolated or purified as it refers to preparations made from biological cells or hosts should be understood to mean any cell extract containing the indicated DNA or protein including a crude extract of the DNA or protein of interest. For example, in the case of a protein, a purified preparation can be obtained following an individual technique or a series of preparative or biochemical techniques and the DNA or protein of interest can be present at various degrees of purity in these preparations. The procedures may include for example, but are not limited to, ammonium sulfate fractionation, gel filtration, ion exchange change chromatography, affinity chromatography, density gradient centrifugation and electrophoresis.

A preparation of DNA or protein that is "pure" or "isolated" should be understood to mean a preparation free from naturally occurring materials with which such DNA or protein is normally associated in nature. "Essentially pure" should be understood to mean a "highly" purified preparation that contains at least 95% of the DNA or protein of interest.

A cell extract that contains the DNA or protein of interest should be understood to mean a homogenate preparation or cell-free preparation obtained from cells that express the protein or contain the DNA of interest. The term "cell extract" is intended to include culture media, especially spent culture media from which the cells have been removed.

Iteron: The term "iteron" refers to a direct repeat motif of 6-12 bp within the common region of the viral genome between the TATA box and the start site for the transcription of for example, the ACl gene. Iterons have been proposed to serve as the high affinity binding sites of the Rep protein and therefore function as origin recognition sequences.

Plant: The term "plant" should be understood as referring to a multicellular differentiated organism capable of photosynthesis including angiosperms (monocots and dicots) and gymnosperms.

Plant cell: The term "plant cell" should be understood as referring to the structural and physiological unit of plants. The term "plant cell" refers to any cell which is either part of or derived from a plant. Some examples of cells encompassed by the present invention include differentiated cells that are part of a living plant; differentiated cells in culture; undifferentiated cells in culture; and the cells of undifferentiated tissue such as callus or tumors.

Plant cell progeny: The term "plant cell progeny" should be understood as referring to any cell or tissue derived from plant cells including callus; plant parts such as stems, roots, fruits, leaves or flowers; plants; plant seed; pollen; and plant embryos.

Propagules: The term "propagules" should be understood as referring to any plant material capable of being sexually or asexually propagated, or being propagated in vivo or in vitro. Such propagules preferably consist of the protoplasts, cells, calli, tissues, embryos or seeds of the regenerated plants.

Polynucleotide: This term generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation single- and double- stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term "polynucleotide" also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications have been made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

Polypeptide: Polypeptide, protein and peptide are used interchangeably. The term polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins Polypeptides may contain amino acids other than the 20 gene- encoded amino acids and include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art Such modifications are well described in basic texts and in more detailed monographs, as well as in the research literature Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide Also, a given polypeptide may contain many types of modifications

Polypeptides may be branched and they may be cyclic, with or without branching Cyclic, branched and branched cyclic polypeptides may result from post-translational modifications or may be made by synthetic methods

Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, Proteins-Structure and Molecular Properties, 2nd Ed , T E Creighton, W H Freeman and Company, New York, 1993 and Wold, F , Posttranslational Protein Modifications Perspectives and Prospects, pgs 1-12 in Posttranslational Covalent Modification of Proteins, B C Johnson, Ed , Academic Press, New York, 1983, Seifter et al, "Analysis for protein modifications and nonprotein cofactors", Methods in Enzymol 181.626-646 (1990) and Rattan et al, "Protein Synthesis Posttranslational Modifications and Aging", Ann NY Acad Sci 663:48-61 (1992)

Promoter: A DNA sequence generally described as the 5 ' region of a gene, located proximal to the start codon The transcription of an adjacent gene(s) is initiated at the promoter region If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter Examples of promoters include, but are not limited to the CMV promoter (InVitrogen, San Diego, CA), the SV40, MMTV, and hMTIIa promoters (U S Pat No 5,457,034), the HSV-1 4/5 promoter (U S

Pat No 5,501,979), the early intermediate HCMV promoter (WO92/17581), ubiquitin, actin, phenylammonia lyase (PAL), CaMV 35S, CsVMW, and RTBV promoters Also, tissue-specific enhancer elements may be employed Additionally, such promoters may include tissue and cell-specific promoters of an organism

Recombinant Host: According to the invention, a recombinant host may be any prokaryotic or eukaryotic host cell which contains the desired cloned genes on an expression vector or cloning vector This term is also meant to include those prokaryotic or eukaryotic cells that have been genetically engineered to contain the desired gene(s) in the chromosome or genome of that organism For examples of such hosts, see Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989) Preferred recombinant hosts are eukaryotic cells transformed with the DNA construct of the invention More specifically, mammalian cells are preferred

Rep-iteron antagonist The term "Rep-iteron antagonist" refers to a defective Rep protein or single or multiple iteron nucleotide sequences that are effective at inhibiting replication of any geminivirus that infects plants

Selective cleavage site: The term "selective cleavage site" refers to an amino acid residue or residues which can be selectively cleaved with either chemicals or enzymes in a predictable manner A selective enzyme cleavage site is an amino acid or a peptide sequence which is recognized and hydrolyzed by a proteolytic enzyme Examples of such sites include, without limitation, trypsin or chymotrypsin cleavage sites

Stringent Hybridization. As used herein "stringent hybridization" conditions should be understood to be those conditions normally used by one of skill in the art to establish at least a 95% homology between complementary pieces of DNA or DNA and RNA

There are only three requirements for hybridization to a denatured strand of DNA to occur (1) There must be complementary single strands in the sample (2) The ionic strength of the solution of single-stranded DNA must be fairly high so that the bases can approach one another, operationally, this means greater than 0 2M (3) The DNA concentration must be high enough for intermolecular collisions to occur at a reasonable frequency The third condition only affects the rate, not whether renaturation/hybridization will occur Conditions routinely used by those of skill in the art are set out in readily available procedure texts, e.g. , Ausubel F etal, Current Protocols in Molecular Biology, Vol I, Chap 2 10, John Wiley & Sons, Publishers (1994) or Sambrook et al, Molecular Cloning, Cold Spring Harbor (1989), the entire contents of which are incorporated herein by reference As would be known by one of skill in the art, the ultimate hybridization stringency reflects both the actual hybridization conditions as well as the washing conditions following the hybridization, and one of skill in the art would know the appropriate manner in which to change these conditions to obtain a desired result

For example, a prehybridization solution should contain sufficient salt and nonspecific DNA to allow for hybridization to non-specific sites on the solid matrix, at the desired temperature and in the desired prehybridization time For example, for stringent hybridization, such prehybridization solution could contain 6x sodium chloride/sodium citrate (lxSSC is 0 15 M NaCl, 0 015 M Na citrate, pH 7 0), 5x Denhardt's solution, 0 05% sodium pyrophosphate and 100 μg perml of herring sperm DNA An appropriate stringent hybridization mixture might then contain 6x SSC, lx Denhardt's solution, 100 μg per ml of yeast tRNA and 0.05% sodium pyrophosphate.

Alternative conditions for DNA-DNA analysis could entail the following: 1 ) prehybridization at room temperature and hybridization at 68 ° C; 2) washing with 0.2x SSC/0.1% SDS at room temperature;

3) as desired, additional washes at 0.2x SSC/0.1% SDS at 42°C (moderate-stringency wash); or

4) as desired, additional washes at O.lx SSC/0.1% SDS at 68 °C (high stringency). Known hybridization mixtures, e.g., that of Church and Gilbert, Proc.

Natl. Acad. Sci. USA 57: 1991-1995 (1984), comprising the following composition may also be used: 1% crystalline grade bovine serum albumin/lmM EDTA 0.5M NaHPO₄, pH 7.2/7% SDS. Additionally, alternative but similar reaction conditions can also be found in Sambrook et a , Molecular Cloning, Cold Spring Harbor (1989). Formamide may also be included in prehybridization/ hybridization solutions as desired. The invention may include DNA sequences that stringently hybridize to specifically disclosed sequences of the invention.

Transgenic plant: The term "transgenic plant" should be understood as referring to a plant having stably incorporated exogenous DNA in its genetic material. The term also includes exogenous DNA which may be introduced into a cell or protoplast in various forms, including, for example, naked DNA in circular, linear or supercoiled form, DNA contained in nucleosomes or chromosomes or nuclei or parts thereof, DNA complexed or associated with other molecules , DNA enclosed in liposomes , spheroplasts , cells or protoplasts . Transgenic plants are also considered to include at least the progeny of such created plants that express the transgene originally inserted into the first generation plant.

It should be understood that these descriptions are not meant to be definitive or limiting and may be adjusted as required by those of ordinary skill in the art to accomplish the desired objective. It has also been discovered that the iteron binding domain is located in the amino-terminus portion of the Rep protein, and in particular, conserved and variable domains in the amino terminus of the Rep protein have been discovered which allow binding to the iteron, and the variable domain maps to residues 1-10 of the Rep protein which defines the sequence specificity of the iteron binding site.

More importantly, it has been discovered that iteron sequences are conserved around residues 11-15 of the Rep protein among large numbers of isolates of geminivirus (see, e.g. Figs. 1 A-1C) such that the Rep protein from one virus can bind the iteron of another virus. A small number of different iteron sequences have been identified which encompass all known geminiviral iteron sequences.

The binding of Rep to its cognate site is therefore now known to be sequence-specific and the efficiency of the binding is related to the sequence of the first 10-12 aa of the Rep protein and to the sequence of the iterons (i.e., a pair of typically 5 nucleotides separated by two to twenty spacer nucleotides). See Figure 1 for the structure and sequence of an iteron and Rep N-terminal sequences.

In vivo experiments are described in the examples in which point mutations in both the Rep and the iterons are made to demonstrate a correlation between these two entities. Furthermore, in vitro DNA binding experiments described in the examples demonstrates the physical interaction between the two entities. In addition, in vivo data described in the examples demonstrates that different wild type and truncated Reps compete in binding to viral iteron sequences and compete in their influence upon virus replication.

The data presented in the Examples (1) explains the principle of replication specificity of geminiviruses, (2) defines the minimum sequences of Rep that are essential for specific recognition between related strains in geminiviruses, (3) establishes the existence of iterons and their role in binding, and (4) provides the molecular basis whereby the two elements, Rep and iterons, can be manipulated to control geminivirus replication. Such explanations, however, are not meant to limit the scope of the invention. Examples of molecular control include expressing truncated Reps in single or multiple combinations, each of which bind to one or more geminivirus iterons, and thereby inhibit viral replication, or expressing nucleotide sequences which contain one or more geminivirus iteron sites that would bind and trap the wild type Rep protein, and thereby interfere with geminivirus replication.

Figs. 1A-1C are a table which shows the amino acid residue sequence of the iteron binding domain of a large number of Rep proteins from different geminivirus isolates, shows the conserved nature of the binding site domain by arranging the geminvirus Rep protein in different conserved groups, and illustrates the conserved relationship between the different geminivirus isolates and the known iterons Figs. 1A-1C also define iteron "classes" which distinguish the Rep protein binding specificity such that a Rep protein from one iteron class will not bind the iteron from another iteron class Because one or more iteron sequences may be targeted by the present invention, the invention describes in one embodiment the use of a single iteron; in another embodiment, the use of two or more iterons, preferably about two to about thirty iterons, although the number can vary widely in view of the fact that Figs. 1A-1C illustrate the iteron sequence for about 28 isolates and because it is likely that additional field isolates will be cloned and sequenced.

The invention therefore describes a method for producing in a plant resistance to infection by a geminivirus The method can be practiced using a variety of approaches based on the basic scientific finding that a conserved iteron binding site is located in the amino terminus of a Rep protein.

The invention describes in one embodiment a method for producing in a plant resistance to a geminivirus comprising introducing a geminivirus replication associated protein (Rep)-iteron antagonist into said plant, where the antagonist is selected from the group consisting of (1) a nucleotide sequence defining a geminivirus iteron capable of binding to a Rep protein, and (2) a defective Rep protein, wherein the defective Rep comprises a conserved geminivirus iteron binding site. As an antagonist, the functional viral Rep protein competes with the viral iteron sequence for binding and therby inhibits viral replication. A Rep- iteron antagonist can be used in a variety of methods and compositions according to the invention In one embodiment for practicing the method, one can introduce into a plant a defective Rep protein which binds viral iterons and, upon binding to the iteron, blocked by competition the binding of wild-type Rep proteins, thereby inhibiting viral replication The defective Rep protein can be any of a variety of polypeptides which possess the ability to bind a geminivirus iteron sequence, but lack any of a variety of other functions required for geminivirus replication, such as the nicking site activity, the NTP binding site, the AC3 protein interaction site, and the like which render the defective Rep protein incapable of supporting replication The Rep protein functions to be deleted or mutated are located in the wild-type protein's carboxy-terminal region, whereas the iteron-binding domain has been discovered to map to the amino-terminal portion of the wild-type Rep protein

Thus, a preferred defective Rep protein of this invention is a truncated Rep protein which contains the amino terminus at least amino acid residues 1-52, preferably amino acid residues 1-52 A typical protein contains a binding site shown in Figs 1 A-1C, comprising a Rep amino acid sequence and has at least 25 to 30 amino acid residues In another embodiment, a preferred Rep protein has amino acid residues 1-52

More preferably, a defective Rep protein of this invention further has the ability to interact with (i.e., bind to) another Rep protein and form a multimer

The ability of Rep proteins to interact is shown in the Examples, and can be measured by any of a variety of methods, including the interactions as measured herein In particular, a Rep protein corresponding to amino acid residues 1 -56 has the ability to bind an iteron and to bind with defective or wild-type Rep protein Thus, the term "defective replication associated protein" or "defective Rep protein" means any of a variety of peptides and proteins including active fragments, fusion proteins containing an active iteron binding site fragment, and derivatives thereof which possess the geminivirus iteron DNA binding activity Exemplary variations include the deletion mutants and fragmented Rep proteins described in the Exhibits A preferred defective Rep protein includes residues 1-56 of the geminivirus Rep protein. In another embodiment, a preferred Rep protein is based on the natural sequence of a tomato leaf curl virus from New Dehli (ToLCV-Nde), which has been described in several strains, particularly the mild and severe isolates. The complete nucleotide sequence of the DNA-A, including the gene which encodes replication associated protein (Rep), of both mild and severe strains of tomato leaf curl virus from New Dehli (ToLCV-Nde) has been determined. The nucleotide sequence for DNA-A of both strains of ToLCV-Nde is deposited with Genebank having accession numbers of UI 5015 and UI 5016 for the severe and mild strains, respectively.

In another embodiment, the invention describes a method for simultaneously inhibiting the infection of plants by a large number of different isolates of geminivirus using a broad spectrum defective Rep protein which binds to a conserved iteron present in different geminivirus isolates. An exemplary "broad spectrum defective Rep protein" comprises an amino-terminal Rep protein amino acid residue sequence shown in Figs. 1 A- 1 C . In a related embodiment, one can use a combination of several different classes of broad spectrum defective Rep proteins, such that one member of each "iteron class" is included in the combination of Rep proteins. An exemplary combination includes a small number of different polypeptides, each of which comprises a different amino acid residue sequence for the iteron binding site selected from the corresponding different iteron binding site classes shown in Figs.1 A-IC. Alternatively, one can use any combination of defective Rep proteins to encompass two or more different iteron classes as defined in Figure lA-lC. The recitation of many different iteron sequences and corresponding N- terminal sequences of the Rep protein from different viruses demonstrates that variability at the iteron level in geminiviruses is limited. Even though the viruses may originate in different geographical regions, or have diverse host ranges, some isolates share the same iteron sequence and have certain amino acid sequences in the N-terminal region of the Rep protein which give them a common iteron target sequence. For instance, all geminiviruses with the iteron sequence GGTAC (SEQ . LD NO: ) have the amino acids FQIN (SEQ. ID NO: ) common between them, other than the motif 1, FLTY (SEQ. ED NO: ) , which is conserved throughout the geminiviridae. For other isolates, the homology is not so remarkable. However, the fourth residue downstream of the conserved Phe (F) residue is almost always identical between different isolates having the same iteron sequence.

Based on the sequences shown in Figs. 1 A-1C, it is seen that a variety of iteron binding sites are contemplated by the present invention. Thus, it is contemplated that a Rep protein can comprise any one of the known iteron binding site sequences shown in Figs. 1A-1C, such as an amino acid residue sequence comprising a formula selected from the group consisting of -FRVQ- (SEQ. LD

NO: 126), -FRVN- (SEQ. ID NO: 127), -FRLN-(SEQ. ID NO: 128), -FRIQ- (SEQ. LD NO:129), -FRLQ- (SEQ. LD NO: 130), -FKVQ- (SEQ. LD NO: 131), -FKIY- (SEQ. ID NO: 132), -FKIN- (SEQ. ID NO: 133), -FRLA- (SEQ. ID NO: 134), FRLN- (SEQ. ID NO: 135), -LKTN- (SEQ. LD NO: 136), -FAIN-(SEQ. LD NO: 137), -FRLT-(SEQ. LDNO:138), -FNLN- (SEQ. IDNO: 139), -FRVN-(SEQ.

ID NO: 140), -FSLN- (SEQ. LD NO: 141), -FKJY- (SEQ. ID NO: 142), -FLLN- (SEQ. LD NO: 143), -FQLN-(SEQ. LD NO: 144), -FQIY-(SEQ. ID NO: 145), - FCLN-(SEQ. ID NO: 146) , -FCVN- (SEQ. ID NO: 147), -FKLN- (SEQ. ID NO: 148), -FSVK- (SEQ. ID NO: 149), -FSVN- (SEQ. LD NO: 150), -FSEK- (SEQ. ID NO: 151) , -FYKK- (SEQ. LD NO: 152), -FQLK-(SEQ. ID NO: 153), -

FQIA-(SEQ. LD NO: 154) , -FRLQTKY (SEQ. ID NO: 154)- , -FRVYSKY- (SEQ. ID NO:156) and -HRNANT- (SEQ. LD NO: 157).

It is also contemplated that the replication associated protein (Rep) may bind to an iteron sequence selected from the group of sequences selected from GGAGAXGGAGA (SEQ LD NO:99), GGTGTXGGTGT (SEQ ID NO: 100) ,

GGTACXGGTAC (SEQ LD NO: 107 ), GGGGAXGGGGA (SEQ ID NO: 109 ), GGGGGXGGGGG (SEQ ID NO: 110), GGTGCGCCCXGGGCGCACC (SEQ ID NO: 101) GCGCCTTCXGAAGGCGCG (SEQ ID NO: 102) GGTTTGCGXCGCAAACC (SEQ ID NO: 103) GGAGGTGCGTCCX- CCTCCACGGG(SEQ tDNO: 105), GGAGTXGGAGT (SEQ LD NO: 106 ) and

GGTACXGGTAC. (SEQ ID NO: 107), GTGAGTGXCACTCAC (SEQ. ED NO: 9), GGTACXGGTAC (SEQ. EDNO:36), GGGGAXGGGGA(SEQ. EDNO:42), GGGGGXGGGGG (SEQ. ED NO:50) wherein "X" is 3-30 nucleotides. Alternatively, the replication associated protein (Rep) may bind to a DNA sequence comprising GGTGTCTGGAGTC (SEQ ID NO: 111). It is also contemplated that the Rep protein can have a modified amino acid residue sequence which comprises a modified (mutated or altered) iteron binding site sequence which is produced using combinatorial library screening methods to produce a sequence which exhibits high efficiency binding to a preselected iteron sequence, or which has been selected for specific binding to one or a few related iteron sequences.

In another embodiment, the invention contemplates a nucleic acid comprising a geminivirus iteron nucleotide sequence that defines a geminivirus iteron capable of binding to a Rep protein. Specifically, the iteron sequence can be used to compete for binding to geminivirus Rep protein, and thereby prevent Rep protein from binding iteron sequences present on infective geminiviral replicative forms, thereby inhibiting virus replication and preventing symptoms of infection in the plant.

Thus, in one embodiment, the invention contemplates a method for producing resistance to geminivirus infection in a plant comprising introducing a nucleotide sequence into the plant, wherein the nucleotide sequence comprises a geminivirus iteron sequence capable of binding a Rep protein.

Various iteron sequences can be used as described herein, including the use of multiple different iteron sequences to bind multiple Rep proteins from multiple strains of different geminiviruses. In one embodiment, a single nucleic acid molecule may contain multiple iteron sequences. In a preferred embodiment, the nucleic acid comprises each class of iteron sequences shown in Figsl A-IC.

Introduction of a defective Rep protein or iteron nucleotide sequence into plants can be accomplished by a variety of methods including standard gene transfer methods, inoculation of the plant with a transfer or carrier vector, "biolistic" (i.e., ballistic) introduction of nucleic acids into mature plant tissue, direct DNA uptake into plant protoplast, transformation of plants with Agrobacterium tumefaciens-based vectors, and the like. The Rep protein is typically expressed using a nucleotide sequence which encodes a defective Rep protein and which contains expression control elements which provide for expression of the protein in plants. Alternatively, the DNA can contain the iteron sequence, and no protein expression is required.

Plant expression elements for a nucleotide sequence are generally well known in the art and are not to be considered limiting to the invention. The nucleotide sequence which encodes the Rep protein can be present on an expression vector, as a DNA fragment, or as a component of a "transfer" or carrier vector such as the infectious Agrobacterium gene transfer system commonly used in plants.

Thus, iteron sequences can be introduced into plants by a variety of methods, and therefore the invention is not to be construed as so limited. The nucleotide sequence can be introduced directly such as by biolistics, can be present on a vector capable of transcribing a nucleic acid copy of the iteron sequence inside a plant cell, or can be present as a component part of the plant genome of a transgenic plant. Preferred iteron sequences are described in the Examples.

The introduction of a Rep-iteron antagonist, ie., a defective Rep protein, single or multiple iteron nucleotide sequence, according to the invention is effective at inhibiting replication of any geminivirus that infects plants and whose replication depends upon the interaction of functional replication associated protein (Rep) with the iteron sequence of the infecting viral genome. Such antagonist may also include mutated Rep or iterons. Preferred viruses are the Geminiviridae family of viruses, which includes Mastrevirus, Curtovirus, Topcuvirus and Begomovirus genera, and the Nanovirus and Circoviridae viruses or other viruses that replicate using Rep proteins that bind to an iteron, wherein use of a Rep-iteron antagonist reduces infection of the organism.

Preferred Mastrevirus genus species are selected from the group consisting of Bajra streak virus, Bean yellow dwarf virus, Bromus striate mosaic virus, Chickpea chlorotic dwarf virus, Chloris striate mosaic virus, Digitaria streak virus,

Digitaria striate mosaic virus, Maize streak virus//Ethiopia, Maize streak virus//Ghanal, Maize streak virus//Ghana2, Maize streak virus//Kenya, Maize streak virus//Komatipoort, Maize streak virus/ZMalawi, Maize streak virus//Mauritius, Maize streak virus//Mozambique, Maize streak virus//Nigerial, Maize streak virus//Nigeria2, Maize streak virus//Nigeria3, Maize streak virus/ZPort Elizabeth, Maize streak virus/ZReunionl , Maize streak virus//Reunion2,

Maize streak virus//Setaria, Maize streak virus//South Africa, Maize streak virus//Tas, Maize streak virus//Uganda, Maize streak virus//Vaalhart maize, Maize streak virus//Vaalhart wheat, Maize streak virus/ΛVheat-eleusian, Maize streak virus//Zaire, Maize streak virus//Zimbabwel, Maize streak virus//Zimbabwe2, Miscanthus streak virus, Panicum streak virus/Karino, Panicum streak virus/Kenya, Paspalum striate mosaic virus, Sugarcane streak virus//Egypt, Sugarcane streak virus/Natal, Sugarcane streak virus/Mauritius, Tobacco yellow dwarf virus, Wheat dwarf virus/Czech Republic (Wheat dwarf virus-CJI, WDV-CJI), Wheat dwarf virus/France and Wheat dwarf virus/Sweden. Preferred Curtovirus genus species are selected from the group consisting of Beet curly top virus-California, Beet curly top virus-California//Logan, Beet curly top virus-CFH, Beet curly top virus//Iran, Beet curly top virus-Worland,Horseradish curly top virus, Tomato leafroll virus and Tomato pseudo-curly top virus. Preferred Begomovirus genus species are selected from the group consisting of Abutilon mosaic virus, Acalypha yellow mosaic virus, African cassava mosaic virus//Ghana, African cassava mosaic virus/Kenya, African cassava mosaic virus/Nigeria, African cassava mosaic virus/Uganda, Ageratum yellow vein virus, Althea rosea enation virus, Asystasia golden mosaic virus, Bean calico mosaic virus, Bean dwarf mosaic virus, Bean golden mosaic virus-Brazil, Bean golden mosaic virus-Puerto Rico, Bean golden mosaic virus-Puerto Rico/Dominican Rep. (Bean golden mosaic virus-Dominican Rep., BGMV-DR), Bean golden mosaic virus-Puerto Rico/Guatemala (Bean golden mosaic virus-Guatemala, BGMV-GA], Bhendi yellow vein mosaic virus, Chino deltomate virus (Tomato leaf crumple virus, TLCrV), Cotton leaf crumple virus, Cotton leaf curl virus-India, Cotton leaf curl virus-Pakistan 1/Faisalabadl (Cotton leaf curl virus-Pakistan2), Cotton leaf curl virus-Pakistan 1/Faisalabad2 (Cotton leaf curl virus-Pakistan3),Cotton leaf curl virus-Pakistan 1/Multan (Cotton leaf curl virus-Pakistan 1 ), Cotton leaf curl virus-Pakistan2/Faisalabad (Pakistani cotton leaf curl virus), Cowpea golden mosaic virus, Croton yellow vein mosaic virus//Lucknow, Dolichos yellow mosaic virus, East African cassava mosaic virus/Kenya, East African cassava mosaic virus/Malawi, East African cassava mosaic virus/Tanzania, East African cassava mosaic virus/Uganda// 1 (African cassava mosaic virus-Uganda variant), East African cassava mosaic virus/Uganda /2, Eclipta yellow vein virus, Eggplant yellow mosaic virus, Eupatorium yellow vein virus, Euphorbia mosaic virus, Honeysuckle yellow vein mosaic virus, Horsegram yellow mosaic virus, Indian cassava mosaic virus,

Jatropha mosaic virus, Leonurus mosaic virus, Limabean golden mosaic virus,

Lupin leaf curl virus, Macroptilium golden mosaic virus-Jamaica//2, Macroptilium goldenmosaicvirus-Jamaica//3, Macrotyloma mosaic virus, Malvaceous chlorosis virus, Melon leaf curl virus, Mungbean yellow mosaic virus, Okra leaf curl virus//Ivory Coast, Okra leaf curl virus//India, Papaya leaf curl virus, Pepper huasteco virus, Pepper golden mosaic virus, (Texas pepper virus), Pepper mild tigrA virus, Potato yellow mosaic virus//Guadeloupe, Potato yellow mosaic virus/Trinidad and Tobago, Potato yellow mosaic virus/Venezuela, Pseuderanthemum yellow vein virus, Rhynchosia mosaic virus, Serrano golden mosaic virus, Sida golden mosaic virus/Costa Rica, Sida golden mosaic virus Honduras, Sida golden mosaic virus/Honduras//Yellow vein, Sida yellow vein virus, Solanum apical leaf curl virus, Soybean crinkle leaf virus, Squash leaf curl virus, Squash leaf curl virus/Extended host, Squash leaf curl virus/Restricted host, Squash leaf curl virus/Los Mochis, Squash leaf curl virus-China, Tomato golden mosaic virus/Common strain, Tomato golden mosaic virus/Yellow vein strain, Tobacco leaf curl virus//India, Tobacco leaf curl virus-China, Tomato leaf curl virus//Solanum species Dl, Tomato leaf curl virus//Solanum species D2,

Tomato leaf curl virus- Australia, Tomato leaf curl virus-Bangalore 1 (Indian tomato leaf curl virus-Bangalorel), Tomato leaf curl virus-Bangalore2, (Indian tomato leaf curl virus, ItoLCV), Tomato leaf curl virus-Bangalore3 (Indian tomato leaf curl virus- Bangalorell), Tomato leaf curl virus-New Delhi/Severe (Tomato leaf curl virus-India2, ToLCV-INl), Tomato leaf curl virus-New Delhi/Mild (Tomato leaf curl virus-India2, ToLCV-IN2), Tomato leaf curl virus-New DelhiLucknow (Indian tomato leaf curl virus), Tomato leaf curl virus//Senegal, Tomato leaf curl virus-Sinaloa (Sinaloa tomato leaf curl virus, STLCV), Tomato leaf curl virus-Taiwan, Tomato leaf curl virus-Tanzania, Tomato mottle virus, Tomato mottle virus-Taino (Taino tomato mottle virus, TTMo V), Tomato severe leaf curl virus//Guatemala, Tomato severe leaf curl virus//Honduras, Tomato severe leaf curl virus//Nicaragua, Tomato yellow dwarf virus, Tomato yellow leaf curl virus-China, Tomato yellow leaf curl virus-Israel, Tomato yellow leaf curl virus-Israel/Mild, Tomato yellow leaf curl virus-Israel Egypt, (Tomato yellow leaf curl virus-Egypt, TYLCV-EG), Tomato yellow leaf curl virus-Israel//Cuba, Tomato yellow leaf curl virus-Israel//Jamaica, Tomato yellow leaf curl virus-Israel//Saudi Arabial , (Tomato yellow leaf curl virus-Northern Saudi Arabia, TYLCV-NSA), Tomato yellow leaf curl virus-Nigeria, Tomato yellow leaf curl virus-Sardinia, Tomato yellow leaf curl, virus-Sardinia/Sicily (Tomato yellow leaf curl virus-Sicily, TYLCV-SY), Tomato yellow leaf curl virus-Sardinia/Spain//l (Tomato yellow leaf curl virus-Spain, TYLCV-Sp), Tomato yellow leaf curl virus- Sardinia/Spain//2 (Tomato yellow leaf curl virus-Almeria, TYLCV-Almeria ) Tomato yellow leaf curl virus- Sardinia/Spain//3 (Tomato yellow leaf curl virus-European strain), Tomato yellow leaf curl virus-Saudi Arabia (Tomato yellow leaf curl virus-Southern Saudi Arabia, TYLCV-SSA), Tomato yellow leaf curl virus-Tanzania, Tomato yellow leaf curl virus-Thailand// 1, Tomato yellow leaf curl virus-Thailand//2 , Tomato yellow leaf curl virus// Yemen, Tomato yellow mosaic virus-Brazil// 1, Tomato yellow mosaic virus-Brazil//2, Tomato yellow mottle virus, Tomato yellow vein streak virus-Brazil, Watermelon chlorotic stunt virus, Watermelon curly mottle virus and Wissadula golden mosaic virus-Jamaica//l.

The invention also contemplates a nucleic acid molecule, such as a DNA expression vector, useful for expression of a Rep protein of this invention in plants. Thus the nucleic acid molecule contains a nucleotide sequence which encodes a geminivirus Rep protein, variant or fragment thereof capable of binding a geminivirus iteron nucleotide sequence, and further contains elements for regulation and control of gene expression in plants Exemplary elements are described in United States Patent Nos 5,188,642, 5,202,422, 5,463,175 and 5,639,947, the disclosures of which are hereby incorporated by reference

Exemplary expression vectors and systems for introduction of a Rep protein into plants are described in the Examples

The invention further contemplates a transgenic plant containing a nucleotide sequence of this invention for expressing the geminivirus Rep protein, variants and fragments thereof Preparation of transgenic plants is well known in the art and described at least in the above-mentioned U S patents

Also contemplated is a composition useful for introducing a nucleotide sequence of this invention into plants The composition comprises an effective amount of the nucleotide sequence for introducing the geminivirus Rep protein into a plant, and depends upon the method used for introducing the protein to the plant For example, using direct DNA uptake by protoplast, the composition is an aqueous solution containing nucleic acid and buffers to facilitate uptake by protoplast, as is well known For transformation by Agrobacterium, the composition contains a suspension of Agrobacteria containing the nucleotide sequence capable of expressing the dsDNA-binding protein

In one embodiment, a vector is employed that is capable of integrating the desired gene sequences into the host cell chromosome. Cells that have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g. , antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. In another embodiment, the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.

DNA encoding the desired protein is preferably operably linked to a promoter region, a transcription initiation site, and a transcription termination sequence, functional in plants. Any of a number of promoters which direct transcription in a plant cell is suitable. The promoter can be either constitutive or inducible. Some examples of promoters functional in plants include the nopaline synthase promoter and other promoters derived from native Ti plasmids, viral promoters including the 35S and 19S RNA promoters of cauliflower mosaic virus (Odell et al., Nature 373:810-812 (1985)), and numerous plant promoters.

Alternative promoters that may be used include nos, ocs, and CaMV promoters. Overproducing plant promoters may also be used. Such promoters, operably linked to the Rep gene, should increase the expression of the Rep protein. Overproducing plant promoters that may be used in this invention include the promoter of the small subunit (ss) of ribulose-l ,5-biphosphate carboxylase from soybean (Berry-Lowe et al. , J. Molecular and App. Gen. 7:483-498 (1982), and the promoter of the chlorophyll a/b binding protein. These two promoters are known to be light-induced in eukaryotic plant cells (see, for example, Genetic Engineering of Plants, an Agricultural Perspective,

A. Cashmore, Plenum, New York 1983, pages 29-38; Corruzi, G. et al. , J. of Biol. Chem. 158: 1399 (1983); and Dunsmuir, P. et al. , J. of Mol. and Applied Genet. 2:285 (1983)).

Genetic sequences comprising the desired gene or antisense sequence operably linked to a plant promoter may be joined to secretion signal sequences and the construct ligated into a suitable cloning vector. In general, plasmid or viral (bacteriophage) vectors containing replication and control sequences derived from species compatible with the host cell are used. The cloning vector may typically carry a replication origin, as well as specific genes that are capable of providing phenotypic selection markers in transformed host cells, typically antibiotic resistance genes.

General methods for selecting transgenic plant cells containing a selectable marker are well known and taught, for example, by Herrera-Estrella, L. and Simpson, J. (1988) "Foreign Gene Expression in Plants" in Plant Molecular Biology, A Practical Approach, Ed. CH. Shaw, IRL Press, Oxford, England, pp. 131-160, Scholthof, H. /. Virol 73:1813-1819 (1999), Yusibov et al, Curr. Top. Microbiol. Immunol. 14081-94 (1999), Houdred et al., Plant Physiol. 119:113-114 (1999).

In another embodiment, the present invention relates to a transformed plant cell comprising exogenous copies of DNA (that is, copies that originated outside of the plant) encoding a Rep gene expressible in the plant cell wherein said plant cell is free of other foreign marker genes (preferably, other foreign selectable marker genes); a plant regenerated from the plant cell; progeny or a propagule of the plant; and seed produced by the progeny.

Plant transformation techniques are well known in the art and include direct transformation (which includes, but is not limited to: microinjection

(Crossway, Mol. Gen. Genetics 202:179-185 (1985)), polyethylene glycol transformation (Krens et al, Nature 196:11-14 (1982)), high velocity ballistic penetration (Klein et al., Nature 317:10-13 (1987)), fusion of protoplasts with other entities, either minicells, cells, lysosomes, or other fusible lip id-surfaced bodies (Fraley et al., Proc. Natl. Acad. Sci. USA 79:1859-1863 (1982)), electroporation (Fromm etal, Proc. Natl. Acad. Sci. USA 82:5824 (1985)) and techniques set forth in U.S. Patent No. 5,231,019)) and Agrobacterium tumefaciens mediated transformation as described herein and in (Hoekema et al., Nature 303:119 (1983), de Framond et al., Bio/technology 7:262 (1983), Fraley et al. WO84/02913, WO84/02919 and WO84/02920, Zambryski et al.

EP 116,718, Jordan et al., Plant Cell Reports 7:281-284 (1988), Leple et al. Plant Cell Reports 77:137-141 (1992), Stomp et al., Plant Physiol. 92:1226- 1232 (1990), and Knauf et al., Plasmid 8:45-54 (1982)). Another method of transformation is the leaf disc transformation technique as described by Horsch et al. Science 227:1229-1230 (1985). The transformation techniques can, for example, utilize a DNA encoding an amino acid sequence of Figs. lA-lC, fragments thereof or the antisense sequence, expressible in plants. Included within the scope of a gene encoding the Rep sequences of Figs. lA-lC are functional derivatives, as well as variant, analog, species, allelic and mutational derivatives of the polypeptides of the invention.

As used herein, modulation of Rep expression may entail the enhancement or reduction of the naturally occurring levels of the protein. Specifically, the translation of RNA encoding Rep may also be reduced or inhibited by the expression of an antisense gene or RNA. In general, antisense cloning entails the generation of an expression module which encodes an RNA complementary (antisense) to the RNA encoding Rep (sense). By expressing the antisense RNA in a cell which expresses the sense strand, hybridization between the two RNA species will occur resulting in the blocking of translation. Alternatively, overexpression of the Rep protein might be accomplished by use of appropriate promoters, enhancers, and other modifications. Those of skill in the art would be aware of references describing the use of antisense genes in plants (van der Krol et al, Gene 72:45-50 (1988); van der Krol et al., Plant Mol. Biol. 74:467-486 (1990); Zhang etal., Plant Cell 4:1575-1588 (1992) and U.S. Pat. 5, 107,065). Other foreign marker genes (i.e., exogenously introduced genes) typically used include selectable markers such as a neo gene (Potrykus et al, Mol. Gen. Genet 799:183-188 (1985)) which codes for kanamycin resistance; a bar gene which codes for bialaphos resistance; a mutant EPSP synthase gene (Hinchee et al, Bio /technology 6:915-922 (1988)) which encodes glyphosate resistance; a nitrilase gene which confers resistance to bromoxynil (Stalker et al., J. Biol. Chem. 263:6310-6314 (1988)); a mutant acetolactate synthase gene (ALS) which confers imidazolinone or sulphonylurea resistance (EP application number 154,204); a methotrexate resistant DHFR gene (Thillet et al, J. Biol. Chem. 263:12500-12508) and screenable markers which include β- glucuronidase (GUS) or an R-locus gene, alone or in combination with a C- locus gene (Ludwig et al, Proc. Natl. Acad. Sci. USA 86:1091 (1989); Paz-

Ares et al, EMBO J. 6:3553 (1987)).

The genetic construct for expressing the desired protein can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA. The genetic material may also be transferred into plant cells using polyethylene glycol to form a precipitation complex with the genetic material that is taken up by cells. (Paszkowski et al, EMBO J. 3:2717-22 (1984)). The desired gene may also be introduced into plant cells by electroporation. (Fromm et al. , "Expression of Genes Transferred into Monocot and Dicot Plant Cells by Electroporation," Proc. Nat' I. Acad. Sci. U. S.A. 82:5824 (1985)). In this technique, plant protoplasts are electroporated with plasmids containing the desired genetic construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of plasmids. Electroporated plant protoplasts reform cell walls, divide, and form plant calli. Selection of the transformed plant cells expressing the desired gene can be accomplished using phenotypic markers as described above.

Alternatively, microprojectile bombardment may be used (Daniel H. Methods Mol. Biol. 62:463-489 (1997).

Another method of introducing the desired gene into plant cells is to infect the plant cells with Agrobacterium tumefaciens transformed with the desired gene. Under appropriate conditions well-known in the art, transformed plant cells are grown to form shoots, roots, and develop further into plants. The desired genetic sequences can be joined to the Ti plasmid of Agrobacterium tumefaciens. The Ti plasmid is transmitted to plant cells on infection by Agrobacterium tumefaciens and is stably integrated into the plant genome. See Horsch et al , "Inheritance of Functional Foreign Genes in Plants," Science

133: 496-498 (1984); Fraley et al , Proc. Nat 'I Acad. Sci. U.S.A. 80: 4803 (1983)); Feldmann, K.A. et al , Mol. Gen. Genet. , 108: 1-9 (1987); Walden, R. et al , Plant J., 1: 281-288 (1991).

Presently there are several different ways to transform plant cells with Agrobacterium: (1) co-cultivation of Agrobacterium with cultured, isolated protoplasts, or (2) transformation of cells or tissues with Agrobacterium. Method (1) requires an established culture system that allows culturing protoplasts and plant regeneration from cultured protoplasts. Method (2) requires that the plant cells or tissues can be transformed by Agrobacterium and that the transformed cells or tissues can be induced to regenerate into whole plants. In the binary system, to have infection, two plasmids are needed: a T- DNA containing plasmid and a vir plasmid.

Routinely, however, one of the simplest methods of plant transformation is explant inoculation, which involves incubation of sectioned tissue with

Agrobacterium containing the appropriate transformation vector (Plant Genetic Transformation and Gene Expression, A Laboratory Manual, Oxford: Blackwell Scientific Publications (1988); Walden, Genetic Transformation in Plants, Milton Koynes: Open University Press (1988)). Methods for inserting viral DNA into plant material are known in the art, see for example, U.S. Patent No. 5,569,597 or Porta C. et al, 5:109-111 (1996).

All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be used for the expression of the desired gene. Suitable plants include, for example but are not limited to, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manicot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis, Nemesia, Pelargonium,

Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura. Additional plant genera that may be transformed by Agrobacterium include Ipomoea, Passiflora, Cyclamen, Malus, Prunus, Rosa, Rubus, Populus, Santalum, Allium, Lilium, Narcissus, Ananas, Arachis, Phaseolus, and Pisum. Plant regeneration techniques are well known in the art and include those set forth in t e Handbook of Plant Cell Culture, Volumes 1-3, Eds. Evans et al. Macmillan Publishing Co. , New York, NY (1983, 1984, 1984, respectively); Predieri and Malavasi, Plant Cell, Tissue, and Organ Culture 77: 133-142 (1989); James, D.J., et al., J. Plant Physiol. 732: 148-154 (1988); Fasolo, F. , et al. , Plant Cell, Tissue, and Organ Culture 76:75-87 (1989);

Valobra and James, Plant Cell, Tissue, and Organ Culture 27:51-54 (1990); Srivastava, P.S. , et al. , Plant Science 41:109-114 (1985); Rowland and Ogden, Hort. Science 27: 1127-1129 (1992); Park and Son, Plant Cell, Tissue, and Organ Culture 75:95-105 (1988); Noh and Minocha, Plant Cell Reports 5:464- 467 (1986); Brand and Lineberger , Plant Science 57: 173-179 (1988); Bozhkov,

P.V. , et al. , Plant Cell Reports 77:386-389 (1992); Kvaalen and von Arnold, Plant Cell, Tissue, and Organ Culture 27:49-57 (1991); Tremblay and Tremblay, Plant Cell, Tissue, and Organ Culture 27:95-103 (1991); Gupta and Pullman, U.S. Patent No. 5,036,007; Michler and Bauer, Plant Science 77: 111-118 (1991); Wetzstein, Η.Y. , et al. , Plant Science 64: 193-101 (1989);

McGranahan, G.H., et al, Bio /Technology 6:800-804 (1988); Gingas, V.M. , Hort. Science 16: 1217-1218 (1991); Chalupa, V. , Plant CellReports 9:398-401 (1990); Gingas and Lineberger, Plant Cell, Tissue, and Organ Culture 17: 191- 203 (1989); Bureno, M.A., et al., Phys. Plant. 85:30-34 (1992); and Roberts, D.R., et al, Can. J. Bot. 68: 1086-1090 (1990).

Plant regeneration from cultured protoplasts is described in Evans et al. , "Protoplast Isolation and Culture, " in Handbook of Plant Cell Culture 7: 124- 176 (MacMillan Publishing Co., New York, 1983); M.R. Davey, "Recent Developments in the Culture and Regeneration of Plant Protoplasts, " Protoplasts, 1983 - Lecture Proceedings, pp. 19-29 (Birkhauser, Basel, 1983);

P.J. Dale, "Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops," in Protoplasts 1983 - Lecture Proceedings, pp. 31-41 (Birkhauser, Basel, 1983); and H. Binding, "Regeneration of Plants, " in Plant Protoplasts, pp. 21-37 (CRC Press, Boca Raton, 1985).

Techniques for the regeneration of plants varies from species to species but generally, a suspension of transformed protoplasts containing multiple copies of the desired gene is first provided. Embryo formation can then be induced from the protoplast suspensions, to the stage of ripening and germination as natural embryos. The culture media will generally contain various amino acids and hormones, such as auxins and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa.

Mature plants, grown from transformed plant cells, are selfed to produce an inbred plant. The inbred plant produces seed containing the recombinant DNA sequences promoting increased resistance to geminivirus infection.

Parts obtained from regenerated plants, such as flowers, seeds, leaves, branches, fruit, and the like are covered by the invention provided that these parts comprise the geminivirus resistant cells. Progeny and variants, and mutants of the regenerated plants are also included within the scope of this invention. As used herein, mutant describes variation as a result of environmental conditions, such as radiation, or as a result of genetic variation in which a trait is transmitted meiotically according to well-established laws of inheritance.

All plants which can be transformed are intended to be hosts included within the scope of the invention (preferably, dicotyledonous plants). Such plants include, but are not limited to, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus,

Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Sencia, Salpiglossis, Cucumis, Browalia, Glycine, Lolium, Zea, Triticum, Sorghum, Malus, Apium, Datura and Monocotyledonous plants such as corn.

Some examples of commercially useful agricultural plants to which methods of the invention may be applied include Abutilon, Acalypha, apple,

Ageratum, Althearosea, Asystasia, Bajra, banana, barley, beans, beet, Blackgram, Bromus, Cassava, chickpea, Chilllies, Chloris, clover, coconut, coffee, cotton, cowpea, Croton, cucumber, Digitaria, Dolichos, eggplant, Eupatorium, Euphorbia, fababean, honeysuckle, horsegram, Jatropha, Leonurus, limabean, Lupin, Macroptilium, Macrotyloma, maize, melon, millet, mungbean, oat, okra,

Panicum, papaya, Paspalum, peanut, pea, pepper, pigeon pea, pineapple, Phaseolus, potato, Pseuderanthemum, pumpkin, Rhynchosia, rice, Serrano, Sida, sorghum, soybean, squash, sugarcane, sugarbeet, sunflower, sweet potato, tea, tomato, tobacco, watermelon, wheat and Wissadula. Additionally, the method may be applied to grains, legumes, vegetables and fruits, including but not limited to wheat, corn, barley, alfalfa, cotton, rapeseed, rye, peas, celery, grapes, cabbage, oilseed, apples, strawberries, mulberries, cranberries and lettuce that become infected with geminivirus.

Other embodiments of the invention will be apparent to one skilled in the art in light of the previous disclosures. Therefore, the previous disclosures as well as the following examples should not be considered to limit the scope of the invention.

Example 1 Characteristics of Replication Associated Protein (Rep)

Initiation of replication is a function of site specific binding of the Rep protein to its cognate site in the common region. In most of the cases studied so far, the binding site comprises of a small sequence of 5-8 bases in the origin of replication that are repeated and occur in close proximity to the TATA box. Each of these repeats is referred to as iterons. For ToLCV-Nde the binding site of the Rep protein has not been determined so far. Electromobility shift assays (EMS A) were used to identify the Rep protein binding site in the intergenic region of the viral genome and determine the specificity of this interaction using two homologous strains of ToLCV-Nde which differ in their binding site sequence in the common region. Transient expression in tobacco protoplasts indicated that the Rep proteins of the two strains are not interchangeable despite 91% sequence identity between the two genes. In addition, this specificity of replication was found associated with the N-terminal sequences of the Rep protein that in turn may influence its interaction with the origin of replication.

Identification of the binding site of replicase associated protein in the intergenic region of mild and severe strain of ToLCV-Nde

Based upon analogy with published reports potential repeat motifs were identified in the common region of mild and severe strain of ToLCV-Nde close to the TATA (SEQ ED NO: 158) box. The ability of the Rep protein to interact with this repeat motif in electrophoretic mobility shift assays (EMS As) was determined.

The binding site sequence in the severe strain was identified as GGTGTCTGGAGTC (SEQ ED NO : 121 ) while in the mild strain the repeat motif was GGCGTCTGGCGTC GGTGTCTGGAGTC (SEQ ED NO: 159). A 13 nucleotide sequence was used comprising this repeat in binding assays. As a control, the 52 bp common region was used as a probe to interact with the Rep protein. Results showed the formation of a similar complex with both, the identified 13 mer sequence as well as the full length common region suggesting that the identified sequence may well be the binding site for the Rep protein. Similarly, the Rep protein of the mild strain could shift either of the probes resulting in complex. The specificity of this binding was confirmed by competition assays. For both the mild and the severe strain, the complex was abolished when an excess of cold 13 -mer probe specific to each strain was added as a specific competitor in the binding reaction. Further, a 1000 fold excess of an unrelated 18-mer oligonucleotide did not affect the formation of complex in either of the strains. Binding of the rep protein to its cognate site is specific between the strains

To determine if the Rep protein of the mild strain can distinguish its cognate binding site from the severe strain, the 19 mer repeat sequence of the severe strain was used as a probe. In gel shifts, the Rep protein of the mild strain did not form any complex with the severe strain binding site. In analogous experiments with the Rep protein of the severe strain, a very weak complex was observed when the binding site sequence of the mild strain was used as a probe. These results indicated that the Rep proteins of the two strains bind specifically to their cognate sites in the origin of replication.

Specificity of binding by the Rep protein may depend on several factors

Earlier results suggested that binding of the Rep protein to its cognate site may be a specific event which prompted us to investigate the factors influencing the specificity of binding. Synthetic oligonucleotides were designed to address several parameters. These were a the sequence of the repeat motif, i.e. changing the 5 ' or the 3 ' iteron from the related strain or any unrelated virus sequence, b) the spacing within the two repeat motifs and c) the numbers of the repeat motifs: one, two or four.

Sequence of the repeat motif:

5' severe, 3' mild. (GGTGTCTGGCGTC) (SEQ ED NO: 160): The Rep protein of the mild strain did not show any complex formation with this probe while the severe strain Rep protein formed a complex as visualized by a shift in the probe.

5' unrelated. 3' mild. (GGAGTCTGGCGTC)(SEQ ED NO: 161): Neither of the

Rep proteins from the mild or the severe strain could generate a complex with this probe. 5' unrelated . 3' severe: fGGAGTCTGGTGTCVSEQ ED NO: 162): The mild strain Rep protein did not form any complex with this probe but the severe strain formed a very weak complex. 5' mild . Υ unrelated: (GGCGTCTGGAGTC)(SEQ ED NO: 163): Very weak complex formation was observed by the Rep proteins in both strains.

Results of the study showed no complex formation was observed if the spacing between the two repeats was either increased or decreased from the 3 bases already known to exist. Additionally, results with the binding assays showed that the Rep protein of the severe strain was unable to bind efficiently if only a single iteron (GGTGTC) (SEQ ED NO: 164) was presented as a probe, but with two iterons (GGTGTC T GGTGTC) (SEQ ID NO: 165) the binding was more specific and a retarded complex was clearly distinguishable. When the number of repeat motifs was doubled (GGTGTC T GGTGTC T GGTGTCT GGTGTC)

(SEQ ID NO: 166), at least three different bands with retarded mobility were observed, suggesting that the Rep protein recognizes and binds to additional sites.

Doubling the number of available binding sites generated multiple bands.

Binding domain of Rep protein may lie on its N-terminus In order to define the minimal size of Rep protein responsible for binding to the origin, three different truncated Rep proteins from the severe strain were purified. T-Repl has only the 1-52 amino acids of the Rep protein and consists of the motif FLTYPKC (SEQ ED NO: 172), a conserved sequence present in all the organisms that replicate via a rolling circle mechanism and the helices α- 1 and -l . T-Rep2 has the 1-111 amino acids from the N-terminus of the Rep protein and comprises of all the three conserved motifs 1, 2 and 3 as well as the helices α-1 and a-l. T-Rep3 has the amino acids 1 - 160 from the N-terminus of the Rep. All three proteins were checked in gel shift assays using the 19 mer repeat sequence to confirm if they still retain DNA binding activity. Of the three truncated Rep proteins, T-Rep 1 and 2 formed a weak complex that appeared as a faint, retarded band, but T-Rep3 generated a complex similar to the wild type full length protein, suggesting that amino acids 1-160 may contain sufficient information to allow in vitro binding of the Rep protein to its cognate site in the origin of replication.

To confirm if the observed bands with retarded mobility were specifically due to binding of the truncated Rep proteins to the 19 mer binding site repeat motif, a supershift assay was performed using the α- Histidine antibody (Ausubel et al Protocols in Molecular Biology) The purified full length and truncated Rep proteins were allowed to incubate with the α-His antibody on ice followed by incubation with the repeat motif as the probe In all the cases, a supershift was observed suggesting that the interaction of the Rep proteins with the binding site was specific Without the α-His-antibody, the complex formed in all cases migrated much faster on the gel indicating that the supershift was specifically due to the binding of the truncated Rep proteins to its iteron sequence as well as to the antibody In all the cases mentioned, the shifts could be abolished by using excess of specific competitor DNA

In another experiment, the full length and truncated rep protein were immunoprecipitated using ACl antibody All the three truncated Rep proteins could be immunoprecipitated with anti His antibody showing that the complex formation and subsequent trapping was due to specific interaction of the Rep protein with its antibody

Southwestern assays were used to confirm the specificity of the shifts observed with the truncated Rep proteins, a southwestern blot was undertaken Full length Rep protein as well as the truncated versions of it were run on an SDS PAGE and transferred on a nitrocellulose membrane The transferred proteins were then detected using the 13 mer repeat motif as the probe All the three truncated Reps could be detected in the southwestern indicating that only 1-56 amino acids of the Rep protein may be enough to bind or recognize the binding site in the origin of replication

Example 2 Sequence Parameters that Determine Rep Binding Specificity

To determine the parameters that may influence specificity of binding of the Rep protein to its cognate site in the on, two homologous strains of tomato leaf curl virus from New Delhi (ToLC-Nde ) were used These strains share 94% sequence identity but cause very different symptoms on tomato and Nicotiana benthamiana plants (Padidam, M , et al, J. Gen. Virol 76 25-35 (1995)) While the severe strain is characterized by severe puckering and downward leaf curling in the plants, the mild strain produces mild symptoms with minor leaf curl and no puckering of the leaves. In addition, the two strains do not support the replication of the heterologous DNA (Chatterji, A, et al, J. Virol 75:5541-5549 (1999)) making them ideal experimental systems to investigate specificity of interaction between the Rep protein and its binding site in the origin of replication.

Previous studies (Chatterji, A., et al, J. Virol 73:5541-5549 (1999)) indicated a possible interaction between amino acid 10, near the N-terminus of the Rep protein and the putative binding site in the origin that may determine specificity of replication between the severe and the mild strains of ToLCV-Nde.

Exchange of the 10th amino acid between the Rep proteins of the two strains of ToLC-NdeV with a concomitant change in the binding site sequence in the origin altered the replication of the two strains suggesting that these components may influence the levels of viral replication and accumulation (Chatterji, A., et al, J. Virol 73:5541-5549 (1999)).

An objective of this study was to determine if the repeat sequences identified earlier in the origin of replication of the two strains function as the binding sites for their respective Rep proteins. Further, it was of interest to define

DNA sequence requirements for specificity of origin recognition in the Rep proteins of the two strains by using chimeric iteron sequences.

Electrophoretic mobility shift assays (EMSAs) were performed using different synthetic oligonucleotides as probes or competitors to show specificity of binding in our assays. The nature and significance of DNA-protein interaction was studied in vivo using transient replication assays in tobacco protoplasts. Alterations were found with respect to sequence, size or number of iterons that reduced binding by the Rep protein and resulted in drastic reduction in virus replication. In addition, it was found that the inability of the Rep protein of the mild strain to accumulate DNA B of the severe strain was related to its inability to recognize the binding site sequence of the severe strain DNA-B. Materials and Methods

A.. Expression of ToLCV Rep proteins The full length ACl gene from the severe and the mild strains of ToLC-NdeV was amplified from pMPAl (DNA-A of the severe strain, ToLC-NdeV) and pMPA2 (DNA-A of the mild strain, ToLC-NdeV) (Padidam,

M., et al, J. Gen. Virol. 76:25-35 (1995)). The amplified sequence was ligated between Bam HI and Hind III sites in baculovirus expression vector, pBAC4x-l (Novagen) resulting in an in-frame fusion of the ACl gene sequence with the vector sequence encoding a methionine and six histidine residues under the polh promoter. The clones were identified and confirmed by restriction digestion and sequence analysis.

Recombinant baculovirus was isolated by co-transfecting 0.5 (g of recombinant plasmid with 1 g of linearized Autographa calif ornica nuclear polyhedrosis virus DNA (Smith, G.E., and Summers, M.D., Virology 59:517-527 (1978)) into Spodopterafrugiperda Sf9 cells (Summers, M.D., and Smith, G.E.,

Texas Agricultural Experiment Station Bulletin No. 1555 (1987)). Recombinant viruses were plaque-purified and a high titer stock was prepared. Large-scale purification of the target protein was done in Trichoplusia ni Tn-5 cells (Invitrogen).

B. Purification of Rep proteins

Tn-5 cells were harvested 60 h post infection by centrifugation at 3000 rpm for 10 minutes. The pellets were washed in IX PBS and suspended in ice cold IX binding buffer (5 mM imidazole, 0.5 M NaCl, and 20 mM Tris, pH 7.9). The cells were lysed by three cycles of freeze-thaw and the lysate was clarified at 15,000 rpm for 30 minutes. The resulting supernatant was loaded on a Ni-NTA column (Novagen) previously equilibrated with binding buffer and washed with 10 column volumes of wash buffer (70 mM imidazole, 0.5 mM NaCl, and 20 mM Tris, pH7.9). The protein was eluted with IM imidazole, 0.5 mM NaCl and 20 mM Tris, pH7.9. The eluted fractions were dialyzed against 20mM Tris, pH7.9, 150mM NaCl to remove imidazole, concentrated using Centricon filters (Amicon) and protein concentration was estimated using Bradford's reagent (Biorad).

C. Electrophoretic mobility shift assays

ToLC-NdeV specific primers were used to amplify a 52 bp fragment from the ER of the virus genome. This fragment contains the iterons, the transcription start site as well as the TATA box and the conserved hairpin sequence. The amplified fragment was end labeled with (³²P ATP and T4 polynucleotide kinase, purified on polyacrylamide gels and was used as a probe in the EMSAs. The 18mer oligonucleotides containing the potential binding sites (underlined) for the Rep proteins of the two strains were synthesized and annealed to their complementary strands. These two oligonucleotide probes were named bs-m, 5'-GGCGTCTGGCGTCT-3 ' (UI 5017) (SEQ ID NO: 180) for the mild strain and bs-s, 5'- GGTGTCTGGAGTCT-3 ' (U15015) (SEQ ED NO: 189) for the severe strain. The final concentrations of the probes were 500 pM (30,000cpm). The concentration of competitor DNA used was 100 pM per reaction. Probe and competitor DNAs were purified on Sephadex G-25 columns, quantified by a scintillation counting followed by dilution to 30,000 cpm for the binding assays.

The binding assays (EMSAs) were performed using the purified Rep protein from the two strains. The binding reactions contained 500 ng of pure protein, 1 ng of labeled DNA and 0.2 μg of poly dl-dC. Binding buffer contained

20mM HEPES pH 7.5, 60 mM KC1, ImM DTT and 15 % glycerol. Reactions were incubated at 25° C for 30 minutes and the complexes were resolved on 4% polyacrylamide gels in 0.25X TBE. The gels were dried on Whatman paper and autoradiographed. Comparative efficiency of binding was analyzed by quantifying the amount of radioactivity in the retarded bands using the phosphorimager

(Molecular Dynamics).

The sequences of the synthetic oligonucleotides used as probes or competitors in the EMSAs are listed below in Table 1. TABLE 1 Comparative levels of in vitro binding and in vivo replication by the origin mutants

Mutant Oligonucleotide sequence Binding³-⁰ Replication^b

ss^α sc^α

CR-s GGTGTCTGGAGTC 100 100 100

(SEQ ED NO: 121) CR-m GGCGTCTGGCGTC 100 100 89.53

(SEQ ED NO: 123)

IT-l/2(s) GGTGTCTGGCGTC 100 100 93.41

(SEQ ID NO: 168)

IT-l/2(m) GGTGTCTGGCGTC <1 <1 <1 (SEQ ED NO: 168)

IT-3/4(s) GGGGTCTGGAGTC 13.4 28.61 2.18

(SEQ ED NO:51)

IT-3/4(m) GGGGTCTGGAGTC <1 <1 <1

(SEQ ED NO: 52)

IT-5/6(s) GGGGTCTGGCGTC <1 3.23 <1

(SEQ ED NO: 170)

IT-5/6(m) GGGGTCTGGCGTC <1 <1 <1

(SEQ ED NO: 169)

IT-7/8(s) GGCGTCTGGGGTC <1 2.84 <1 (SEQ ID NO: 171)

IT-7/8(m) GGCGTCTGGGGTC 18.41 <1 <1

(SEQ ED NO: 171)

IT-9/10(S) GGTGTCTGGTGTC 102.6 108 103.9 (SEQ ED NO: 53)

IT-11/12(S) GGAGTCTGGAGTC 87.21 98.63 98.82

(SEQ ED NO:58)

IT-13/14(S) GGTGTCTTTTTTGGAGTC <1 4.47 <1 (SEQ ID NO:63)

IT-13/14(M) GGCGTCTTTTTTGGCGTC >1 <1 <1

(SEQ ED NO: 192)

IT-15/16(S) GGTGTCGGAGTC >1 <1 <1 (SEQ ID NO: 174)

IT-15/16(M) GGCGTCGGCGTC >1 <1 1.53

((SEQ ED NO: 175)

IT-17/18(S) GGTGTC 29.47 24.63 <1

(SEQ ED NO: 164)

IT-17/18(M) GGCGTC 8.53 17.64 0.13

(SEQ ED NO: 176)

IT-19/20(S) GGTGTCTGGTGTCTGGTG- 75.94 47.13 TCTGGTGTC (SEQ ED NO:191) IT-19/20(M) GGCGTCTGGCGTCTGGCGTCTGGCGTC - 62.24 36.53

(SEQ ED NO: )

^aThe values shown represent the amount of radioactivity (%) bound in the shifted DNA-protein complex band as a result of the Rep protein binding to the ³²P labeled DNA protein in gel shift assays. ^bThe values shown are average (%) amounts of single stranded (ss) and supercoiled (sc) viral DNA detected in four independent protoplasts transfections per mutant. Protoplasts prepared from N. tabacum BY2 cells were transfected with 2 μg of DΝA-A and harvested 48h after electroporation. Viral DΝA was quantitated using a phosphorimager. Standard error values between different transfections were in the range of ± 2-5%.

The amount of radioactivity bound in the complex shifted as a result of Rep protein binding to its respective CR sequences was assigned a value of 100. ^dThe amounts of viral DΝA observed in protoplasts inoculated with the wild type DΝA-A of the severe or the mild strain were assigned a value of 100.

D. Immunoprecipitation assays

Rep protein was immunopreciptated from 50 (μg of Sf9 cell extract using

10 (μg of anti-ACl antiserum and rabbit anti-mouse IgG coupled to Sepharose beads. The immuno complexes were resolved by SDS-PAGE and detected by immunoblotting (Towbin, H., et al, Proc. Natl Acad. Sci. USA 76:4350-4354

(1979)) using a rabbit polyclonal anti-ACl antiserum and an anti-rabbit goat antibody conjugated with horseradish peroxidase The peroxidase activity was detected using chemiluminescent Supersignal substrate (Pierce)

E Construction of mutants

A 146 bp common region fragment from Nco I to Ssp I from both severe and mild strains of ToLC-NdeV was amplified and cloned in pBS (SK-) vector

Site directed mutations were made in the iterated sequences using overlapping PCR (Horton, R M , "In vitro recombination and mutagenesis of DNA", Methods in Molecular Biology, PCR cloning protocols, B A White, ed , Humana Press Inc , Totowa, New Jersey (1994), vol 67, pp 141-150) Following confirmation of mutations by sequencing, the common region fragments were recloned into the

DNA-A of the respective strains For convenience, the mutants were given names identical to the synthetic oligonucleotides used to create the nucleotide changes in the iteron sequence

F Transient Replication assays Protoplasts isolated from Nicotiana tabacum BY2 cells were electroporated and cultured according to published methods (Watanabe, Y , etal, FEBSLett 119 65-69 (1987)) Transfections were done using 2 (μg each of the wild type or mutant replicon DNA containing a partial tandem copy of pMPAl or pMPA2 (Chatterji, A , etal, J. Virol 73 5541-5549 (1999)) The mutant replicon contained sequence alterations in the iteron sequences of the Rep protein in the viral origin Total DNA from the protoplasts was extracted 48 hours after transfection (Dellaporta, S L , et al, Plant Mol. Biol. Reporter 1 19-21 (1983), Mettler, I J etal, PlantMol Biol. Rep. 5 346-349 (1987)) and analyzed for viral DNA accumulation by Southern blotting (Chatterji, A , et al, J Virol 73 5541-5549 (1999)) Results

Expression and purification of the Rep protein from mild and severe strains of ToLCV

Previous studies (Chatterji, A, et al, J. Virol 73 5541-5549 (1999)) showed that the severe (pMPAl) and the mild (pMPA2) strains of ToLC-NdeV exhibit specificity in replication of their cognate DNA This selectivity is determined by interaction between amino acid residues at the N-terminus of the ACl and the Rep protein binding site in the intergenic region (ER) In this study, the nature of this interaction was directly examined in vitro by competitive DNA binding assays and in vivo by transient replication assays in tobacco protoplasts

To isolate large amounts of Rep protein, the relevant sequences encoding the ACl gene of the mild and the severe strains of ToLC-NdeV were expressed from a polyhedrin promoter in a baculovirus expression vector The recombinant baculovirus was used to infect Sf9 cells to obtain a high titer virus stock and standardize optimal expression of the target protein Cells were harvested at different time points after inoculation and protein was extracted after three cycles of freeze-thaw The Rep protein from both the severe as well as the mild strain of ToLC-NdeV was detected in the crude insect cell lysates by immuno-precipitation using the polyclonal antiserum to ACl gene (Fig 2 A, lanes 1, 2)

The high titer virus stock was used to infect Tn-5 cells for large-scale purification of the target protein The soluble protein extracts were loaded on a Ni-NTA column and the eluted fractions were analyzed by SDS PAGE The purified Rep protein had an estimated MW of 41 KD in coomassie stained polyacrylamide gels (Fig 2B, lanes 2, 5) and its identity was further confirmed by immunoblotting using ACl polyclonal antibody (Fig 2C, lanes 1 to 7)

B Rep proteins of the two strains bind a specific DNA sequence in the origin of replication

Based upon analogy with published reports describing binding sites of the Rep protein (Arguello-Astorga, G R , etal, Virology 203 90- 100 (1994), Fontes, E.P.B., et al, J. Biol. Chem. 269:8459-8465 (1994a)), potential repeat motifs close to the TATA box in the common regions of mild and severe strains of ToLCV were identified. The ability of the Rep protein from the two strains to interact with their respective repeat motifs was determined in EMSAs. The potential repeat sequence in the severe strain was identified as

GGTGTCTGGAGTC (nts 2640-2653) (U15015) ( SEQ ED NO: 121) while in the mild strain the repeat motif identified was GGCGTCTGGCGTC (nts 2640-2653) (SEQ ED NO: 122 ) (U15017) (Chatterji, A., etal, J. Virol 73:5541-5549 (1999)). The 13-bp sequence (nts 2640-2653) containing the repeat motifs was used as the probe in EMSAs with the purified Rep protein. As a control, the 52-bp fragment of the common region (nts 2614 to 2666) from the respective strains was used as a probe in similar assays.

A distinct DNA-protein complex was observed using both the common region (52-bp) fragment as well as the 13-bp repeat sequence (Fig. 3, lanes 1 and 2). This data suggests that the 13-bp sequence may represent the binding site for the Rep protein of the severe strain. Similarly, the Rep protein of the mild strain formed a complex with its respective 52-bp common region fragment as well as the 13-bp repeat motif sequence (Fig. 3, lanes 6 and 7) from the common region.

The specificity of binding by the Rep proteins was tested in competition assays with radiolabelled probes and an excess of unlabeled DNA. Incubation of the Rep proteins with 50 fold molar excess of DNA of the same sequence abolished the formation of the complex completely (Fig. 3, lanes 3 and 8). On the other hand, a 1000 fold molar excess of an unrelated 13-bp sequence derived from the pUC 18 vector DNA did not affect the formation of the complex (Fig. 3, lanes 4 and 9). These results indicate that the interaction of Rep protein with its 13-bp sequence is highly specific. Since no obvious difference in terms of the mobility of the shifted complex was observed as a result of binding of the Rep protein to the 13 -bp oligonucleotide or to the larger common region fragment containing the viral origin, the data suggested that the repeat sequences may constitute the high affinity binding site for the Rep protein. C Rep proteins of two strains exhibit specificity in binding to their cognate iterons

To determine whether the Rep protein from the mild strain could bind to the iteron sequence of the severe strain and vice-versa, purified proteins from the two strains were incubated with labeled 13-bp sequence containing the heterologous repeat motifs in EMSAs No DNA-protein complexes were observed when the severe strain Rep protein was incubated with the oligonucleotide containing the binding site sequence of the mild strain (Fig 3 , lane 5) Furthermore, the mild strain Rep protein did not bind to the 13-bp sequence comprising the repeat motifs of the severe strain (Fig 3 , lane 10) of ToLC-NdeV

D Specificity of binding is related to the sequence, spacing and number of iterons

Unlike the iteron sequences of the mild strain of ToLC-NdeV, the sequence of iterons that constitute the binding site of the severe strain are not identical To better understand the basic DNA sequence requirements that contribute to or influence specificity of origin recognition, chimeric iteron sequences were made by exchanging individual motifs in the binding site of the two strains and determined the ability of Rep protein to bind them In addition, the effect of each of these mutations on virus multiplication was determined in transient assays by assessing the ability of mutant DNA-A components to replicate in tobacco protoplasts

Sequence of the repeat motif: Since the iterons constituting the binding site are repeated, they are referred to as 5' or 3' depending upon their position in the common region Mutant oligonucleotides were synthesized to determine whether the 5' and the 3' iterons contributed equally to the binding efficiency of the Rep protein and if binding specificity between the strains could be altered by exchanging the appropriate iterons These oligonucleotides were used as probes in EMSAs to test their ability to form a complex with the purified Rep proteins

5 ' severe, 3 ' mild: (IT ^X GGTGTCTGGCGTC) (SEQ ED NO 160) The Rep protein of the severe strain formed a strong complex with this sequence (Fig 4 lane 1) However, the Rep protein of the mild strain did not bind very efficiently to this probe resulting in a very weak complex (Fig 4A, lane 7) En protoplasts, the severe strain background with this mutant origin was able to support replication of the virus to wild type levels but the mild strain accumulated very low levels of viral DNA (Fig 5, lanes 2 and 13)

5 ' unrelated, 3 ' severe (IT % GGGGTCTGGAGTC) (SEQ ED NO 182) ) Neither of the Rep proteins from the mild or the severe strain generated a complex with this probe in vitro (Fig 4A, lanes 2 and 8) Also, in tobacco protoplasts, the accumulation of viral DNA was low (Fig 5, lane 3) in the case of severe strain mutant but the mild strain mutant did not replicate any viral DNA at all (Fig 5, lane 14)

5 ' unrelated, 3 ' mild: (IT 5/6 GGGGTCTGGCGTC) (SEQ ED NO 169)

Neither of the Rep proteins bind this sequence (Fig 4 A, lanes 6 and 10) Very low levels of viral DNA accumulation was detected in tobacco protoplasts when inoculated with the severe strain DNA-A component but the mild strain failed to replicate any viral DNA (Fig.5, lanes 4, 15)

5 ^' mild, 3 ' unrelated: (IT 7/8 GGCGTCTGGGGTC) (SEQ ED NO 171 ) Neither of the Rep proteins formed a complex with this probe (Fig 4 A, lanes 6 and 10) In tobacco protoplasts, both mutants failed to accumulate viral DNA (Fig 5, lanes 5 and 16)

The iterons comprising the binding site of the severe strain Rep protein are not identical repeats To determine if either of the iteron sequences influence the binding efficiency, synthetic oligonucleotides were designed with GGTGTCTGGTGTC TIT 9/10) (SEQ ED NO 165)and GGAGTCTGGAGTC TIT 1 1/12) (SEQ ID NO 193) as perfect repeats and tested in EMSAs for their capacity to bind the Rep protein of the severe strain In gel shifts, the Rep protein bound to GGTGTCTGGTGTC (SEQ ID NO 194)as visualized by a retarded band but the binding to GGAGTCTGGAGTC (SEQ ID NO 195)was weaker in comparison to the mutant, 9/10 (Fig 4A, lanes 3 and 4) In protoplasts, both mutants replicated viral DNA indistinguishable from the wild type controls (Fig

5 lanes 6 and 7) Spacing within the iterons: The iterated motifs in both the severe

(GGTGTCTGGAGTC) (SEQ ED NO: 196) and the mild (GGCGTCTGGCGTC)

(SEQ ED NO: 197) strain of ToLCV-Nde are separated by a single nucleotide.

To find out if the spacing between the iterons was significant for origin recognition by the Rep protein, the distance between the iterons was either increased to six nucleotides (IT 13/14) or reduced to none by deleting the single

T nucleotide (IT 15/16) between the iterons. DNA-protein complex were not observed when the spacing between the two repeats was either increased to six bases or decreased to zero (Fig. 3B lanes 1 and 2). Similar results were obtained with the Rep protein from the mild strain (Fig. 4B lanes 3 and 4). In protoplasts, neither of the two mutants accumulated viral DNA (Fig. 5, lanes 8, 9, 17, and 18).

Number of iterons: The Rep protein of the severe strain was unable to bind if only a single iteron (IT 17/18 GGTGTC) (SEQ ED NO: 198)was used as a probe

(Fig. 4B,lane 5). When the number of repeat motifs was doubled(IT 19/20 GGTGTCTGGAGTCTGGTGTCTGGAGTC (SEQ ED NO : 199), multiple bands with retarded mobility were observed (Fig. 3B, lane 7). The Rep protein of the mild strain did not bind as efficiently to a monomer, GGCGTC (IT 17/ 18).

Doubling the number of repeat sequences (IT 1 9/20

GGCGTCTGGCGTCTGGCGTCTGGCGTC) (SEQ ED NO:200) did not improve efficiency of binding (Fig. 3B, lane 8). In transient assays, the virus mutant origins containing a single iteron did not replicate very well and only little accumulation of ss DNA (Fig. 5, lanes 10 and 19) was observed. But the mutants containing twice the number of repeat motifs in their origin were able to support virus replication and accumulated viral DNA similar to wild type levels (Fig. 5, lanes 11 , and 20).

These results suggest that binding of the Rep protein to its cognate site is highly specific and the sequence of both the 5' and the 3' iteron is important for binding to occur. Secondly, while both the iterons are required for binding, they contribute differently to the efficiency of binding. The 5' iteron appears to be more important for binding, yet, substitution by an unrelated 3' iteron did not allow efficient complex formation. Third, the efficiency of binding of the Rep protein to its cognate site can be correlated to the levels of virus replication and accumulation in protoplasts. Doubling the number of available binding sites generated multiple bands in gel shift assays but did not impact virus replication.

Discussion

This example defines DNA sequences in the viral origin of replication that are specifically recognized by the respective Rep protein of two strains of ToLC-NdeV and demonstrates that binding of the Rep protein to their cognate sequences is essential for viral replication. In addition, the binding of the Rep proteins to their cognate iterons is found to be highly specific between the strains and is dependent upon several criteria including the sequence, spacing and the number of iterons. Further, evidence is provided that any mutation in the iteron that affects DNA binding in vitro impacts viral DNA accumulation in vivo.

Previous studies (Chatterji, A, et al, J. Virol 73:5541-5549 (1999)) identified 13 -mer repeat sequences in the common region of ToLC-NdeV involved in interaction with the Rep protein and are essential for virus replication.

Competitive DNA binding assays using purified Rep proteins established that the

Rep protein of ToLC-NdeV (severe strain) specifically binds to the iterated sequence, 5' GGTGTCTGGAGTC (U15015) (SEQ ED NO: 121) located on the

5' end of the TATA box between positions 2640 and 2653 and is conserved between the DNA-A and DNA-B components. Similar iterated sequences have been found in the common region of several geminiviruses (Arguello-Astorga,

G.R., et al, Virology 203:90-100 (1994)) and some have been verified in biochemical assays to act as Rep protein specific binding sites (Fontes, E.P.B., et al, Plant Cell 4:597-608 (1992); Fontes, E.P.B., et al, J. Biol. Chem. 269:8459-8465 (1994a); Behjatania, S.A., et al, Nucleic Acids Res.

26:925-931(1998)). The specificity of binding was confirmed by competition assays. A 50 fold molar excess of the homologous probe abolished the formation of a DNA-protein complex, yet, a 1000 fold molar excess of heterologous DNA

(pUC 18) did not affect the binding of the Rep protein indicating specificity of the complex formed. The binding efficiency of the Rep protein to the 13 bp oligonucleotide or to the 52 bp common region fragment was indistinguishable. This data suggested that the complex observed as a result of Rep protein binding to the 13 bp sequence is authentic and the two repeat sequences represent the binding site of the Rep protein in both the mild and severe strains of ToLC-NdeV.

The sequence of the repeat motifs which constitute the binding site differ between the mild and the severe strain by two base pairs, GGTGTCTGGAGTC

(U15015) (SEQ ID NO:121) for the severe strain versus GGCGTCTGGCGTC

(UI 5017) (SEQ ED NO: 122) for the mild strain. In EMSAs the Rep protein of the severe strain did not form a complex with either the 13 -mer DNA sequence of the mild strain or the 52 bp fragment from the common region of the mild or severe strain, suggesting a high degree of specificity between the Rep protein of the two strains for their cognate sites. This observation is not surprising since there are several examples among geminiviruses documenting high specificity in replication of cognate genomes. As well, pseudorecombinants can only be formed between related viruses or strains of a specific virus (Lazarowitz, S.G., et al, Plant Cell 4:799-809 (1992); Stanley, J., etal, Proc. Natl. Acad. Sci. U.S.A. 57:6291-6295 (1990); Frischmuth, T., et al, Virology 796:666-673 (1993); Fontes, E.P.B., et al, Plant Cell 6:405-416 (1994b)) reported that the Rep protein of bean golden mosaic virus (BGMV) can specifically recognize its cognate site but is unable to bind to the corresponding binding site of the related virus like TGMV. Similarly, the inability of Worland, CFH and the Logan strains of beet curly top virus (BCTV) to transcomplement replication of each other might be related to specific recognition of the binding site by the corresponding Rep proteins (Choi, I.R., and Stenger D.C., Virology 106:904-911 (1995); Stenger, D.C., Phytopathology

55:1174-1178 (1998)). Together, these results highlight the importance of specific interaction between the Rep protein and its cognate site in the replication of geminiviruses. In addition, it is important to emphasize the fact that precise recognition motifs of Rep proteins may be different between the viruses and are not interchangeable thereby maintaining specificity between closely related viruses and strains of the same virus. Direct evidence for the essential role of Rep protein binding to the repeat sequences in geminivirus replication was obtained by the mutation of these sequences in the viral origin and testing their ability to replicate in tobacco protoplasts It was found that the ability of the Rep protein to bind to its cognate site was correlated to the ability of the virus DNA to replicate in transient assays

Mutants that did not form a complex in the EMSAs did not replicate viral DNA suggesting that recognition or binding is a prerequisite for replication and viral DNA accumulation to occur

The sequence requirements which govern the recognition specificity of the Rep protein for its cognate site was determined by altering sequence, spacing, and number of iterons Mutagenesis of the Rep protein binding site showed that both repeat motifs are essential for DNA binding to occur, yet, the efficiency of binding was related to the sequence of its cognate 5' iteron These results were also reflected in the reduced levels of virus accumulation in protoplasts transfected with mutant viral DNA suggesting that replication is directly correlated to the Rep protein binding to its specific site in the origin

Furthermore, recognition of the binding site by the Rep protein was also found to be sensitive to any changes made in the spacing between the iterons Drastically reduced levels of virus accumulation were observed in protoplasts when the spacing between the two iterons was changed to either six nucleotides or reduced to none Because the Rep protein may bind as a dimer (Fontes, E P B , et al, J. Biol Chem. 169 8459-8465 (1994a), Fontes, E P B , et al, Plant Cell 6405-416 (1994b)), it is possible that the proximity of the two repeat motifs is congenial for binding to occur and any alterations with respect to spacing between the two iterons does not allow efficient recognition

Our results indicate that the two iterons, even when they are identical make different contributions to the efficiency of binding as observed in the case of mild strain of ToLC-NdeV and has also been shown for TGMV (Fontes, E P B., et al, J. Biol. Chem. 169 8459-8465 (1994a)) However, our results suggest that the 5' iteron is more important than the 3' one as far as binding is concerned, in case of TGMV (Fontes, E P B , et al, J. Biol. Chem. 269:8459-8465 (1994a);Fontes, E.P.B., etα/., R/α«tCe// 6:405-416 (1994b)) and BCTV(Choi, I.R., and Stenger D.C., Virology 116:11-116 (1996)), the 3' iteron has been shown to be more important for efficient binding as compared to the 5' repeat. In gel shift assays, the Rep protein of the severe strain was able to bind strongly to the sequence GGTGTCTGGTGTC (SEQ ED NO:201)than when

GGAGTCTGGAGTC (SEQ ID NO:202) sequence was used as a probe. Also, the probe GGTGTCTGGTGTC (SEQ ED NO: 203) was able to compete out any complex with the wild type repeat sequence, GGTGTCTGGAGTC (SEQ ED NO: 121) abolishing the binding of the severe strain Rep protein with its wild type iteron sequence. This data suggests that 5' iteron sequences are necessary for efficient binding of the Rep protein to its binding site.

The effect of deletion of a single base pair between the two repeat sequences resulted in negligible virus replication levels indicating that the ToLC-NdeV origin is sensitive to changes in the spacing of the iterons. Doubling the number of iterons resulted in the appearance of multiple bands which may indicate protein-protein interaction facilitated as a result of multimerization of the Rep protein, without causing any effect on accumulation of viral DNA. These results imply that recognition of cognate iterons may represent an essential step in replication process, yet, other interactions between these elements and possibly, yet-to-be-identified proteins that recognize or bind them.

Summary

The DNA binding sites for the replication associated protein (Rep) of two strains of tomato leaf curl virus from New Delhi (TolC-NdeV) were identified using electrophoretic mobility shift assays (EMSAs). The Rep proteins of the two strains were found to exhibit strict sequence specificity in recognition of their cognate repeat motifs (iterons) in the origin despite the fact that they share 91% sequence identity between them. Using a series of synthetic oligonucleotides as probes in EMSAs, the interaction of Rep protein with its binding site was found to be dependent upon number, size and sequence of the two iterons. Mutations in the sequence of the repeat motifs or alterations in the arrangement of the motifs resulted in loss of DNA binding and accumulation of viral DNA in protoplasts suggesting that binding of Rep protein to its cognate iterons is an essential step in virus replication. In addition, a difference in sequence of two base pairs in the binding site of two ToLC-NdeV strains was found to affect DNA binding by the corresponding Rep protein and replication of the virus DNA in protoplasts.

Example 3 Truncated Replication-Associated Proteins (Rep)

Previous studies indicated a possible interaction between amino acid residues 9-10, specifically, amino acid 10 for ToLCV-Nde, at the N-terminus of the Rep protein and its binding site in the origin of replication that in turn determines specificity of replication between two strains. Mutagenesis and exchange of the 10th amino acid between the mild and the severe strain with concomitant change in the binding site sequence of ToLCV-Nde altered the replication ability of the two strains suggesting that these two components may influence the levels of viral replication and accumulation.

To better understand the significance of earlier observations which suggested an interaction between the N-terminal amino acid residues (9 and 10) of the Rep protein and the iteron sequences in the origin of replication, comparison was made of the ori and the N-terminal Rep protein sequences from a few geminiviruses, like Tomato golden mosaic virus (TGMV; iteron sequence,

GGTAG), Beangoldenmosaicvirus(BGMV; iteronsequence, TGGAG), Tomato leaf curl virus (ToLCV-Nde: iteron sequence, GGTGT) and Potato yellow mosaic virus (PYMV; iteron sequence, GGGGG). Differences at amino acid positions 9 and 10 on the N-terminal of their Rep proteins were investigated. Differences were found in the viruses at position 10 as shown in Table 2.

The Rep sequence variation in the four cases stated in table 2 at amino acid 9 did not appear very significant (isoleucine and valine are both neutral amino acids), but at position 10 differences were observed with respect to different iterons. Since the arrangement, sequence and orientation of iterons Table 2. Comparison of iterons and the Rep N-terminal amino acids at position 9 and 10 in four different geminiviruses

Virus Rep⁹'¹⁰ Iteron Rep N-termianl Sequ

TGMV I, N GGTAG FRIN

BGMV V, Q TGGAG FRVQ

ToLCV-Nde V, N GGTGT FRVN

PYMV I, K GGGGG FSEK

PaLCV I, N GGGGA FCEN

ITmLCV I, N GGTGG FNTN

within the members of the family geminiviridae reported so far are limited to only eight different types (Arguello-Astroga et al., 1994: listed in Figs. 1A-1C) one would expect that the variation at the N-terminus of Rep might be limited too. Results as outlined in the examples have shown that at least in case of ToLCV, these iterons bind the Rep protein specifically acting as Rep protein binding sites.

It has also been shown that binding is sequence specific so that the Rep protein from two highly homologous strains of ToLCV that differ in their iteron sequence binds only to cognate iterons. Results have demonstrated that viral replication is compromised if iteron sequences are exchanged between the strains without changing the Rep sequence. Taken together, these results strongly suggested that interaction between the Rep protein and its iteron sequence is the first crucial step in recognition and initiation of virus replication. Therefore, this can be exploited as a means to block or completely shut down viral replication in plants.

The ability of truncated and full length Rep proteins to compete for the repeat sequence (iterons) constituting the binding site of the protein was determined in vivo by transfecting tobacco protoplasts with 4 μg each of viral DNA-A and the truncated or full length rep protein on an expression vector. Protoplasts were harvested 48h after inoculation and assayed for virus replication by analyzing total DNA on Southern blots.

Results suggest that in vivo, the truncated Rep proteins tend to compete with the full length Rep protein for the same binding site sequence, as evident by a reduction in the levels of viral DNA accumulation in case of both the mild and the severe strains. Co-inoculation of viral DNA-A with any of the three truncated Rep proteins appear to reduce virus accumulation but, in both mild and the severe strain Rep proteins, the T-Rep3 causes maximum reduction in the level of virus accumulation. Results of the transfections are provided in Tables 3 and 4.

Table 3. Competition between the full length (Al or A2) and truncated Rep proteins (T-Repl, T-Rep2, T-Rep3) for the same iteron sequences

Virus % replication

1. Al alone 100

2. A2 alone 100

3. Al+T-Repl 90

4. Al+T-Rep2 45

5. Al+T-Rep3 48

6. A2+T-Repl 40

7. A2+T-Rep2 50

8. A2+T-Rep3 5

Since this strategy is based upon a very fundamental step in virus infection and establishment in the host and because there appear to be limited types of iterons present within the Begomoviruses, it offers a broad application in control of geminiviruses by combining different types of truncated Rep proteins recognizing different types of iterons Based on this experiment, it is possible to compete with the wild type Rep protein by expressing a truncated Rep that will

Table 4. Co-inoculation experiments to determine if the severe strain (Al) is dominant over the mild strain (A2) with respect to recognition of the cognate iteron

Virus % replication

1 Al+T-Repl of A2 50

2 Al+T-Rep2 of A2 50

3 A2+T-Repl of Al 5

4 A2+T-Rep2 of Al 8

5 Al+FLRep of Al 100- 125

6 Al+FL Rep of A2 80 7 A2+FL Rep ofA2 100-120

8 A2+FL Rep ofAl 80

interfere with normal virus replication and in one case could reduce it to as low as 5% of the wild type levels

Example 4

Expression of N-Terminal Sequences of the Replication-Associated Protein (Rep)

The binding sites of ToLCV-Nde Rep are not known Potential binding site sequences were identified in the common region of ToLCV-Nde genome by site directed mutagenesis (Chatterji, A. , et al. , J. Virol 73 5481 -5489 ( 1999)) and further confirmed by gel shift assays using purified Rep protein (Chatterji et al , in preparation) The ToLCV-Nde Rep protein binds to the iterated motifs, GGTGTCTGGAGTC (nts 2640-2653) (U15015) (SEQ ED NO 121) in the origin of replication In this study the minimal binding domain on the ACl gene of ToLCV was mapped by determining the ability of truncated Rep protein to bind origin DNA sequences using electrophoretic mobility shift assays (EMSAs) The effect of transient expression of truncated and full length ACl sequences on viral

DNA replication in Nicotiana tabacum BY2 protoplasts and N benthamiana plants was also tested Next, the ability of the truncated Rep protein of ToLCV- Nde to bind the iteron sequences of other geminiviruses in EMSAs was tested and therefore, the effect of its expression on replication of other geminiviruses was determined The Rep protein of ToLCV-Nde specifically binds to its origin recognition sequence, GGTGTCTGGAGTC (SEQ ED NO 121) in EMSAs and the expression of its N-terminal sequences inhibits the replication of viral DNA Further, transient expression of the ToLCV-Nde truncated Rep protein encoding the minimal binding domain could inhibit the replication of other geminiviruses that have similar iteron sequences

Materials and Methods

Plasmid Constructs

A) Mapping the minimal binding domain of ToLCV ACl gene

Coding sequences corresponding to ACl were PCR amplified and cloned in bacterial expression vector pET 28a (Novagen) and overexpressed in E coli cells The recombinant proteins were named according to their C- and N- terminal amino acids The C-terminal truncations were made by inserting an in-frame stop codon at positions 2436, p AC 1-1 ₍₁.₅₂₎, 2250, p AC 1 -2_{( 14)}, and 2110, pAC 1 -3 _{( 60)} The truncated AC 1 sequences were sub-cloned as Bam HI to Hind III fragments in pET 28 A vector digested similarly to give pACl-1 and pACl-2 and pACl-3 respectively At the N-terminus only one truncation was made by deleting the first 21 amino acids and inserting a Nco I site to create an in-frame start codon The truncated fragment was cloned as a Nco I to Hind III fragment in the vector pET 28 a to produce pACl-4_(22.360) B) Construction of plant expression cassettes

For expression of a truncated AC 1 gene in plant cells the C-terminal truncations described above were sub-cloned as Bam HI fragments in a similarly digested plant expression vector, plau 2 (pELTAB 350) under a cassava vein mosaic virus (CsVMV) promoter to produce the gene expression cassettes, pELTAB 401, pILTAB 402 and pILTAB 403 respectively. Constructions of infectious clones of pMPAl and pMPBl have been previously described

(Padidam, M., et al, J. Gen. Virol. 76:25-35 (1995)). Full-length infectious dimers of African cassava mosaic virus (ACMV-kenya), pCLV 1.3 A and pCLV 2B (Stanley, J., Nature 305:643-645 (1983)) and infectious monomers of pepper hausteco virus, PHV (Bonilla-Ramirez, G.M., et al, J.Gen Virol. 75:947-951

(1997)) were obtained. The potato yellow mosaic virus clones have been described (Umaharan et al, 1999).

C) Electrophoretic mobility shift assays (EMSA)

EMSA were performed similar to the method in Example 2

D) Transient Replication assays in protoplasts and plants Transient replication assays in protoplasts were performed similar to

Example 2

Transient replication assays in plants were performed as follows. Two week old seedlings of N benthamiana were grown in magenta boxes and inoculated with partial tandem dimers of viral DΝA using a Bio Rad helium driven particle gun (Padidam, M., et al, J. Gen. Virol. 76:25-35 (1995)). Ten plants were inoculated for each mutant using 0.5μg each of DΝA-A and DΝA-B genomic components per plant. Plants were observed for symptom development and the newly emerging leaves were harvested for Southern blot analysis after 3 -4 weeks post inoculation. E) Southern blot analysis

DNA extractions from systemically infected leaf samples were done as described in Dellaporta, S.L., et al, Plant. Mol. Biol. 7: 19-21 (1983) and from protoplasts by following the procedure of Mettler (Plant. Mol. biol. Rep. 5:346- 349 (1987). Total DNA (4μg) was electrophoresed on 1% agarose gels without ethidium bromide and transferred on nylon membranes. Viral DNA was detected using a component specific radiolabelled probe (a 900bp, Aflll -Pstl fragment containing the ORFs ACl, AC2 and AC3), or a probe specific for the B component (878 bp PCR amplified BC1 ORF). The amount of viral DNA was quantified as previously described (Chatterji, A., et al, J. Virol 73:5481-5489

(1999)) by exposing the Southern blots to phosphor screens and counting on a phosphorimager (Molecular Dynamics).

F) DNA and polypeptide sequences or accession numbers used in the example. Gene bank accession numbers of the DNA used in this study are as follows: pMPAl-U15015, pMPBl-U15017, pMPA2-U15016, ACMV-K-J02057, J02058, PHV-mex-X70418, 70419 and PYMV/TT-AF039031.

The peptide sequence of the full length Rep protein of ToLCV-Nde is: MASPRRFRVNAKNYFLTYPKCSLTKEEALSQLQTLETPTKKKFIKICRE LHEDGSPHEHVLIQFEGKFQCKN RFFDLVSPSRSAHFHPNIQGAKSAS

DVKNYIAKDGDVLEWGVFQIDGRSARGGQQTANDAYAQAENTGNKD DALRVLKELAPKDYVLQFHNLNTNLDREFQPPSEVYVSPFSISSFDRVPA DLVDWVSSNWCAAARPFRPISIVIEGDSRTGKTMWARCLGPHNYLCG HLDLSPKVYSNDAWYNΛTDDVDPHYLKHFKEFMGAQRDCQSNTKYG KP VMEKGGIPTEFLCNKGPNS S YKEYLDEEKNAALKQWAEKNAVFITLE

EPLYSGREMALPEEEEEHSQEAS (SEQ ED NO: 183) (U15015)

TRepl/pACl-1 has the first 52 amino acids from the N-terminus cloned in plau 6 and the sequence is as follows: MASPRRFRVNAKNYFLTYPKCSLTKEEALSQLQTLETPTKKKFIKICRE LHE (SEQ ED NO: 184) (U15015) TRep2/pACl-2 has the first 114 amino acids from the N-terminus cloned in plau 6 and the sequence is as follows:

MASPPvRFRVNAKNYFLTYPKCSLTr^EALSQLQTLETPTKKKFEKICRE LHEDGSPHIHVLIQFEGKFQCKNNRFFDLVSPSRSAHFHPNIQGAKSAS DVKNYIAKDGDVLEWG (SEQ ED NO:185) (U15015)

TRep3/pACl-3 has the first 160 amino acids from the N-terminus cloned in plauό and the sequence is as follows:

MASPRRFRVNAKNYFLTYPKCSLTKEEALSQLQTLETPTKKKFEKICRE LHEDGSPHIHVLIQFEGKFQCKNNRFFDLVSPSRSAHFHPNIQGAKSAS D VKNYIAKDGD VLEWGVFQIDGRS ARGGQQTANDAYAQAINTGNKD

DALRVLKELAPKDYVL (SEQ ED NO: 186 (U15015)

Results

A) Determination of the minimal binding domain of the Rep protein of ToLCv-Nde The AC 1 binds specifically to a directly repeated motif DNA sequence in the common region of the ToLCV-Nde genome. Purified Rep proteins were truncated at amino acids 160, 114 and 52 to map the C-terminal boundary of the ACl DNA binding domain in vitro. As a control, a full length Rep protein (encoding amino acids 1-360 of the ACl gene) was used in all assays. The truncated and full length Rep proteins were over-expressed in E. coli under a T7 promoter and purified on a nickel affinity column. The affinity-purified proteins were highly enriched as determined by coomassie stained SDS PAGE gels and were detected in immuno-blots using the anti- histidine antibody.

His tagged AC 1 proteins were tested for their ability to bind a radiolabeled 13 mer (nts 2640-2653) that includes the ACl binding site sequence,

5 GGTGTCTGGAGTC3' (U15015) (SEQ IDNO: 121). Shifted complexes were observed with full length (1-360) and C-terminal truncated proteins, pAC 1 -3 ₁₆₀. No binding was observed for pACl-l₍₁.₅₂₎ or pACl-2₍₁.₁₁₄₎. Together these results located the C-terminal boundary of the ACl DNA binding domain to lie between amino acids 115 to 160. The N-terminal boundary of the ACl binding domain was determined in vitro by comparing the activity of the full length AC1₍₁.₃₆₀₎ and N-terminal truncated AC1₍₂₂.₃₆₀₎ to bind the 13 bp sequence, 5 GGTGTCTGGAGTC3' (U15015) (SEQ ED NO 121) in gel shift assays The shifted complex was observed only with the full-length Rep protein and no bound DNA was detected for AC 1 _(22-36o₎ These results demonstrated that the sequences within the first 21 amino acids of AC 1 are essential for protein DNA interactions Put together, these results limited the DNA binding domain of ToLCV-Nde Rep protein to lie between amino acids 1-160

B ToLCV-Nde replication is inhibited by transiently expressed AC 1 The effect of ACl on viral DNA replication was investigated by co-inoculation of N tabacum BY2 protoplasts with DΝA-A and various expression cassettes expressing truncated ACl gene sequences from a CsVMV promoter ToLCV-Νde DΝA-A alone replicated in B Y-2 cells and accumulated

Table 5: Virus replication in BY2 protoplasts and Nicotiana benthamiana plants co-inoculated with truncated Rep protein constructs and viral DΝA (Al).

Virus construct Symptom expression* Replication* Plants Protoplasts

Al+B Severe, 10/10 100 100

A2+B Mild, 10/10 55 48-50

Al+B+T-Repl 9/10 mild, 1/10 severe 100 90

Al+B+T-Rep2 Mild, 10/10 90 45

Al+B+T-Rep3 Mild, 10/10 30-40 45

Al+B+T-Repl/A2 Severe, 10/10 100 94

Al+B+T-Rep2/A2 Severe, 10/10 100 89

Al+B+T-Rep3/A2 mild-intermediate, 10/10 60 45-50

A2+B+T-Rep3/A2 No symptoms, 10/10 50 56

# A total often plants were inoculated Shows the number of plants infected / number of plants inoculated Plants were scored for symptom expression three weeks post inoculation * The numbers refer to the amount (m percentage) of viral DNA replication in protoplasts electroporated with similar constructs The viral DNA was quantified using a phosphoπmager (Molecular Dynamics) high levels of single stranded (ss) and supercoiled (sc) DNA (Fig 6 A, lanes 1-4) but a significant decrease in the level of viral DNA replication (50-60% drop) occurred in the presence of truncated AC 1 expressed from pAC 1 -3 (Fig 6B, lanes 1-12, Table 5). Reduction in replication levels was estimated by quantifying the amount of radioactivity using a phosphorimager (Storm 860, Molecular Dynamics). The levels of reduction in viral replication was not as dramatic in the presence of expressed plasmids p AC 1 - 1 and p AC 1 -2. pAC 1 - 1 encodes 52 amino acids of the N-terminal of ACl followed by an in-frame stop codon immediately after the 52 amino acids. pACl-2 has the capacity to encode 114 N-terminal amino acids of the ACl gene. Earlier experiments using the gel shift assays showed that the pACl-1 and pACl-2 do not contain an intact DNA binding domain as compared to p AC 1 -3 , therefore, these results implied that presence of an intact DNA binding domain is essential to observe the inhibitory effect of truncated ACl on viral replication.

Analogous truncations made in the ACl gene of the mild strain of ToLCV-Nde exhibited similar inhibitory effect on viral DNA accumulation in B Y2 protoplasts (Table 6).

Table 6: Virus replication in BY2 protoplasts and Nicotiana benthamiana plants co-inoculated with truncated Rep protein constructs and viral DNA (A2).

Virus construct Symptom expression* Replication*

Protoplasts Plants

Al+B Severe, 10/10 100 100

A2+B Mild, 10/10 55 52

A2+B+T-Repl Mild, 10/10 55 56

A2+B+T-Rep2 Mild, 10/10 45 42-45

A2+B+T-Rep3 Mild, 10/10 32-35 42

A2+B+T-Repl/Al mild, 10/10 50 60

A2+B+T-Rep2/Al mild 10/10 52 58

A2+B+T-Rep3/Al mild,10/10 50 58 C. Transformation of N. benthamiana

Two week old seedlings of N benthamiana plants were co-bombarded with 2μg each of infectious dimers of DΝA-A and DΝA-B as well as truncated ACl gene sequences expressed from CsVMV promoter. The plants were regularly screened for symptom development . Symptom severity was graded from asymptotic (score 0) to mild (very minor leaf curl, no puckering of leaves, no stunting of plants, score 1), intermediate (leaf curl symptoms but no adverse effect on growth, score 2) and severe (severe leaf curl, prominent blistering on the leaves and extreme stunting of plants, score 3). All (10/10) inoculated wild type plants developed severe symptoms five days after inoculation. In contrast, plants co-inoculated with pACl-3 were less susceptible to ToLCV infection (Table 6) . Four out often plants were asymptomatic, 3/10 showed mild symptoms and only 2/10 plants expressed intermediate symptoms of leaf curl (Table 6). None of the 10 plants showed severe infection or stunted growth typically expressed by wild type inoculated plants. Most of the plants inoculated with pACl-1 andACl-2 developed severe symptoms seven days post inoculation (Table 6).

The level of viral DNA in ToLCV infected plants was analyzed by southern blot analysis of young leaves sampled 21 days post inoculation using DNA-A (ACl) and DNA-B (BC1) specific probes (Fig 7). The viral DNA levels were variable ranging from undetectable to very low (an average of 15% of the

WT levels) in asymptomatic plants but the accumulation of both genomic components increased with increasing severity of symptom expression. Plants co-bombarded with expression cassettes pACl-1 and pACl-2 developed intermediate to severe symptoms in majority of the cases.

D. Transiently expressed N-Rep of ToLCV reduces viral DNA accumulation in other geminiviruses

To further investigate the potential of truncated Rep protein to inhibit the replication of other geminiviruses, the effect of pACl-3 expression on viral DNA accumulation of ACMV, PHV and PYMV/TT was studied. The rationale in choosing theses viruses was based on the sequence of their putative origin recognition motifs present on their respective viral genome. If p AC 1-3 binds to its homologous recognition sequence, GGTGTC (U15015) (SEQ ED N204O:205 ) in the origin, it might bind to similar or identical sequences even when they are present in the origin of other geminiviruses. Further, if binding to origin sequences is correlated to replication of the virus genome (as was observed in case of ToLCV-Nde), then it was predicted that as long as the truncated viral Rep pACl-3 can bind to the origin function sequences of other geminiviruses it can potentially interfere in the replication of other geminiviruses as well.

The binding site sequences of ToLCV-Nde are not identical repeats, (GGTGTCTGGAGTC) (U15015) (SEQ ID NO:206). In case of ACMV, the putative iteron was identified as GGAGA (J02057) (SEQ ED NO:207); for PHV, the putative origin recognition motif may be GGTGA (SEQ ED NO: ) (X70418) and in case of PYMV, the potential binding sites may be GGTGT (SEQ ED NO: ) (AF039031) .

Table 6: Regulation of virus DNA replication in BY2 protoplasts by the N-terminal sequences of ACl gene of ToLCV-Nde

Virus Iteron N-Rep sequence EMSA pACl-3

ACMV GGAGA MRTPPRFRIQANKYFLTYPKC + 33-37

(SEQ ID NO: ) (J02057)

PHV GGTGA MPLPKRFRLNAKNYFLTYPQC +/- 46-60

(SEQ ID NO: ) (X70418)

PYMV GGTGT MP-PKRFRENANKYFLTYPKC + 47-50

(SEQ ID NO: ) (AF039031)

Only T-Rep3 was tested in competition experiments. The numbers indicate virus replication levels as determined by southern blotting and phosphorimage analysis. Since any virus that had identical iteron sequences as the severe strain of ToLCV-Nde was not tested, it can only conclude that replication levels of the virus may go down in cases where competition is afforded by the truncated Rep. And that competition will result in cases where the origin sequences can be recognized by the truncated rep protein.

Co-inoculation of wild type DNA-A components of ACMV, PHV and PYMV with pACl-3 of ToLCV-Nde Rep protein caused a decrease in the viral replication levels (Table 6) even though the decrease in replication was not as significant as the inhibition observed in the case of p AC 1-3 for its homologous ToLCV DNA.

Discussion

The minimal DNA binding domain of the ACl gene of ToLCV to amino acids 1-160 was mapped and it was shown that the transient expression of these N-terminal sequences of the AC 1 significantly inhibits ToLCV DNA accumulation in tobacco protoplasts and plants. It was found that the sequences comprising the first 1 -52 or 1 - 114 amino acids of the Rep protein cannot effectively compete with the full length Rep protein to cause a reduction in virus accumulation even though a minor reduction in virus replication was observed. However, the pACl-3 truncation afforded maximum competition to the full length Rep protein in terms of binding to the ori sequences as well as in reducing viral replication in protoplasts and plants. Comparison of competition experiments done between the mild and the severe strains of ToLCV-Nde in protoplasts and plants suggested that only homologous Rep sequences can compete and influence virus accumulation since the pACl-3 from the severe strain did not affect the virus replication of the mild strain and vice versa.

Geminivirus Rep proteins are multifunctional and are involved in both replication and regulation of gene expression. The ACl protein of TGMV has been known to bind with high affinity to repeat motifs located between the conserved TATA box and the initiation site of ACl transcription (Fontes, E.P.B., et al, Plant Cell 6:405-416 (1994a); Fontes, E.P.B., et al, J. Biol. Chem. 269:8459-8465 (1994b)). It is presumed that this binding to the origin recognition sequence is responsible for the repression of AC 1 transcription in TGMV (Sunter,

G., et al, Virology 795:275-280 (1993); Eagle, P.A., et al, Plant Cell 6: 1157-1170 (1994)) and ACMV (Hong, Y., and Stanley, J., J.Gen. Virol. 76:2415-2422 (1995)). On the basis of current understanding of Rep functions, there are several possible explanations for the down regulation of virus replication levels by the AC 1. Constitutive expression of the truncated viral rep protein could compete with the incoming Rep for binding to the viral origin of replication and influence viral accumulation levels thereby acting as a dominant negative mutant (Herskowitz, I., Nature 319:119-111 (1987)). Or it could adversely affect the integrity of the viral DNA by introducing nicks at cryptic motifs as described for wheat dwarf virus (Heyraud, F., etal, EMBOJ. 72:4445-4452 (1993)). Another possibility is that the truncated Rep protein could repress transcription of the AC 1 gene of the incoming virus by interacting with the upstream regulatory sequences in the origin. The NTP binding domain on the Rep protein of ToLCV was not mapped and it is possible that pACl-3 lacks this domain and therefore inhibits viral DNA replication.

Based on the strength of the results obtained with homologous Rep and ori gin recognition sequences of ToLCV strains, the question is raised whether the same truncated Rep protein can interfere with the replication of related geminiviruses that have similar origin recognition sequences in their origin of replication. The results suggest that the transient expression of the ToLCV Rep protein encoding the DNA binding domain can reduce the replication of AVMV, PHV and PYMV to approximately 40-45% in tobacco protoplasts. Since the truncated Rep protein could also down regulate virus replication levels in other geminiviruses competition afforded by the p AC 1 -3 (competent for DNA binding) may be responsible for observed reduction in viral accumulation. Virus having identical iteron sequences as the severe strain of ToLCV-Nde was not tested, therefore it may be concluded that replication levels of the virus may go down in cases where competition is afforded by the truncated Rep. Several approaches to control of geminiviruses have been developed.

Transgenic N benthamiana plants expressing defective interfering DΝA (Stanley et al., 1990, Frishmuth, T., and Stanley, J., Virology 753:539-544 (1991)) of ACMV were less susceptible to ACMV infection but resistance was confined to closely related strains of ACMV owing to the need for ACl mediated trans complementation of the Dl DΝA. Transgenic N tabacum expressing antisense

RΝA targeted against TGMV AL1 (Day, A.G., et al, Proc. Natl. Acad. Sci. USA 88 6721 -6725 ( 1991 )) or tomato yellow leaf curl (Bendahamane and Gronenborn, 1997) showed that specificity of resistance depended on the level of homology between the antisense RNA and the target sequence Finally, the potential of expressing a full length AC 1 transgene in ACMV (Hong, Y , and Stanley, J , Mol. Plant Microbe Int. 9 219-225 ( 1996)) and the N-terminal sequences of TYLCV rep (Noris, E , et al, Virology, 214 130-138 (1996)) in virus resistance have also been observed The current studies have not only extended our understanding of ACl mediated resistance in terms of DNA-protein interactions but provided a means to exploit these interactions for achieving resistance in other geminiviruses that is not confined to related strains or viruses even when they belong to different geographical boundaries

Example 5 Identification of Replication Specificity Determinants

Two strains of Tomato leaf curl virus from New Delhi (ToLCV-Nde) were used to further study specificity in replication of viral genomes Rresults showed that the two strains specifically replicate their cognate DNAs and that specificity is determined in part by the amino acid at position 10 of the Rep protein and the corresponding binding site sequence in the on In addition, evidence is presented that the amino acid at position 10 may interact with the 3rd nucleotide of the 13- mer Rep protein binding site in the on.

Material and Methods

A Construction of Mutants

The cloning of DNA-A (pMPAl, accession no , U15015) and DNA-B

(pMPB, Accession no , U 15017) of the severe strain and DNA-A (pMPA2, accession no , U 15016) of the mild strain have been described previously

(Padidam, M , et al, J. Gen. Virol 76 25-35 (1995)) The genome organization of the severe strain is shown in Figure 1 A DNA-A of the mild strain has the same genome organization as DNA-A of the severe strain. The mutations analyzed in this study were targeted in the Rep gene and the common region of the viral genome. A brief description of each mutant used in this study and the method of construction is provided in Table 7. Full length Rep gene or its fragments were exchanged between the mild and the severe strains by utilizing the available restriction enzyme sites. In cases where convenient restriction sites were not present, oligonucleotide-directed mutagenesis (Horton, R.M., "In vifro recombination and mutagenesis of DNA," in PCR cloning protocls., vol 67, White, B.A., ed., Humana Press, Inc., Totowa, NJ (1994), pp. 141-150) was used to substitute the fragments. For creating amino acid substitutions in the Rep protein, mutagenic oligonucleotides were designed to substitute codons. All mutants were confirmed by DNA sequencing. In case of substitutions made by oligonucleotide directed mutagenesis, a small restriction fragment containing the mutation was recloned into the unmutagenised A component at the respective site to avoid incorporation of second site mutations. Partial tandem dimers of the mutants were used to infect Nicotiana tabacum protoplasts and N benthamiana. lants.

B. Protoplast and Plant Inoculations

Protoplasts derived from N tαbαcum B Y-2 suspension cultures were used for transfection with viral DΝA (Watanbe, E75S Letters 279:65-69 (1989)). One million protoplasts were inoculated by electroporation (250 V, 500 μF) with 2 mg each of DΝA-A and DΝA-B and 40 mg of sheared herring sperm DΝA. Protoplasts were collected from cultures 48h post inoculation for DΝA isolation and analysis. Table 7. Description of mutants used in this study

Construct Method of construction

A2-RepAl DNA-A of mild strain containing the full length Rep gene from the severe strain. An Nco I site was introduced at the initiation codon of both the mild and the severe strain to facilitate the exchange of the ACl gene. Full length Rep from the severe strain was digested with Nco I and Bel I and cloned at respective site in the mild strain.

A2-cRepAl DNA-A of the mild strain containing the 3 ' sequences coding for 256 amino acids from the C-terminal region derived from the severe strain. The C-terminal amino acids were cloned as Cla I to Cla I fragment from the severe strain.

A2-nRepAl DNA-A of the mild strain containing the 5' sequences coding for amino acids 1-110 from the N-terminal region of Rep gene. Constructed by cloning the Nco I to Xba I fragment (coding for aa 1-110) from the severe strain into the mild at same sites.

A2-CRB 1 DNA-A of the mild strain containing the common region of severe strain DNA B. The common region of DNA B was amplified as Xba I to Spe I fragment. The primers designed to amplify common region from DNA-B had 18 nucleotides of the mild strain DNA-A at their ends in addition to DNA-B specific sequences. These sequences of the mild strain were later used as primers to extend and exchange the CR of A2. Construct Method of construction

A2- A double mutant of the mild strain containing two amino RepMl/CRAl acid substitutions, Val9 and Asp 10 to Asn in Rep gene and the CR of Al Amino acid substitutions were made by oligonucleotide-directed mutagenesis

A2RepMl/CR A double mutant of the mild strain containing a mutated Rep

BI gene (Val9 to He and Asp 10 to Asn) and the CR of BI

A2- A double mutant of the mild strain containing Val9 to He and

RepMl/CRM3 Asp 10 to Asn in Rep gene as well as two point mutations in its CR The point mutations in CR were made at positions

2642 (C to A) and 2649 (C to T) to make the binding site identical to Al

A2- A double mutant of the mild strain containing only Asp 10 to RepM2/CRM3 Asn in Rep gene and the two point mutations in its CR at positions 2642 (C to A) and 2649 (C to T) respectively, rendering the binding site identical to Al

Al-RepA2 DNA-A of the severe strain containing the full length Rep from the mild strain The Rep gene was isolated from the mild strain as Nco I to Bel I fragment and cloned at same sites in Al

Al-cRepA2 DNA-A of the severe strain containing 3 ' sequences coding for 256 amino acids from the C-terminal region of the Rep gene from the mild strain The fragment was cloned between the two Cla I sites

Al-nRepA2 DNA-A of the severe strain containing 5 ' sequences coding for amino acids 1 to 110 in the N-terminal region of the Rep gene from the mild strain The fragment was clined as Nco

I to Xba I between the strains Construct Method of construction

Al-RepM3 DNA-A of the severe strain containing a substitution at AsnlO to Asp in Rep gene.

A1-CRM1 DNA-A of the severe strain carrying a single nucleotide deletion in the CR at position 2642. The wild type sequence of the putative binding site GGTGTCTGGAGTC is changed to GGGTCTGGAGTC due to this deletion.

A1-CRM2 DNA-A of the severe strain containing a single nucleotide deletion in the CR at position 2649. The wild type sequence in the putative binding site, GGTGTCTGGAGTC is changed to GGTGTCTGGGTC due to this deletion.

A1-CRM4 DNA-A of the severe strain containing a substitution of 3^rd nucleotide in the putative binding site sequence GGTGTCTGGAGTC at 2642 GGCGTCGGAGTC.

Al- DNA-A of the severe strain containing substitution of Asn 10 RepM4/CRM4 to Asp in Rep gene and a single nucleotide change in the binding site sequence at 2642, making the repeat motif as GGCGTCGGAGTC.

Two weeks old seedlings of N benthamiana grown in magenta boxes were inoculated with partial tandem dimers of viral DΝA using a Bio Rad helium driven particle gun (Padidam, M., et al, J. Gen. Virol 76:25-35 (1995)). Ten plants were inoculated for each mutant using 0.5 mg each of DΝA-A and DΝA-B per plant. Plants were observed for symptom development and the newly emerging leaves were harvested for Southern blot analysis 21 days after inoculation. B. Southern Blot Analysis

DNA extractions from systemically infected leaf samples were carried out as described in Dellaporta etal, (Dellaporta, S.J., etal, PlantMol Biol. 7: 19-21 (1983)), and from protoplasts by following the procedure of Mettler (Mettler, I. J., PlantMol Biol Rep. 5:346-349 (1987)). Total DNA (4 μg) was electrophoresed on 1% agarose gels without ethidium bromide and transferred to nylon membranes. Viral DNA was detected using DNA-A specific radiolabelled probe (a 900 bp, Afl ll-Pst I fragment containing the Rep, REn and TrAP genes), or a probe specific for the DNA-B (878 bp PCR amplified movement protein gene). The amount of viral DNA was quantified as previously described (Padidam, M., et al, Virology 114:390-404 (1996)) by exposing the Southern blots to phosphor screens and counting the radioactivity on a phosphorimager (Molecular Dynamics).

Results

A. The Mild Strain Does Not Replicate Efficiently

The two strains of ToLCV-Nde used in this study have been described previously (Padidam, M, et al, J. Gen. Virol. 76:25-35 (1995)). The DNA-A (pMPAl) and DNA-B (pMPB) of the severe strain and DNA-A (ρMPA2) of the mild strain have been cloned. All the three clones were obtained from the same DNA sample prepared from collection of several diseased plants in the same field.

Inoculation of N benthamiana plants with DΝA-A and DΝA-B of the severe strain produced severe symptoms with characteristic leaf curl but plants inoculated with DΝA-A of the mild strain and DΝA-B of the severe strain developed mild infection with minor leaf curl symptoms. For the sake of brevity, hereafter, the severe strain DΝA-A and DΝA-B will be referred to as Al and B 1 and the mild strain DΝA-A will be designated as the A2. Al and A2 DΝAs have the same length (2739 nt.) and share 94% sequence identity. Their CRs are 81% identical (Padidam, M., et al, J. Gen. Virol. 76:25-35 (1995)). The amino acid sequence identity between individual genes in Al and A2 ranged from 91-99% with the greatest similarity in the coat protein gene. The nucleotide sequence identity between the CRs of Al and BI is 97% as compared to 79% between the CRs of A2 and BI. It was not known whether the mild phenotype in plants inoculated with A2 and BI is due to inefficient replication of the virus or because of its inability to spread in the plant. To compare the replication levels of the two strains, BY-2 protoplasts were transfected with Al or A2 DNAs and viral DNA replication was quantitated 48h after transfection. A2 does not accumulate to the same level as the Al in protoplasts. The replication efficiency of A2 DNA varied between 45-58% compared to the Al DNA levels (Table 8). Next the ability of A2 to replicate BI in tobacco protoplasts was tested. In transient assays, A2 replicated B 1 to barely detectable levels (<1% ) (Fig. 9B, lanes 1, 10; Table 8). InN benthamiana plants inoculated with A2 and BI DΝA, very mild symptoms mostly limited to mild chlorosis and slight curling were observed three weeks post inoculation. Southern analysis of total DΝA isolated from infected plants showed very low levels (5- 10% compared to the Al) of DΝA-B accumulation (Fig.10A, lanes 1, 8 and 3B, lanes 1, 10; Table 8). In plants, replication of DΝA B is required to cause a systemic infection (Padidam, M., et al, J. Gen. Virol 76:25-35 (1995)) and these data suggested that low levels of DΝA-B replication coupled with less efficient replication of A2 may account for mild symptoms observed in inoculated plants. The apparent inability of A2 to replicate BI of the severe strain provided the first evidence that the two strains may have different replication requirements. Therefore, the incompatibility of A2 and BI was focused on by making sequence comparisons between the Rep gene of Al and A2 and the CR sequences between the A2 and BI . B . N-terminal D omains of Rep Protein Are Not Interchangeable B etween the Strains

The low levels of DNA-B accumulation in protoplasts and plants inoculated with A2 and B 1 indicated that the mild strain Rep protein replicated B 1 DNA inefficiently. To test if replication levels of DNA-B can be increased by replacing the mild strain Rep protein with its equivalent from Al , a full length Rep gene (from Nco I to Bel I) was exchanged between the two strains (Fig. 8B, Table 7). The mild strain DNA-A containing a full length Rep gene of the severe strain (A2-RepAl) did not replicate efficiently in tobacco protoplasts (Fig. 9B, lanes 2, 11; Table 8). Similarly, the severe strain DNA-A containing the Rep gene from the mild strain (Al -RepA2) failed to accumulate to high levels in protoplasts (Fig. 9A, lanes, 2, 11; Table 8) suggesting that the Rep proteins are not interchangeable. Similar results were obtained in N benthamiana plants inoculated with these mutants (Fig. 10A, lanes 2, 9 and 3B, lanes 2, 11; Table 8). To determine whether the specificity of the Rep protein for the DΝA templates was associated with C- or Ν-terminal regions of the Rep protein, we exchanged both the 5' and the 3^' parts of the Rep gene between the strains. The 3' region of the Rep gene coding for 256 amino acids (containing 18 of the 22 amino acid differences between the two Rep proteins) from the carboxyl terminal end of Rep (Cla l-Cla I fragment) was exchanged between the strains (A2- cRepAl and Al-cRepA2; Fig. 8B; Table 7) and determined the ability of the Rep chimera to replicate in tobacco protoplasts. Unlike the exchange of full length Rep genes, the hybrid Rep proteins were functional in both strains but did not change the phenotype of the two strains. The severe strain mutant, Al-cRepA2 accumulated both DΝA-A and DΝA-B at levels similar to the wild type (Al ) virus

(Fig. 9 A, lanes 3, 12; Table 8). In contrast, the mild strain mutant, A2-cRepAl replicated DΝA-A at moderate levels and very low levels of DΝA-B was detected (Fig. 9B, lanes 3, 12; Table 8). Inoculation of N benthamiana seedlings with these hybrid Rep gene constructs showed that the plants infected with the A2- cRepAl produced mild symptoms while plants inoculated with Al-cRepA2 developed typical leaf curl symptoms within ten days (Table 8; Fig. 10A, lanes 3, 10 and 10B, lanes 3, 12).

It should be noted that by exchanging the full length Rep gene between the strains the overlapping TrAP gene was also transferred; however, the results described above eliminated the possibility that differences in the TrAP gene having an effect on replication of the chimeric viruses.

S equences from the 5 ' region of the Rep gene encoding amino acids 1-110 (Nco l-Xba I fragment) between the strains were exchanged and assayed for replication of viral DNA. The severe strain mutant, Al-nRepA2 contains the N- terminal sequences from the mild strain and led to accumulation of very low levels of viral DNA (Fig. 9A, lanes 4, 13; Table 8). Similarly, the mutant A2-cRepAl resulted in negligible levels of virus replication (Fig. 9B, lanes 4, 13; Table 8). None of the N. benthamiana plants inoculated with either Al-nRepA2 or A2- nRepAl mutants developed symptoms and accumulated very low levels of viral DΝA (Table 8; Fig. 10A, lanes 4, 11 and 10B, lanes 4, 13) indicating the region spanning amino acids 1-110 in the Rep gene may contain residues that determine specificity of replication between the two strains and are not interchangeable.

C. Exhange of the intergenic region (ER) between the virus strains.

The ERs of pMPAl and pMPA2 are only 80% identical while those of pMPAl and pMPB are 97% identical. Further, pMPA2 and pMPB share only

77% sequence homology. It is not known if these differences in the ER were significant in determining the replication specificity observed earlier in the experiments and so, the IR of the pMPA2 (mild) was replaced with that of pMPB. This exchange however, did not improve the replication levels of DΝA-B and the plants remained very mildly symptomatic. Infact, both ss and ds forms of DΝA-A Table 8. Replication and Infectivity of ToLCV mutants in N. tabacum protoplasts and N. benthamiana plants

Protoplast inocluations³ Plant inoculations¹¹

DNA-A DNA-B DNA-A DNA-B

MUTANT" ss ds ss ds ss ds ss ds

AΓ 100 100 100 100 100 100 100 100

A2 41 44 <1 <1* 58 55 4 2

A2-RepAl 4 <1 <1 <1 5 <1 <1 <1

Al-RepA2 <1 <1 <1 <1 12 5 <1 <1

A2-cRepAl 39 33 <1 <1 59 54 <1 <1

Al-cRepA2 94 89 92 91 89 84 94 91

A2-nRepAl 5 <1 <1 <1 4 <1 <1 <1

Al-nRepA2 <1 <1 <1 <1 6 <1 6 5

A2-CRB1 <1 <1 <1 <1 <1 <1 <1 <1

A2- 91 92 84 82 94 92 98 97

RepMl/CRAl

A2- 92 84 90 87 92 89 106 104

RepMl/CRBl

A2- 102 96 94 97 98 95 104 98

RepMl/CRM3

A2- 106 98 89 84 92 79 85 71

RepM2/CRM3

Al-RepM3 4 5 <1 <1 14 8 <1 <1

Al-CRMl <1 <1 <1 <1 3 2 <1 <1

A1-CRM2 4 3 8 6 <1 <1 <1 <1

A1-CRM4 12 <1 <1 <1 nd^d nd nd nd

Al- 98 92 104 108 nd nd nd nd

RepM4/CRM4

RepM and CRM denote mutations in the Rep gene or in the CR respectively. The values shown are average (%) amounts of single stranded (ss) and double stranded (ds) viral DNA detected in sixteen independent protoplasts transfections per mutant Protoplasts prepared from N tabacum BY2 cells were transfected with 2 μg each of DNA-A and DNA-B and harvested 48^th after electroporation Viral DNA was quantitated using a phosphorimager Standard error values between different transfections were the range of ±2-5% b The values show average amount of viral DNA in ten inoculated N benthamiana plants per mutant The plants were inoculated with 0 5 μg each of DNA-A and DNA- B using a particle acceleration gun Standard error values ranged from 2-5% between different plants ^c The amounts of viral DNA observed in protoplasts and plants inoculated with the severe strain were assigned a value of 100 ^d Not determined

Too low for accurate quantification because of background error

were sharply reduced to the limits of detection These results established that the

ACl protein of the two strains displays strict specificity in recognising their respective replication origins and the interaction of the two may be important in driving strain specific replication

D Rep Proteins Display Specificity in Recognizing Replication Origins

Geminivirus replication requires a functional interaction of Rep protein with specific sequences in the on (Fontes, E P.B , etal, J. Biol Chem. 169 8459- 8465 (1994), Fontes, E P B , et al, Plant Cell 6 405-416 (1994)) Studies described above identified the N-terminal region of Rep protein as being important in determining replication of the two strains The common region (CR) of the three DNA components, Al , A2 and B 1 was compared to look for differences that may contribute to template specificity The CRs of Al and A2 are 81% identical, while the CRs of A2 and BI share only 79% sequence homology, by comparison, Al and B 1 are 97% identical To determine whether sequence differences in the CR of mild strain restricts its replication, the CR of A2 was replaced with that of BI (mutant A2-CRB1, Table 1) and tested for its ability to replicate in BY-2 protoplasts and N benthamiana plants As shown in Table 8, exchange of the common region of A2 with BI did not increase the replication levels of DΝA-B in protoplasts. Rather, the replication levels of both DNA-A and DNA-B were drastically reduced (Fig. 9B, lanes, 5, 14; Table 8). Similarly, this mutant did not replicate in N benthamiana (Fig. 10B, lanes 5, 14; Table 8). These results demonstrated that the Rep protein of A2 does not recognize the common region sequences of B 1 suggesting that the two strains specifically recognize unique sequences in their origin of replication.

The experiments provided information to delimit two essential features that influence replication specificity of two strains, the common region sequences and the Ν-terminal residues in Rep protein. Mutations in the Ν-terminal region (amino acids 1-110) of Al and A2 Rep proteins were next introduced with concomitant changes in their viral origin of replication to analyze the precise determinants of a functional, replication competent interaction.

The Rep protein in geminiviruses shares sequence similarities with other initiator proteins that follow rolling circle mode of replication. Based on comparison of sequences of these proteins, Koonin and Ilyina (Koonin, E. V. and

Ilyina, J.V., J. Gen. Virol 73:2763-2766 (1992)) identified a domain amongst geminiviruses in the Ν-terminal region of Rep protein that may be involved in initiating rolling circle replication. In this region, at least three motifs have been identified: Motif III, [xxYxxK] is involved in DΝA nicking and closing activities (Laufs, J.S., et al, FEBS Lett. 377.15S-161 (1995); Hoogstraten, R.A, et al,

Mol Plant. Microbe Interactions 9:594-599), Motif II, HEHxUUQ (U= a bulky hydrophobic residue) which has structural features similar to the Mg2+binding sites of metallozymes (Koonin, EN. and Ilyina, J.V., J Gen. Virol. 73:2763-2766 (1992)) and Motif I, FLTYPqC (q = a basic or a polar amino acid) whose function has not been established yet. The three motifs described are present within the amino acids 1-110 region and are identical between the Rep proteins of Al and A2.

To define the amino acids involved in recognition of the ori, the region between amino acids 1-110 was examined for sequence variation between Rep proteins of Al and A2. Four amino acid differences were identified between the two proteins in this region. The Al strain contains amino acids He, Asn, Lys, and Glu at positions 9, 10, 40 and 52 respectively while the A2 strain has Val, Asp, Ala, and Asp at these positions Two amino acids, Ile9 and AsnlO are immediately adjacent to Motif I To determine if Ile9 and AsnlO had a role in replication, Val9 and Asp 10 in Rep protein of A2 were changed to Ile9 and AsnlO Simultaneously, the common region of the A2 DNA was replaced with either Al (A2-RepMl/CRAl) or BI (A2-RepMl/CRBl) sequences (Fig IOC) In protoplasts, both mutants, A2-RepMl/CRAl (Fig 9B, lanes 6, 15) and A2- RepMl/CRBl (Fig 9B, lanes 7, 16) accumulated viral DNA to similar levels as the severe Al strain N benthamiana plants inoculated with both of these mutants developed severe infection ten days after inoculation and accumulated high levels of viral DΝA as analyzed by Southern hybridization (Fig 10B, lanes 6, 7, 15, 16, Table 8) These data suggested that Ile9 and AsnlO are involved in determining interaction of Rep protein with specific sequences in the CR

E Putative Rep Binding Sites Are Different Between the Two Strains

Rep protein in geminiviruses is known to bind with high affinity to its binding site in the on Based on comparison of many on sequences in geminiviruses (Arguello-Astorga, G.R , et al, Virology 203 90-100 (1994), Behjatnia, S A , et al, Nucleic Acids Res. 26.925-931 (1998), Fontes, E P B , et al, J. Biol Chem. 169 8459-8465 (1994)) putative Rep binding sites in the CRs of Al , A2 and B 1 DΝAs were looked for A 13-bp sequence was identified in on close to the TATA box in the CRs of the three DΝAs In Al and B 1, the repeat sequence GGTGTCTGGAGTC (SEQ ID NO 121 ) was identified while A2 DNA had the repeat sequence GGCGTCTGGCGTC (SEQ ED NO 122)

To determine if these sequences represent potential binding sites for Rep proteins, we introduced mutations in the 13-bp sequence Deletions were made at the 3rd or the 10th nucleotide of the 13-nucleotide sequence since they are different between the two strains Both mutants, Al -CRM 1 (deletion for the 3rd nucleotide) and Al -CRM2 (deletion for the 10th nucleotide) showed dramatically reduced levels of viral DNA replication in infected protoplasts (Fig 9A, lanes 6, 7, 15, 16; Table 8). Inoculated N benthamiana plants did not show any symptoms three weeks post inoculation and replication of both viruses was reduced to minimal levels (Fig. 10A, lanes 6, 7, 13, 14; Table 8). These results demonstrated that the 13-bp sequence in Al is essential for virus replication and may represent the Rep protein binding site.

F. Asnl 0 May Determine Specific Interaction with the Viral Origin

Earlier experiments showed that amino acids He 9 and AsnlO in Rep protein of Al may be involved in specific interaction with the CR. To determine if these amino acids are involved in recognition of the potential binding site in the ori, mutations were made in A2 Rep gene to change Val 9 to He and Asp 10 to

Asn. Simultaneously, the 3rd and the 10th nucleotides on the potential binding site in A2 (GGCGTCTGGCGTC) (SEQ ED NO: 122)were mutated to T and A respectively (GGTGTCGGAGTC) (SEQ ID NO: 121) to make the repeat sequence identical to that of Al (mutant A2-RepMl/CRM3, Fig. 8C). As expected, the mutant A2-RepMl/CRM3 was functional in protoplasts and replicated DNA A and B to wild type levels (Fig. 9B, lanes 8, 17; Table 8). Also, plants inoculated with mutant A2-RepMl/CRM3 and BI produced severe symptoms within two weeks after inoculation accumulating high levels of viral DNA ( Fig. 10B, lanes 8, 17; Table 8). These results implied that the 13 -mer sequence identified in the ori of A2 is the putative binding site and plays a significant role in replication, and that He9 and Asnl 0 may be involved in specific interaction between Rep and ori.

Of the two amino acid changes made, Asp 10 to Asn is expected to be more significant than Val9 to Ile9; we therefore made another mutant A2- RepM2/CRM3, where only the Asp 10 to Asn of Rep protein in A2 is changed together with 3rd (C to T) and the 10th (C to A) nucleotides of the ori to make it identical to Al (Fig. 10C). In an analogous experiment, the AsnlO to Asp was made in Rep protein of Al (Al-RepM3) without any changes in its ori (Fig. 8C). Protoplasts transfected with A2-RepM2/CRM3 accumulated high levels of viral DNA (Fig. 9B, Lanes 9, 18; Table 8) comparable to the wild type severe strain. The corresponding mutant, Al-RepM3 with AsnlO to Asp change in the Rep protein of Al, replicated to very low levels in protoplasts (Fig. 9 A, lanes 5, 14; Table 8). N benthamiana plants inoculated with the mutant A2-RepM2/CRm3 developed severe symptoms and accumulated increased levels of viral DΝA (Fig.

10B, lanes 9, 18; Table 8), showing that AsnlO alone is sufficient to facilitate specific replication of the virus. In contrast, plants inoculated with Al-RepM3 which contains a substitution in the 10th residue of its Rep protein did not replicate viral DΝA with high efficiency (Fig. 10A, lanes 5, 12; Table 8). AsnlO may interact with the 3rd nucleotide of the 5' iteron, GGTGTC

While the above experiment showed that AsnlO in Rep protein may be involved in specific recognition of the ori, it was essential to determine if both the 3 'and the 5' repeats in the 13-mer binding site contributed to the specificity of recognition. To address this question, the Al DΝA was mutated at the 3rd nucleotide in this sequence substituting C for T (Al -CRM4) so that the altered 5 ' iteron is GGCGTCTGGAGGTC. The mutant A1-CRM4 did not replicate efficiently in protoplasts and very low levels of viral DΝA accumulated (Fig. 9 A, lanes 8, 17; Table 8), suggesting that Rep protein may be unable to recognize the modified binding site. This mutant was further modified by substituting Asnl 0 to Asp (Al-RepM4/CRM4, Fig. 10C). The mutant A l-RepM4/CRM4 accumulated wild type levels of DΝA-A in protoplasts (Fig. 9 A, lanes, 9, 18; Table 8) indicating that the Asp 10 may indeed interact with the 3rd base of the iteron, GGCGTC sequence.

Additionally, in order to better define the ACl domain involved in specific recognition of origin, the region between aa 1-110 was scanned for functional variation in the two Rep proteins(data not shown). A stretch often amino acids was identified at the beginning of the ACl very close to the conserved motif 1 (Koonin and Illiyana, 1991 ; Arguello-Astroga et al,1995) and switched this domain in pMPA2 already containing the pMPB ER. This double mutant seemed functional in protoplasts and both forms of viral DΝA components could be detected in same amounts as the severe pMPAl strain. These data demonstrated that the aa 1 - 10 at the AC 1 n-terminal region must be provided in trans for specific recognition of its binding site on the ER and consequent replication of the viral DNA.

Discussion

The specificity of interactions between Rep proteins and ori sequences in two related strains of Tomato leaf curl virus-New Delhi was investigated. The studies showed that the amino acid at position 10 in Rep protein coupled with a change in the binding site sequence may determine whether or not the viral DNA is replicated. Substitution of Asp 10 to Asn in Rep protein of the mild strain accompanied by exchange of the 13-mer binding site (making it identical to the severe strain) altered its replication leading to increased accumulation of viral DNA. In addition, the mild strain thus modified could replicate heterologous strain DNA-B indicating that the interaction of Rep protein with its binding site may be essential for replication of viral DNA. Based on site directed mutations, it is proposed that Asnl 0 specifically recognizes the 3rd base pair of the 5 ' iteron

GGTGTC in the severe strain.

Even though the DNA-A component of the severe (Al) and mild (A2) strains share 94% sequence identity, A2 did not replicate efficiently in protoplasts or plants, nor did it support the replication of DNA- B 1. These results support the hypothesis that the mild symptoms in plants inoculated with A2 and B 1 are caused by low levels of replication of DNA-A combined with very low levels of DNA-B. Comparison of amino acid sequences in Rep proteins of the two strains revealed 22 amino acid differences, 18 of which are located in the C-terminal region of Rep protein. The results of experiments in which sequences of Rep proteins were exchanged indicated that the region encoding 256 amino acid residues from the

C-terminal end did not affect viral replication. On the contrary, exchange of amino acids 1 to 110 of Rep was deleterious to virus accumulation suggesting that this region may contain sequences crucial for virus replication. However, N- terminal sequences in Rep protein alone may not account for the replication specificity was shown by the mild strain mutant A2-CRB 1 which contained the exchanged CR but failed to replicate viral DNA. These results imply that Rep proteins exhibit specificity in their interaction with their template and are not interchangeable. Similar results have been obtained with strains of TYLCV (Jupin, I., et al, FEBS Lett. 262: 116-120 (1995)), TGMV (Gladfelter, H.J., et al,

Virology 239: 186-197 (1997); Orozco, B.M., et al, J. Biol. Chem. 272:9840- 9846 (1997)) and beet curly top virus, BCTV (Choi, I-R., and Stenger, D.C., Virology 106:904-911 (1995); Choi, I-R., and Stenger, D.C., Virology 116:11- 126 (1996); Watanabe, Y., et al, FEBSLett 279:65-69 (1987)). Of the four amino acid differences in the N-terminal region (amino acids

1-110) between the Rep genes of Al and A2, we chose to mutate Ile9 and AsnlO because of their proximity to Motif I (FLTYPKC), a conserved element found in all the initiator poteins that replicate via a rolling circle mechanism (Koonin, EN. and Ilyina, J.V., J. Gen. Virol 73:2763-2766 (1992)). Replication assays in protoplasts and in plants obtained with the mild strain mutants A2RepM 1 /CRAl , and A2-RepMl/CRB 1 provided evidence that these amino acids may be involved in specificity because of their interaction with the sequences in the CR.

The binding site of Rep protein of ToLCV-Νde has not been biochemically determined. Site directed mutagenesis was used to determine whether or not the 13 nucleotide sequence identified in the ori interacts in a functional way with the Rep protein. The severe strain mutants Al-CRMl and Al -CRM2 which contain single nucleotide deletions in the 13 -mer sequence, failed to accumulate viral DΝA demonstrating that the CR sequence is essential for virus replication. Since these deletions also affected spacing of the putative binding site, these results indicate that both sequence and spacing may contribute to specificity.

Mutational analysis of the Rep binding site in TGMV showed that both spacing and sequence of the binding site are important for replication (Orozco, B.M., et al, Virology 141:346-356 (1998)).

The mutant A2-RepM2/CRM3 which contains an Asp 10 to Asn mutation in the Rep protein and corresponding changes in the potential binding site sequence GGCGTCTGGCGTC (SEQ ID NO: 122) to GGTGTCTGGAGTC (SEQ ID NO: 121) (identical to the severe strain) restored the replication efficiency of A2 DNA. These results indicated that AsnlO may differentiate between Al and A2 strains and determine the specificity in recognizing ori sequences. In addition, the fact that replication of BI was restored by changing the putative binding site sequence of A2 coupled with mutation of the Rep protein support the conclusion that both components are key factors that determine which DNA template is to undergo replication. The role of the other two amino acids Lys40 and Glu54, that are different between the two Rep proteins was not studied and therefore do not conclude that Asn 10 is solely responsible for strain specificity.

Since the iteron sequences in the binding sites of Al and A2 are different with respect to the 3rd and the 10th nucleotides, we examined the significance of these differences in context of AsnlO in Rep protein. These studies led to the conclusion that a GGCGTC iteron in the putative binding site may be correlated with the presence of Asp in Rep protein at position 10 of A2. Likewise, in related experiments we showed that AsnlO may recognize the 3rd base pair in the 5' iteron GGTGTC. It is possible that AsnlO is a part of the DNA-binding domain of Rep protein which allows appropriate structural presentation of the Rep protein that potentiates recognition with the iteron sequences by Rep protein and facilitates replication. In TGMV, the deletion of the first 29 amino acids abolished

DNA binding and DNA cleavage demonstrating that an intact N-terminus is required for both activities (Orozco, B.M., et al, J. Biol. Chem. 272:9840-9846 (1997)). The inability of the Cal/Logan strain to replicate the Worland or the CFH strain of BCTV (Watanabe, Y., et al, FEBS Lett 279:65-69 (1987)) was correlated to the differences in the 3rd base pair of the 5' iteron sequence supporting our observations that variation in this region of the sequence may be crucial in determining the replication ability between the strains. Similar reports of incongruity have been shown for different strains of TYLCV (Jupin, 1., et al, FEBS Lett. 262: 116-120 (1995)) whose Rep proteins share more than 76% anmino acid identity but are not interchangeable and between TGMV and BGMV

(Fontes, E.P.B., et al, Plant Cell 6:405-416 (1994)), probably beacuse of differences in their iteron sequences, supporting the importance of specific interaction between the Rep protein and its recognition sequence.

The iteron sequences of Al and BI are not identical. Point mutations introduced in the 5^' iteron of the severe strain (mutant A1-CRM4) to make it identical to its 3' homolog (A1-CRM4) resulted in drastic reduction in virus replication indicating that the two iterons do not contribute equally to the recognition process. Yet, a deletion of the 10th nucleotide of the repeat motif (A1-CRM2) reduced the replication levels in Al suggesting that both iterons are required for replication. Similarly, differential contributions of the two iterons have been reported for TGMV (Fontes, E.P.B., et al, Biol. Chem. 269:8459-

8465 (1994)) and BCTV (Choi, I-R., and Stenger, D.C., Virology 226:11-116 (1996)). Unlike the reports of Fontes etal, (Fontes, E.P.B., etal, J. Biol. Chem. 269:8459-8465 (1994)) and Choi and Stenger, (Choi, I-R., and Stenger, D.C., Virology 116:11-116 (1996)), our studies indicated that the 5' iteron contributes to replication more than the 3' iteron, for example, in mutant A1-CRM4. It is possible that in the case of ToLCV, the Rep protein has a stronger affinity for the

5 ' versus 3 ' iteron, but detailed in vitro binding assays that examine the interaction of Rep protein with each of the iterons are required to confirm this suggestion.

The studies showed a correlation between the amino acid at the 10th position in Rep protein and the 3rd nucleotide of the 5' iteron in the binding site and specificity of replication between the strains. This suggestion was supported by several observations: A2 DNA mutated at Asp 10 to Asn in the Rep protein with concomitant change in the 3rd nucleotide of the putative binding site (A2- RepM2/CRM3) resulted in increased levels of virus accumulation. Similarly, the mutant, Al-RepM4, containing a substitution of AsnlO to Asp in Rep protein without any change in its binding site accumulated very low levels of virus DNA. Further, substitution of AsnlO to Asp accompanied by a change in the 5' iteron of the binding site (mutant Al-RepM4/CRM4), restored virus replication suggesting that the correlation between the amino acid at position 10 and the 3rd nucleotide of the iteron may indeed be related to specificity of replication. The observed specificity of the Rep protein with sequences in the on accounts for selective replication of Al and A2 strains While earlier work (Choi, I-R , and Stenger, D.C , Virology 106 904-912 (1995), Choi, I-R , and Stenger, D.C , Virology 116 11-116 (1996), Jupin, I , et al, FEBS Lett. 262.116-120 (1995), Lazarowitz, S G , et al, Plant Cell 4 799-809 (1992)) has shown that specificity of replication may reside within the N-terminal sequences of the Rep, our work delimits the specificity determinants to amino acid AsnlO of Rep protein in case of the two strains of ToLCV-Nde Since the Asnl 0 is very closely associated with the conserved Motif I sequence, FLTYPKC (Koonin, E V and Ilyina, J V , J. Gen. Virol 73.2763-2766 (1992)) in Rep protein, we suggest that it may function as a part of the Motif I to mediate specific replication of the cognate genomes

All publications, patents and patent applications cited herein are fully incorporated by reference into the disclosure Having now fully described the invention by way of illustration and example for purposes of clarity and understanding, it will be apparent to those of ordinary skill in the art that certain changes and modifications may be made in the disclosed embodiments , and such modifications are intended to be within the scope of the present invention

Claims

What Is Claimed Is:

1 A method for producing resistance in a plant to a geminivirus comprising introducing a geminivirus replication associated protein (Rep)-ιteron antagonist into a plant, plant cell or propagule, wherein said antagonist is selected from the group consisting of a nucleotide sequence of a geminivirus iteron capable of binding to a Rep protein and a defective Rep, wherein said defective Rep comprises a conserved geminivirus iteron binding site

2 The method of claim 1 wherein said Rep protein comprises an amino acid sequence selected from the group consisting of MPPPKKFRVQAKNYFLTYP (SEQ ID NO 1), MRTPRFRIQAKNVFLTYP

(SEQ ID NO 2), MRTPRFRVQAKNVFLTYP (SEQ ID NO 3),

MRTPRFRVQAKNVFLTYP (SEQ ID NO 4), MPPPKKFRVQSRNYFLTYP

(SEQ ID NO 5), MPPPQRFRVQSKNYFLTYP (SEQ ED NO 6),

MPPPKRFRTNAKNYFLTYP (SEQ ED NO 7), MPPPQRFRVQSKNYFLTYP (SEQ ED NO 8), MAPPKRFKVQAKNYFITYP (SEQ ID NO 10),

MAPPKRFKVQ AKNYFITYP (SEQ ED NO 11 ), MAPPKRFKVQAKNYFITYP

(SEQ ED NO 12), MPSKPRRFRVQAKNEFLTYP (SEQ ID NO 13),

MPPPKKFRVQSKNHFLTYP (SEQ ED NO 14), MPPPKKFRVQSKNFEFLTYP

(SEQ ED NO 15), MPPPKRFRVNSKNYFLTYP (SEQ ID NO 16), MPPPKKFRVQSKNHFLTYP (SEQ ED NO 17), MPPPKRFRVNSKNYFLTYP

(SEQ ED NO 18), MPPPKKFRVQSKNYFLTYP (SEQ ED NO 19),

MRTPRFRIQAKNVFLTYP (SEQ ED NO 20), MAPPKRFKIYAKNYFLTYP

(SEQ ED NO 21), MPPPKRFKENAKNYFLTYP (SEQ ED NO 22),

MPRNPKSFRLAARNEFLTHL (SEQ ED NO 23), MPRALKTNAKNYFLTYP (SEQ ED NO 24), MPRSGAFRVNAKNTFATYP (SEQ ED NO 25),

MPRLGRFAENAKNYFLTYP (SEQ ED NO 26), MPLPKRFRLNAKNYFLTYP

(SEQ ID NO 27), MPPKRFRTNSKNYFLTYP (SEQ ID NO 28),

MPRNPNSFRLTARNEFLTYP (SEQ ED NO 29), MAPPNKFRENAKNYFLTYP

(SEQ ED NO 30), MTRPKSFRTNAKNYFLTYP (SEQ ID NO 31), MPRPGRFNTNAKNYFLTYP (SEQ ID NO 32), MASPRRFRVNAKNYFLTYP (SEQ ED NO 33), MPRAGRFSENARNYFLTYP (SEQ ED NO 34), MHPLNKFRINAKNYFLTYP (SEQ ID NO 35), MPRLFKIYAKNYFLTYP (SEQ ID NO 37), MPRLFKIYAKNYFLTYP (SEQ ID NO 38), MPRLFKIYAKNYFLTYP (SEQ ED NO 39), MPPSKKFLINAKNYFLTYP

(SEQ ED NO 40), MPPSKKFLINAKNYFLTYP (SEQ ED NO 41), NPvSFRHRNANTFLTYS (SEQ ED NO 43), SNRQFSHRNANTFLTYP (SEQ ID NO 44), SNRQFSHRNANTFLTYP (SEQ ID NO 45), SNRQFSHRNANTFLTYP (SEQ ID NO 46), SNRQFSHRNANTFLTYP (SEQ ID NO 47), SNRQFSHRNANTFLTYP (SEQ ID NO 48),

SNRQFSHRNANTFLTYP (SEQ ID NO 49), SNRQFSHRNANTFLTYP (SEQ ID NO 54), SNRQFSHRNANTFLTYP (SEQ ID NO 55), SNRQFSHRNANTFLTYP (SEQ ED NO 56), SNRQFSHRNANTFLTYP (SEQ ID NO 57), SNRQFSHRNANTFLTYP (SEQ ID NO 59), SNRQFSHRNANTFLTYP (SEQ ED NO 60), SNRQFSHRNANTFLTYP (SEQ

ID NO 61), SNRQFSHRNANTFLTYP (SEQ ID NO 62), HSVRSFRHRNANTFLTYS (SEQ ID NO 64), HSVRSFRHRNANTFLTYS (SEQ ID NO 65), PSRRFKHRNVNTFLTYS (SEQ ID NO 66), TKSFRLQTKYVFLTYP (SEQ ID NO 67), PRFRVYSKYLFLTYP (SEQ ED NO 68), PRFRVYSKYLFLTYP (SEQ ID NO 69),

MPPTKRFRIQAKNIFLTYPQ (SEQ ID NO 70), MPRTPKRFRIQAKNTFLTYPQ,(SEQ ED NO 71) MPFYKKAKNFFLTYPQ (SEQ ED NO 72), MPRSPSFQEKAKNIFLTYPR (SEQ ED NO 73), MPRQPNSFRIQARNIFLTYPQ (SEQ ID NO 74), MPSNPKRFQIAAKNYFLTYPN (SEQ ID NO 75)

MPPPKRFQENSKNYFLTYP (SEQ ED NO 76), MAPPKRFQINAKNYFLTYP (SEQ ED NO 77), MPRAGRFQENAKNYFITYP (SEQ ED NO 78), MPRAGRFQENAKNYF VTYP (SEQ ID NO 79), MPRAGRFQENAKNYFITYP (SEQ ED NO 80), MSPPKRFQINAKNYFLTYP (SEQ ED NO 81), MAPPKQFQIYAKNYFITYP (SEQ ED NO 82), MPPKRFLENSKNYFLTYP

(SEQ ED NO 83), MPSHPKRFQENAKNYFLTYP (SEQ ID NO 84), MAPPKRFQENCKNYFLTYP (SEQ EDNO:85), MAQPKRFQINAKHYFLSFP (SEQ ID NO: 86), MAQPKRFQINAKHYFLTFP (SEQ ED NO: 87), MPRAGRFQENAKNYFITYP (SEQ ED NO: 88), MPRNNSFCINAKNEFLTFP (SEQ ID NO:89), MPRNNSFCENAKNEFLTFP (SEQ ID NO:90), MPRLNSFCVNAKNEFLTYP (SEQ EDNO:91), MAAPNRFKLNAKNYFLTYP

(SEQ ED NO:92), MPRKGSFSVKAKNYFLTYP (SEQ ED NO:93), MPPPKRFSVNAKNFFLTYP (SEQ ED NO.94), MPRKGSFSEKAKNYFLTYP (SEQ ED NO:95), MPRKGSFSEKAKNYFLTYP (SEQ ED NO:96), M P RK GYF S VK AKNYF L T YP ( S E Q I D N O : 9 7 ) a n d MPRSGRFSEKAKNYFLTYP (SEQ ID NO:98).

3. The method of claim 1 wherein said protein forms a dimer with wild type geminivirus Rep protein.

4. The method of claim 1 wherein said protein comprises from two to thirty different conserved iteron binding sites.

5. The method of claim 1 wherein said Rep protein includes the amino- terminal Rep protein sequence according to a formula selected from the group consisting of -FRVQ- (SEQ. ED NO: 126), -FRVN- (SEQ. ID NO: 127), -FREN- (SEQ. ED NO: 128), -FRIQ- (SEQ. ED NO: 129), -FRLQ- (SEQ. ID NO: 130), - FKVQ- (SEQ. ID NO: 131), -FKIY-(SEQ. ED NO: 132), -FKEN- (SEQ. ED NO: 133), -FRLA- (SEQ. ED NO: 134), FRLN- (SEQ. ED NO: 135), -LKTN-

(SEQ. ED NO: 136), -FAEN-(SEQ. ED NO: 137), -FRLT-(SEQ. ID NO: 138), - FNEN- (SEQ. ID NO: 139), -FRVN- (SEQ. ED NO: 140), -FSEN- (SEQ. ED NO: 141), -FKIY- (SEQ. ID NO: 142), -FLEN- (SEQ. ED NO: 143), -FQEN-(SEQ. ID NO: 144), -FQIY-(SEQ. ID NO: 145), -FCIN-(SEQ. ED NO: 146) , -FCVN- (SEQ. ID NO: 147), -FKLN- (SEQ. ED NO: 148), -FSVK- (SEQ. ID NO: 149),

-FSVN- (SEQ. ED NO: 150), -FSIK-(SEQ. ID NO: 151) , -FYKK- (SEQ. ED NO: 152), -FQIK-(SEQ. ID NO.153), -FQIA-(SEQ. ED NO: 154) , -FRLQTKY (SEQ. ED NO: 155)- , -FRVYSKY- (SEQ. ID NO: 156) and -HRNANT- (SEQ. ED NO:157).

6. The method of claim 1 wherein said defective replication associated protein (Rep) is selected from the group consisting of truncated geminivirus Rep protein, a modified Rep protein capable of binding a geminivirus iteron sequence, or a Rep protein fragment capable of binding a geminivirus iteron sequence.

7. The method of claim 1 wherein introduction of said Rep-iteron antagonist comprises preparing a transgenic plant containing a gene that expresses said replication associated protein or fragment thereof.

8. The method of claim 1 wherein introduction of said Rep-iteron antagonist comprises preparing a transgenic plant having a nucleic acid sequence of a geminivirus iteron capable of binding to a Rep protein.

9. The method of claim 1 wherein introduction of said Rep-iteron antagonist comprises contacting said plant with a composition comprising a nucleic acid molecule selected from the group consisting of an expression vector capable of expressing said replication associated protein and a geminivirus iteron sequence.

10. The method of claim 8 or 9, wherein said contacting comprises biolistic gene transfer or direct DNA uptake into protoplasts.

11. The method of claim 8 or 9, wherein said contacting comprises infection of said plant with a carrier vector.

12. The method of claim 11 wherein said carrier vector is Agrobacterium.

13 The method of claim 1 wherein said expression vector is present in a virus particle capable of infecting said plant and expressing said replication associated protein

14 The method of claim 1 wherein said geminivirus is selected from the group consisting of Mastrevirus, Curtovirus, Begomovirus and Topcuvirus genera

15 The metho d of claim 14 wherein said Mastrevirus sp ecies is selected from the group consisting of Bajra streak virus, Bean yellow dwarf virus, Bromus striate mosaic virus, Chickpea chlorotic dwarf virus, Chloris striate mosaic virus, Digitaria streak virus, Digitaria striate mosaic virus, Maize streak virus/ZEthiopia,

Maize streak virus//Ghanal, Maize streak virus//Ghana2, Maize streak virus//Kenya, Maize streak virus//Komatipoort, Maize streak virus/ Malawi, Maize streak virus//Mauritius, Maize streak virus//Mozambique, Maize streak virus//Nigerial, Maize streak virus//Nigeria2, Maize streak virus//Nigeria3, Maize streak virus//Port Elizabeth, Maize streak virus//Reunionl, Maize streak virus//Reunion2, Maize streak virus//Setaria, Maize streak virus//South Africa, Maize streak virus//Tas, Maize streak virus//Uganda, Maize streak virus//Vaalhart maize, Maize streak virus//Vaalhart wheat, Maize streak virus/AVheat-eleusian, Maize streak virus//Zaire, Maize streak virus//Zimbabwel, Maize streak virus//Zimbabwe2, Miscanthus streak virus, Panicum streak virus/Karino, Panicum streak virus/Kenya, Paspalum striate mosaic virus, Sugarcane streak virus/ Egypt, Sugarcane streak virus/Natal, Sugarcane streak virus/Mauritius, Tobacco yellow dwarf virus, Wheat dwarf virus/Czech Republic (Wheat dwarf virus-CJI, WDV-CJI) Wheat dwarf virus/France and Wheat dwarf virus/ Sweden

16 The method of claim 14 wherein said Curtovirus species is selected from the group consisting of Beet curly top virus-California, Beet curly top virus-California//Logan, Beet curly top virus-CFH, Beet curly top virus//Iran, Beet curly top virus-Worland, Horseradish curly top virus and Tomato leafroll virus

17. The method of claim 14 wherein said Begomovirus species is selected from the group consisting of Abutilon mosaic virus, Acalypha yellow mosaic virus, African cassava mosaic virus//Ghana, African cassava mosaic virus/Kenya, African cassava mosaic virus/Nigeria, African cassava mosaic virus/Uganda, Ageratum yellow vein virus, Althea rosea enation virus, Asystasia golden mosaic virus, Bean calico mosaic virus, Bean dwarf mosaic virus, Bean golden mosaic virus-Brazil, Bean golden mosaic virus-Puerto Rico, Bean golden mosaic virus-Puerto Rico/Dominican Rep. (Bean golden mosaic virus-Dominican Rep., BGMV-DR), Bean golden mosaic virus-Puerto Rico/Guatemala (Bean golden mosaic virus-Guatemala, BGMV-GA), Bhendi yellow vein mosaic virus,

Chino del tomate virus (Tomato leaf crumple virus, TLCrV), Cotton leaf crumple virus, Cotton leaf curl virus-India, Cotton leaf curl virus-Pakistan 1/Faisalabadl (Cotton leaf curl virus-Pakistan2), Cotton leaf curl virus-Pakistan 1/Faisalabad2 (Cotton leaf curl virus-Pakistan3), Cotton leaf curl virus-Pakistani /Multan (Cotton leaf curl virus-Pakistani), Cotton leaf curl virus-Pakistan2/Faisalabad

(Pakistani cotton leaf curl virus),Cowpea golden mosaic virus, Croton yellow vein mosaic virus//Lucknow, Dolichos yellow mosaic virus, East African cassava mosaic virus/Kenya, East African cassava mosaic virus/Malawi, East African cassava mosaic virus/Tanzania,East African cassava mosaic virus/Uganda// 1 (African cassava mosaic virus-Uganda variant), East African cassava mosaic virus/Uganda//2, Eclipta yellow vein virus, Eggplant yellow mosaic virus, Eupatorium yellow vein virus, Euphorbia mosaic virus, Honeysuckle yellow vein mosaic virus, Horsegram yellow mosaic virus, Indian cassava mosaic virus, Jatropha mosaic virus, Leonurus mosaic virus, Limabean golden mosaic virus, Lupin leaf curl virus, Macroptilium golden mosaic virus-Jamaica//2, Macroptilium golden mosaic virus-Jamaica//3 , Macrotyloma mosaic virus, Malvaceous chlorosis virus, Melon leaf curl virus, Mungbean yellow mosaic virus, Okra leaf curl virus//Ivory Coast, Okra leaf curl virus//India, Papaya leaf curl virus, Pepper huasteco virus, Pepper golden mosaic virus, (Texas pepper virus), Pepper mild tigrA virus, Potato yellow mosaic virus//Guadeloupe, Potato yellow mosaic virus/Trinidad and Tobago, Potato yellow mosaic virus/Venezuela,

Pseuderanthemum yellow vein virus, Rhynchosia mosaic virus, Serrano golden mosaic virus, Sida golden mosaic virus/Costa Rica, Sida golden mosaic virus/Honduras, Sida golden mosaic virus/Honduras//Yellow vein, Sida yellow vein virus, Solanum apical leaf curl virus, Soybean crinkle leaf virus, Squash leaf curl virus, Squash leaf curl virus/Extended host, Squash leaf curl virus/Restricted host, Squash leaf curl virus/Los Mochis, Squash leaf curl virus-China, Tomato golden mosaic virus/Common strain, Tomato golden mosaic virus/Yellow vein strain, Tobacco leaf curl virus//India, Tobacco leaf curl virus-China, Tomato leaf curl virus//Solanum species Dl, Tomato leaf curl virus//Solanum species D2,

Tomato leaf curl virus- Australia, Tomato leaf curl virus-Bangalore 1 (Indian tomato leaf curl virus-Bangalorel), Tomato leaf curl virus-Bangalore2, (Indian tomato leaf curl virus, ItoLCV], Tomato leaf curl virus-Bangalore3 (Indian tomato leaf curl virus- Bangalorell), Tomato leaf curl virus-New Delhi/Severe (Tomato leaf curl virus-India2, ToLCV-INl), Tomato leaf curl virus-New Delhi/Mild

(Tomato leaf curl virus-India2, ToLCV-IN2) Tomato leaf curl virus-New

Delhi/Lucknow (Indian tomato leaf curl virus), Tomato leaf curl virus//Senegal,

Tomato leaf curl virus-Sinaloa (Sinaloa tomato leaf curl virus, STLCV), Tomato leaf curl virus-Taiwan, Tomato leaf curl virus-Tanzania, Tomato mottle virus, Tomato mottle virus-Taino (Taino tomato mottle virus, TTMoV)], Tomato severe leaf curl virus//Guatemala, Tomato severe leaf curl virus//Honduras, Tomato severe leaf curl virus//Nicaragua, Tomato yellow dwarf virus, Tomato yellow leaf curl virus-China, Tomato yellow leaf curl virus-Israel, Tomato yellow leaf curl virus-Israel/Mild, Tomato yellow leaf curl virus-Israel Egypt, (Tomato yellow leaf curl virus-Egypt, TYLCV-EG), Tomato yellow leaf curl virus-Israel//Cuba,

Tomato yellow leaf curl virus-Israel//Jamaica, Tomato yellow leaf curl virus-Israel//Saudi Arabial , (Tomato yellow leaf curl virus-Northern Saudi Arabia,

TYLCV-NSA), Tomato yellow leaf curl virus-Nigeria, Tomato yellow leaf curl virus-Sardinia, Tomato yellow leaf curl, virus-Sardinia/Sicily (Tomato yellow leaf curl virus-Sicily, TYLCV-SY), Tomato yellow leaf curl virus-Sardinia/Spain//l

(Tomato yellow leaf curl virus-Spain, TYLCV-Sp), Tomato yellow leaf curl virus-Sardinia/Spain//2 (Tomato yellow leaf curl virus- Almeria, TYLCV- Almeria), Tomato yellow leaf curl virus-Sardinia/Spain//3 (Tomato yellow leaf curl virus-European strain), Tomato yellow leaf curl virus-Saudi Arabia (Tomato yellow leaf curl virus-Southern Saudi Arabia, TYLCV-SSA), Tomato yellow leaf curl virus-Tanzania, Tomato yellow leaf curl virus-Thailand// 1, Tomato yellow leaf curl virus-Thailand//2 , Tomato yellow leaf curl virus//Yemen, Tomato yellow mosaic virus-Brazil//l, Tomato yellow mosaic virus-Brazil /2, Tomato yellow mottle virus, Tomato yellow vein streak virus-Brazil, Watermelon chlorotic stunt virus, Watermelon curly mottle virus and Wissadula golden mosaic virus- Jamaica// 1.

18. The method of claim 1 wherein said plant is selected from the group consisting of Abutilon, Acalypha, apple, Ageratum, Althearosea, Asystasia, Bajra, banana, barley, beans, beet, Blackgram, Bromus, Cassava, chickpea, Chilllies, Chloris, clover, coconut, coffee, cotton, cowpea, Croton, cucumber, Digitaria, Dolichos, eggplant, Eupatorium, Euphorbia, fababean, honeysuckle, horsegram,

Jatropha, Leonurus, limabean, Lupin, Macroptilium, Macrotyloma, maize, melon, millet, mungbean, oat, okra, Panicum, papaya, Paspalum, peanut, pea, pepper, pigeon pea, pineapple, Phaseolus, potato, Pseuderanthemum, pumpkin, Rhynchosia, rice, Serrano, Sida, sorghum, soybean, squash, sugarcane, sugarbeet, sunflower, sweet potato, tea, tomato, tobacco, watermelon, wheat and Wissadula.

19. The method of claim 1 wherein said replication associated protein (Rep) binds to an iteron sequence selected from the group of sequences selected from GGAGAXGGAGA (SEQ ED NO : 99), GGTGTXGGTGT (SEQ ED NO : 100) , GGTACXGGTAC (SEQ ID NO: 107 ), GGGGAXGGGGA (SEQ ED NO: 109 ), GGGGGXGGGGG (SEQ ED NO: 110), GGTGCGCCCXGGCGCACC (SEQ

ID NO: 101) GCGCCTTCXGAAGGCGCG (SEQ ID NO: 102) GGTTTGCGXCGCAAACC (SEQ ED NO: 103) GGAGGTGCGTCCX- CCTCCACGGG (SEQ ED NO: 105), GGAGTXGGAGT (SEQ ED NO: 106 ) and GGTACXGGTAC. (SEQ ED NO: 107), GTGAGTGXCACTCAC (SEQ. ED NO: 104), GGTACXGGTAC (SEQ ED NO 108), GGGGAXGGGGA (SEQ ED NO 109), GGGGGXGGGGG (SEQ ED NO 110) wherein "X" is 3-30 nucleotides

20 The method of claim 1 wherein said replication associated protein (Rep) binds to a DNA sequence comprising GGTGTCTGGAGTC (SEQ ED NO

111)

21 The method of claim 7 wherein said gene comprises a nucleic acid sequence encoding a polypeptide comprising a sequence selected from the group consisting of -FRVQ- (SEQ ED NO 130), -FRVN- (SEQ ED NO 131), -FREN- (SEQ ID NO 132), -FRIQ- (SEQ ED NO 133), -FRLQ- (SEQ ED NO 134), -

FKVQ- (SEQ ED NO 135), -FKIY-(SEQ ID NO 136), -FKEN- (SEQ ED NO 137), -FRLA- (SEQ ED NO 138), FRLN- (SEQ ID NO 139), -LKTN- (SEQ ED NO 140), -FAEN-(SEQ ED NO 141), -FRLT-(SEQ ID NO 142), - FNEN- (SEQ ED NO 143), -FRVN- (SEQ ED NO 144), -FSIN- (SEQ ED NO 145), -FK1Y- (SEQ ID NO 146), -FLEN- (SEQ ED NO 147), -FQEN-(SEQ

ED NO 148), -FQIY-(SEQ ED NO 149), -FCEN-(SEQ ED NO 150) , -FCVN- (SEQ ED NO 151), -FKLN- (SEQ ID NO 152), -FSVK- (SEQ ED NO 153), -FSVN- (SEQ ED NO 154), -FSEK-(SEQ ED NO 155) , -FYKK- (SEQ ED NO 156), -FQEK-(SEQ ED NO 157), -FQIA-(SEQ ID NO 158) , -FRLQTKY (SEQ ED NO 159)- , -FRVYSKY- (SEQ ID NO 160) and -HRNANT- (SEQ

ED NO 161)

22 A vector containing a nucleotide sequence that encodes a defective geminivirus replication associated protein, wherein said encoded protein comprises a polypeptide having an amino acid sequence of a conserved geminivirus iteron binding site or a mutant thereof

23 The vector of claim 22 wherein said vector is an expression vector that is expressed in plants

24 The vector of claim 22 wherein said polypeptide comprises an amino acid residue sequence shown in Figure 1

25 The vector of claim 22 wherein said encoded polypeptide forms a dimer with wild type geminivirus Rep protein

26 The vector of claim 22 wherein said polypeptide defines from two to thirty different conserved iteron binding sites

27 The vector according to claim 22 wherein said nucleotide sequence encodes at least two different replication associated proteins

28 A composition for producing resistance to a geminivirus that infects plants comprising an effective amount of the DNA expression vector of claim 22

29 A transgenic plant containing a DNA expression vector according to claim 22

30 An isolated nucleic acid molecule comprising a nucleotide sequence ofa conserved geminivirus iteron

31 The nucleic acid molecule of claim 30 wherein said nucleotide sequence comprises at least two geminivirus iterons

32 The nucleic acid molecule according to claim 30 wherein said nucleotide sequence comprises from two to thirty different classes of geminivirus iteron shown in Figure 1 A-1C

33 A composition for producing resistance to a geminivirus that infects plants comprising an effective amount of the nucleic acid molecule of claim 30

34 A transgenic plant comprising a nucleic acid molecule having a nucleotide sequence comprising a conserved geminivirus iteron

35 The method of claim 1 wherein said antagonist is the nucleic acid of claim 30

36 An isolated DNA sequence comprising GGTGTCTGGAGTC (SEQ

ID NO )

37 The progeny of the transgenic plant of claim 29 or 34, wherein said progeny expresses a defective Rep protein or a conserved geminivirus iteron

38 A seed of the transgenic plant of claim 29 or 34

39 An isolated polypeptide selected from the group consisting of

ACl_1-2ι, AClj.₆₀, AClj.₅₂,

AC1 ₁₁ ACl _{14 and} AC1 ₆₀

40 An isolated nucleic acid comprising a sequence encoding a protein of claim 39

41 A truncated Rep protein

42 The truncated protein of claim 41 wherein said protein comprises least ACl _!.₁₆₀

43 A method for inhibiting geminivirus replication in a plant comprising introducing a geminivirus replication associated protein (Rep)-iteron antagonist into said plant, said antagonist selected from the group consisting of a nucleotide sequence defining a geminivirus iteron capable of binding to a Rep protein and a defective Rep, wherein said defective Rep comprises a conserved geminivirus iteron binding site

44. A method for providing resistance to infection by geminiviruses in a susceptible plant comprising: a) transforming susceptible plant cells with a DNA molecule that comprises operatively linked in sequence in the 5' to 3' direction: i) a promoter region that functions in plant cells to cause the production of an RNA sequence; and ii) a gene encoding a defective Rep protein, wherein said defective Rep comprises a conserved geminivirus iteron binding site; b) selecting said plant cells that have been transformed; c) regenerating said plant cells to provide a differentiated plant; and d) selecting a transformed plant that expresses said defective Rep gene at a level sufficient to render the plant at least partially resistant to infection by the geminivirus.

45. An at least partially virus-resistant transformed plant normally susceptible to infection by a geminivirus having inserted into its genome a DNA molecule that comprises operatively linked in sequence in the 5' to 3' direction: i) a promoter region that functions in plant cells to cause the production of an RNA sequence; and ii) a gene encoding a defective Rep protein, wherein said defective Rep comprises a conserved geminivirus iteron binding site.

46. The truncated Rep protein of claim 41 which comprises an amino acid sequence corresponding to amino acids 1 - 160 wherein said truncated Rep protein inhibits at least partially, geminivirus infection or geminivirus replication in a transgenic plant.

47. The method of claim 46, wherein said truncated Rep protein at least partially inhibits geminivirus infection or geminivirus replication in Nicotiana.

48. The method of claim 14 wherein said topcuvirus species is Tomato pseudo-curly top virus.

49. The nucleic acid molecule according to claim 30 wherein said nucleotide sequence comprises from two to thirty of the same classes of geminivirus iteron shown in Figure 1 A-IC.

50. The truncated Rep protein of claim 41 which comprises an amino acid sequence corresponding to amino acids 1-52 wherein said truncated Rep protein inhibits at least partially, geminivirus infection or geminivirus replication in a transgenic plant.

51. A method for producing at least partial resistance to a virus or a method for reducing replication of a virus in a plant, plant cell, propagule, animal or animal cell, comprising introducing a replication associated protein (Rep)-iteron antagonist into a plant, plant cell, propagule, animal or animal cell, wherein said antagonist is selected from the group consisting of a nucleotide sequence of an iteron capable of binding to a Rep protein and a defective Rep, wherein said defective Rep comprises a conserved iteron binding site, and wherein said Rep- iteron antagonist renders the infected plant, plant cell, propagule, animal or animal cell at least partially resistant to the infection.

52. The method of claim 51 wherein the Rep-iteron antagonist sequence is at least 50% identical to those found in Figs 1 A-IC (SEQ ED NOS: 1-8, 10-35,

37-41, 43-49, 54-57, 59-62, 64-107).

53. The method of claim 51 wherein the Rep-iteron antagonist sequence is at least 60%, 70%, 80%, 90% 95% or 99% identical.to those found in Figs 1 A- 1C (SEQ ID NOS: 1-8, 10-35, 37-41, 43-49, 54-57, 59-62, 64-107).

54. The method of claim 51 wherein the viral infection is from a Nanovirus or Circoviridae.

55. The method of claim 51 wherein said virus is a virus that replicates in a manner similar to the geminivirus, i.e. dependent on the binding of a Rep protein to an iteron.

56. A composition for producing at least partial resistance to a virus or for reducing replication of a virus in a plant, plant cell, propagule, animal or animal cell wherein said composition is used in any of the methods of claims 51-55.

57. A Rep-iteron antagonist comprising a nucleic acid sequence encoding a protein that binds to an iteron wherein viral infection or DNA replication of the virus causing the infection is reduced following said antagonist binding to an iteron..

58. A Rep-iteron antagonist comprising a nucleic acid sequence that competes for binding of a Rep protein with the iteron of a virus causing the infection, wherein viral infection or DNA replication of the virus causing the infection is reduced following said antagonist binding to the Rep protein.

59. A Rep-iteron antagonist comprising a polypeptide or the nucleic acid sequence encoding a polypeptide comprising the sequence FLTY or KAYTDK.

60. A Rep-iteron antagonist comprising a polypeptide or the nucleic acid sequence encoding a polypeptide selected from the group consisting of FLTYPqC wherein q is a basic or a polar amino acid, H HxUUQ wherein U is a bulky hyrophobic residue and xxYxxK wherein x may be any amino acid.

61. A vector comprising a nucleic acid sequence encoding any of the Rep- iteron antagonists of claims 57-60.