Identification of Replication Specificity Determinants in Two Strains of Tomato Leaf Curl Virus from New Delhi

Abstract

We used two strains of tomato leaf curl virus from New Delhi to investigate specificity in replication of their cognate genomes. The strains share 94% sequence identity and are referred to as severe and mild on the basis of symptoms on tomato and tobacco. Replication assays in tobacco protoplasts and plants showed that a single amino acid change, Asn10 to Asp in the N terminus of Rep protein, determines specificity for replication of the two strains based upon its interaction with the origin of replication (ori) sequences. The change of Asp10 to Asn in Rep protein of the mild strain coupled with point mutations at the 3rd and 10th nucleotides of the 13-mer binding site altered its replication ability, resulting in increased levels of virus accumulation. Similarly, changing Asn10 to Asp in Rep protein of the severe strain impaired replication of the virus and altered its severe phenotype in plants. Site-directed mutations made in ori and Asn10 of Rep protein suggested that Asn10 recognizes the third base pair of the putative binding site sequence GGTGTCGGAGTC in the severe strain.

Geminiviruses are characterized by having small circular single-stranded DNA genomes encapsidated in twinned icosahedral particles (21, 30) that replicate via double-stranded replicative intermediates by using a rolling circle mechanism (29, 31). They 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. DNA-A encodes the replication-associated protein (Rep), the replication enhancer (REn), the transcriptional activator protein (TrAP), and the coat protein, while the movement functions are located on DNA-B. Genetic studies have shown that both the movement protein and the nuclear shuttle protein encoded by DNA-B are necessary for systemic infection (3, 9). In DNA-A and DNA-B, the open reading frames (ORFs) are arranged in two divergent clusters separated by an intergenic region. The intergenic region 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 (11, 12). The ori for squash leaf curl virus has been mapped to a 90-nucleotide DNA segment within the CR (22) and within a 100-bp fragment in tomato golden mosaic virus (TGMV) (10).

The only geminivirus protein that is indispensable for virus multiplication is the Rep protein. 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 (14, 19, 20, 24). 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 (7, 8, 12).

Rep proteins of different geminiviruses show specificity for replication of their cognate genome despite the strong sequence homology and functional conservation between them (5, 11, 17, 22). This specificity is determined in part by the high-affinity binding site of the Rep protein located within the origin and a replication specificity domain localized in the N-terminal region of the Rep protein. In tomato yellow leaf curl virus (TYLCV), the virus-specific origin recognition is mapped to first 116 amino acids of the Rep protein (17). A similar function was mapped to the first 211 amino acids of the TGMV Rep protein (13). Recently, the functional domains of TGMV Rep protein responsible for Rep oligomerization (amino acids 121 to 181), DNA binding (amino acids 1 to 181), and DNA cleavage (amino acids 1 to 120) have been identified (25).

We previously characterized two strains of tomato leaf curl virus from New Delhi (ToLCV-Nde) that share 94% sequence identity between them but cause significantly different symptoms on tomato and Nicotiana benthamiana plants (27). The severe strain causes severe puckering and downward leaf curling in plants with marked reduction in leaf size and internodal length. The mild strain produces minor leaf curl and no puckering of leaves. We used these two strains to study specificity in replication of viral genomes. Our results show that 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 ori. In addition, we present evidence that the amino acid at position 10 may interact with the third nucleotide of the 13-mer Rep protein binding site in the ori.

MATERIALS AND METHODS

Construction of mutants.

The cloning of DNA-A (pMPA1; accession no. U15015) and DNA-B (pMPB; accession no. U15017) of the severe strain and DNA-A (pMPA2; accession no. U15016) of the mild strain have been described previously (27). The genome organization of the severe strain is shown in Fig. 1A. 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 CR of the viral genome. A brief description of each mutant used in this study and the method of construction is provided in Table 1. Full-length Rep gene or its fragments were exchanged between the mild and severe strains by utilizing the available restriction enzyme sites. In cases where convenient restriction sites were not present, oligonucleotide-directed mutagenesis (16) 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 unmutagenized 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 plants.

FIG. 1.

Genome organization of ToLCV-Nde showing mutations made in the Rep gene and in the CR. (A) Genome maps of DNA-A and DNA-B of the severe strain. The genes encoding conserved proteins in geminiviruses are shown as solid arrows. Rep (AC1), TrAP (AC2), REn (AC3), and coat protein (AV1) on DNA-A are shown. BC1 and BV1 on DNA-B represent the movement protein and the nuclear shuttle protein, respectively. The genome organizations of DNA-A of the severe and the mild strain are identical. Relevant restriction sites used for mutagenesis are indicated. (B) Schematic representation of mutants made in the Rep gene of mild and severe strain DNA-A. Fragments were exchanged at the N (NcoI to XbaI) or C (ClaI to ClaI) terminus of the Rep gene between the strains. The ToLCV severe strain is indicated by white hatched lines, whereas the mild strain is shown by black lines. (C) Schematic showing the organization of oris in geminiviruses (not to scale). The mutations made in the putative binding site of Rep protein and the N-terminal region of the Rep gene are shown. The hairpin, TATA box, and 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-terminus of the Rep gene of the mild and the severe strain together with point mutations made in the Rep protein binding sites are presented. The panel on the left shows the sequence of the first 10 amino acids on the Rep protein of A1 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).

TABLE 1.

Description of mutants used in this study

Construct	Method of construction
A2-RepA1	DNA-A of the mild strain containing the full-length Rep gene from the severe strain. An NcoI site was introduced at the initiation codon of both the mild and the severe strain to facilitate the exchange of the AC1 gene. Full-length Rep from the severe strain was digested with NcoI and BclI and cloned at corresponding sites in the mild strain.
A2-cRepA1	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 a ClaI-to-ClaI fragment from the severe strain.
A2-nRepA1	DNA-A of the mild strain containing the 5′ sequences coding for amino acids 1–110 from the N-terminal region of the Rep gene. Constructed by cloning the NcoI-to-XbaI fragment (coding for amino acids 1–110) from the severe strain into the mild strain at same sites.
A2-CRB1	DNA-A of the mild strain containing the CR of severe strain DNA-B. The CR of DNA-B was amplified as an XbaI-to-SpaI fragment. The primers designed to amplify the CR 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.
A2-RepM1/CRA1	Double mutant of the mild strain containing two amino acid changes, Val9 to Ile and Asp10 to Asn, in the Rep gene and the CR of A1. Amino acid substitutions were made by oligonucleotide-directed mutagenesis.
A2RepM1/CRB1	Double mutant of the mild strain containing a mutated Rep gene (Val9 to Ile and Asp10 to Asn) and the CR of B1.
A2-RepM1/CRM3	Double mutant of the mild strain containing changes of Val9 to Ile and Asp10 to Asn in the 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 A1.
A2-RepM2/CRM3	Double mutant of the mild strain containing only a change of Asp10 to Asn in the 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 A1.
A1-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 an NcoI-to-BclI fragment and cloned at the same sites in A1.
A1-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 ClaI sites.
A1-nRepA2	DNA-A of the severe strain containing 5′ sequences coding for amino acids 1–110 in the N-terminal region of the Rep gene from the mild strain. The fragment was cloned as an NcoI-to-XbaI fragment between the strains.
A1-RepM3	DNA-A of the severe strain containing a change of Asn10 to Asp in the 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 change of the 3rd nucleotide in the putative binding site sequence, GGTGTCTGGAGTC at 2642 to GGCGTCGGAGTC.
A1-RepM4/CRM4	DNA-A of the severe strain containing a change of Asn10 to Asp in the Rep gene and a single nucleotide change in the binding site sequence at 2642, making the repeat motif GGCGTCGGAGTC.

Protoplast and plant inoculations.

Protoplasts derived from N. tabacum BY-2 suspension cultures were used for transfection with viral DNA (33). One million protoplasts were inoculated by electroporation (250 V, 500 μF) with 2 μg each of DNA-A and DNA-B and 40 μg of sheared herring sperm DNA. Protoplasts were collected from cultures 48 h postinoculation for DNA isolation and analysis.

Two-week-old seedlings of N. benthamiana grown in magenta boxes were inoculated with partial tandem dimers of viral DNA by using a Bio-Rad helium-driven particle gun (27). Ten plants were inoculated for each mutant, using 0.5 μg each of DNA-A and DNA-B per plant. Plants were observed for symptom development, and the newly emerging leaves were harvested for Southern blot analysis 21 days after inoculation.

Southern blot analysis.

DNA extractions from systemically infected leaf samples were carried out as described in reference 6 and from protoplasts by the procedure of Mettler (23). Total DNA (4 μg) was electrophoresed on 1% agarose gels without ethidium bromide and transferred to nylon membranes. Viral DNA was detected by using a DNA-A-specific radiolabeled probe (a 900-bp AflII-PstI 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 (28) by exposing the Southern blots to phosphor screens and counting the radioactivity on a PhosphorImager (Molecular Dynamics).

RESULTS

The mild strain does not replicate efficiently.

The two strains of ToLCV-Nde used in this study have been described previously (27). The DNA-A (pMPA1) and DNA-B (pMPB) of the severe strain have been cloned and characterized, but only the DNA-A (pMPA2) of the mild strain has been cloned; the DNA-B of the mild strain has not been isolated to date. 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 DNA-A and DNA-B of the severe strain produced severe symptoms with characteristic leaf curl, but plants inoculated with DNA-A of the mild strain and DNA-B of the severe strain developed mild infection with minor leaf curl symptoms. For the sake of brevity, hereafter, the severe strain DNA-A and DNA-B will be referred to as A1 and B1 and the mild strain DNA-A will be designated A2. A1 and A2 DNAs have the same length (2,739 nucleotides) and 94% sequence identity. Their CRs are 81% identical (27). The amino acid sequence identity between individual genes in A1 and A2 ranged from 91 to 99%, with the greatest similarity in the coat protein gene. The nucleotide sequence identity between the CRs of A1 and B1 is 97%, compared to 79% between the CRs of A2 and B1.

It was not known whether the mild phenotype in plants inoculated with A2 and B1 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 A1 or A2 DNA and viral DNA replication was quantitated 48 h after transfection.

We observed that A2 does not accumulate to the same level as the A1 in protoplasts. The replication efficiency of A2 DNA varied between 45 and 58% compared to the A1 DNA levels (Table 2). Next we tested the ability of A2 to replicate B1 in tobacco protoplasts. In transient assays, A2 replicated B1 to barely detectable levels (<1%) (Fig. 2B, lanes 1 and 10; Table 2). In N. benthamiana plants inoculated with A2 and B1 DNAs, very mild symptoms mostly limited to mild chlorosis and slight curling were observed 3 weeks postinoculation. Southern analysis of total DNA isolated from infected plants showed very low levels (5 to 10% compared to the A1 DNA) of DNA-B accumulation (Fig. 3A, lanes 1 and 8; Fig. 3B, lanes 1 and 10; Table 2). In plants, replication of DNA-B is required to cause a systemic infection (27), and these data suggested that low levels of DNA-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 B1 of the severe strain provided the first evidence that the two strains may have different replication requirements. Therefore, we focused on the incompatibility of A2 and B1 by making sequence comparisons between the Rep genes of A1 and A2 and the CRs of A2 and B1.

TABLE 2.

Replication and infectivity of ToLCV mutants in N. tabacum protoplasts and N. benthamiana plants

Mutanta	Avg % detectedb
	Protoplast inoculationsc				Plant inoculationsd
	DNA-A		DNA-B		DNA-A		DNA-B
	ss	ds	ss	ds	ss	ds	ss	ds
A2	41	44	<1	<1e	58	55	4	2
A2-RepA1	4	<1	<1	<1	5	<1	<1	<1
A1-RepA2	<1	<1	<1	<1	12	5	<1	<1
A2-cRepA1	39	33	<1	<1	59	54	<1	<1
A1-cRepA2	94	89	92	91	89	84	94	91
A2-nRepA1	5	<1	<1	<1	4	<1	<1	<1
A1-nRepA2	<1	<1	<1	<1	6	<1	6	5
A2-CRB1	<1	<1	<1	<1	<1	<1	<1	<1
A2-RepM1/CRA1	91	92	84	82	94	92	98	97
A2-RepM1/CRB1	92	84	90	87	92	89	106	104
A2-RepM1/CRM3	102	96	94	97	98	95	104	98
A2-RepM2/CRM3	106	98	89	84	92	79	85	71
A1-RepM3	4	5	<1	<1	14	8	<1	<1
A1-CRM1	<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	ND	ND	ND
A1-RepM4/CRM4	98	92	104	108	ND	ND	ND	ND

^aRepM and CRM denote mutations in the Rep gene and the CR, respectively. 

^bAverage amounts of single-stranded (ss) and double-stranded (ds) viral DNAs detected. The amount of viral DNA observed in protoplasts and plants inoculated with the severe strain A1 were assigned a value of 100. ND, not determined. 

^cAmount detected in 16 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 h after electroporation. Viral DNA was quantitated with a PhosphorImager. Standard error values between different transfections were in the range of ±2 to 5%. 

^dAmount detected in 10 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 to 5% between different plants. 

^eToo low for accurate quantification because of background error. 

FIG. 2.

Southern blot analysis of viral DNA in N. tabacum protoplasts inoculated with different mutants of ToLCV. Total DNA was extracted from protoplasts 48 h after transfection, electrophoresed through 1% agarose gel without ethidium bromide, transferred to a nylon membrane, and hybridized with ³²P-labeled DNA-A- and DNA-B-specific probes. (A) Replication of mutants made in severe strain DNA-A and probed with A-component (lanes 1 to 9)- and B-component (lanes 10 to 18)-specific probes; (B) replication of mutants made in the mild strain DNA-A probed with A-component (lanes 1 to 9)- and B-component (lanes 10 to 18)-specific probes. The positions of single-stranded (ss) and supercoiled (sc) viral DNAs are indicated. Each lane contains 4 μg of DNA obtained from protoplasts in a single transfection.

FIG. 3.

Southern blot analysis of viral DNA in N. benthamiana plants inoculated with ToLCV mutants. Total DNA was extracted from newly emerging leaves 3 weeks after bombardment and electrophoresed in 1% agarose gels without ethidium bromide, transferred to a nylon membrane, and hybridized with ³²P-labeled DNA-A- and DNA-B-specific probes. (A) Replication of severe strain mutants probed with A-component (lanes 1 to 7)- and B-component (lanes 8 to 14)-specific probes; B replication of mutants made in the mild strain DNA-A and probed with A-component (lanes 1 to 9)- and B-component (lanes 10 to 18)-specific probes. The positions of single-stranded (ss) and supercoiled (sc) viral DNAs are indicated.

N-terminal domains of Rep protein are not interchangeable between the strains.

The low levels of DNA-B accumulation in protoplasts and plants inoculated with A2 and B1 indicated that the mild strain Rep protein replicated B1 DNA inefficiently. To test if replication levels of DNA-B can be increased by replacing the mild strain Rep protein with its equivalent from A1, a full-length Rep gene (from NcoI to BclI) was exchanged between the two strains (Fig. 1B; Table 1). The mild strain DNA-A containing a full-length Rep gene of the severe strain (A2-RepA1) did not replicate efficiently in tobacco protoplasts (Fig. 2B, lanes 2 and 11; Table 2). Similarly, the severe strain DNA-A containing the Rep gene from the mild strain (A1-RepA2) failed to accumulate to high levels in protoplasts (Fig. 2A, lanes 2 and 11; Table 2), suggesting that the Rep proteins are not interchangeable. Similar results were obtained in N. benthamiana plants inoculated with these mutants (Fig. 3A, lanes 2 and 9; Fig. 3B, lanes 2 and 11; Table 2).

To determine whether the specificity of the Rep protein for the DNA templates was associated with C- or N-terminal region of the Rep protein, we exchanged both the 5′ and 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 (ClaI-ClaI fragment) was exchanged between the strains (A2-cRepA1 and A1-cRepA2 [Fig. 1B; Table 1]) 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, A1-cRepA2, accumulated both DNA-A and DNA-B at levels similar to the wild-type (A1) virus (Fig. 2A, lanes 3 and 12; Table 2). In contrast, the mild strain mutant A2-cRepA1 replicated DNA-A at moderate levels and DNA-B at very low levels (Fig. 2B, lanes 3 and 12; Table 2). Inoculation of N. benthamiana seedlings with these hybrid Rep gene constructs showed that the plants infected with A2-cRepA1 produced mild symptoms whereas plants inoculated with A1-cRepA2 developed typical leaf curl symptoms within 10 days (Table 2; Fig. 3A, lanes 3 and 10; Fig. 3B, lanes 3 and 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 had an effect on replication of the chimeric viruses.

We then exchanged sequences from the 5′ region of the Rep gene encoding amino acids 1 to 110 (NcoI-XbaI fragment) between the strains and assayed for replication of viral DNA. The severe strain mutant A1-nRepA2 contains the N-terminal sequences from the mild strain and led to accumulation of very low levels of viral DNA (Fig. 2A, lanes 4 and 13; Table 2). Similarly, the mutant A2-cRepA1 resulted in negligible levels of virus replication (Fig. 2B, lanes 4 and 13; Table 2). None of the N. benthamiana plants inoculated with either the A1-nRepA2 or A2-nRepA1 mutant developed symptoms and accumulated very low levels of viral DNA (Table 2; Fig. 3A, lanes 4 and 11; Fig. 3B, lanes 4 and 13), indicating that the region spanning amino acids 1 to 110 in the Rep gene may contain residues that determine specificity of replication between the two strains and are not interchangeable.

Rep proteins display specificity in recognizing replication origins.

Geminivirus replication requires a functional interaction of Rep protein with specific sequences in the ori (11, 12). Studies described above identified the N-terminal region of Rep protein as being important in determining replication of the two strains. We compared the CRs of the three DNA components, A1, A2, and B1, to look for differences that may contribute to template specificity. The CRs of A1 and A2 are 81% identical, while the CRs of A2 and B1 share only 79% sequence homology; by comparison, A1 and B1 are 97% identical. To determine whether sequence differences in the CR of the mild strain restricts its replication, the CR of A2 was replaced with that of B1 (mutant A2-CRB1 [Table 1]) and tested for its ability to replicate in BY-2 protoplasts and N. benthamiana plants. As shown in Table 2, exchange of the common region of A2 with B1 did not increase the replication levels of DNA-B in protoplasts. Rather, the replication levels of both DNA-A and DNA-B were drastically reduced (Fig. 2B, lanes 5 and 14; Table 2). Similarly, this mutant did not replicate in N. benthamiana (Fig. 3B, lanes 5 and 14; Table 2). These results demonstrated that the Rep protein of A2 does not recognize the CR sequences of B1, suggesting that the two strains specifically recognize unique sequences in their oris.

The experiments done thus far helped to delimit two essential features that influence replication specificity of two strains, the CR sequences and the N-terminal residues in Rep protein. We next introduced mutations in the N-terminal region (amino acids 1 to 110) of A1 and A2 Rep proteins with concomitant changes in their viral oris to analyze the precise determinants of a functional, replication-competent interaction.

The Rep protein in geminiviruses show sequence similarities with other initiator proteins that follow a rolling circle mode of replication. Based on comparison of sequences of these proteins, Koonin and Ilyina (18) identified a domain among geminiviruses in the N-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, which is involved in DNA nicking and closing activities (15, 19); motif II, HIHxUUQ (U is a bulky hydrophobic residue), which has structural features similar to those of the Mg² binding sites of metalloenzymes (18); and motif I, FLTYPqC (q is a basic or a polar amino acid), whose function has not been established yet. The three motifs described are present within the region spanning amino acids 1 to 110 and are identical between the Rep proteins of A1 and A2.

To define the amino acids involved in recognition of the ori, the region between amino acids 1 and 110 was examined for sequence variation between Rep proteins of A1 and A2. Four amino acid differences were identified between the two proteins in this region. The A1 strain contains amino acids Ile, 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 Asn10, are immediately adjacent to motif I. To determine if Ile9 and Asn10 had a role in replication, Val9 and Asp10 in Rep protein of A2 were changed to Ile9 and Asn10. Simultaneously, the CR of the A2 DNA was replaced with either A1 (A2-RepM1/CRA1) or B1 (A2-RepM1/CRB1) sequence (Fig. 1C). In protoplasts, both mutants A2-RepM1/CRA1 (Fig. 2B, lanes 6 and 15) and A2-RepM1/CRB1 (Fig. 2B, lanes 7 and 16) accumulated viral DNA to similar levels as the severe A1 strain. N. benthamiana plants inoculated with both of these mutants developed severe infection 10 days after inoculation and accumulated high levels of viral DNA as determined by Southern hybridization (Fig. 3B, lanes 6, 7, 15, and 16; Table 2). These data suggested that Ile9 and Asn10 are involved in determining interaction of Rep protein with specific sequences in the CR.

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 ori. Based on comparison of many ori sequences in geminiviruses (1, 2, 11), we looked for putative Rep binding sites in the CRs of A1, A2, and B1 DNAs. A 13-bp sequence was identified in ori close to the TATA box in the CRs of the three DNAs. In A1 and B1, the repeat sequence GGTGTCTGGAGTC was identified, while A2 DNA had the repeat sequence GGCGTCTGGCGTC.

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 A1-CRM1 (deletion at the 3rd nucleotide) and A1-CRM2 (deletion at the 10th nucleotide) showed dramatically reduced levels of viral DNA replication in infected protoplasts (Fig. 2A, lanes 6, 7, 15, and 16; Table 2). Inoculated N. benthamiana plants did not show any symptoms 3 weeks postinoculation, and replication of both viruses was reduced to minimal levels (Fig. 3A, lanes 6, 7, 13, and 14; Table 2). These results demonstrated that the 13-bp sequence in A1 is essential for virus replication and may represent the Rep protein binding site.

Asn10 may determine specific interaction with the viral origin.

Earlier experiments showed that amino acids Ile9 and Asn10 in Rep protein of A1 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 Val9 to Ile and Asp10 to Asn. Simultaneously, the 3rd and 10th nucleotides on the potential binding site in A2 (GGCGTCTGGCGTC) were mutated to T and A, respectively (GGTGTCGGAGTC) to make the repeat sequence identical to that of A1 (mutant A2-RepM1/CRM3 [Fig. 1C]). As expected, the mutant A2-RepM1/CRM3 was functional in protoplasts and replicated DNA-A and DNA-B to wild-type levels (Fig. 2B, lanes 8 and 17; Table 2). Also, plants inoculated with mutant A2-RepM1/CRM3 and B1 produced severe symptoms within 2 weeks after inoculation, accumulating high levels of viral DNA (Fig. 3B, lanes 8 and 17; Table 2). 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 Ile9 and Asn10 may be involved in specific interaction between Rep and ori.

Of the two amino acid changes made, Asp10 to Asn is expected to be more significant than Val9 to Ile9; we therefore made another mutant, A2-RepM2/CRM3, where only the Asp10 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 A1 (Fig. 1C). In an analogous experiment, the Asn10-to-Asp change was made in Rep protein of A1 (A1-RepM3) without any changes in its ori (Fig. 1C). Protoplasts transfected with A2-RepM2/CRM3 accumulated high levels of viral DNA (Fig. 2B, lanes 9 and 18; Table 2) comparable to the wild-type severe strain. The corresponding mutant, A1-RepM3 with the Asn10-to-Asp change in the Rep protein of A1, replicated to very low levels in protoplasts (Fig. 2A, lanes 5 and 14; Table 2). N. benthamiana plants inoculated with the mutant A2-RepM2/CRm3 developed severe symptoms and accumulated increased levels of viral DNA (Fig. 3B, lanes 9 and 18; Table 2), showing that Asn10 alone is sufficient to facilitate specific replication of the virus. In contrast, plants inoculated with A1-RepM3, which contains a substitution in the 10th residue of its Rep protein, did not replicate viral DNA with high efficiency (Fig. 3A, lanes 5 and 12; Table 2).

Asn10 may interact with the third nucleotide of the 5′ iteron, GGTGTC.

While the above experiment showed that Asn10 in Rep protein may be involved in specific recognition of the ori, it was essential to determine if both the 3′ and 5′ repeats in the 13-mer binding site contributed to the specificity of recognition. To address this question, the A1 DNA was mutated at the third nucleotide in this sequence, substituting C for T (A1-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 DNA accumulated (Fig. 2A, lanes 8 and 17; Table 2), suggesting that Rep protein may be unable to recognize the modified binding site. This mutant was further modified by changing Asn10 to Asp (A1-RepM4/CRM4 [Fig. 1C]). The mutant A1-RepM4/CRM4 accumulated wild-type levels of DNA-A in protoplasts (Fig. 2A, lanes 9 and 18; Table 2), indicating that Asp10 may indeed interact with the third base of the iteron GGCGTC sequence.

DISCUSSION

We investigated the specificity of interactions between Rep proteins and ori sequences in two related strains of ToLCV-Nde. 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 the viral DNA is replicated. Change of Asp10 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, we propose that Asn10 specifically recognizes the third base pair of the 5′ iteron GGTGTC in the severe strain.

Even though the DNA-A component of the severe (A1) 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-B1. These results support the hypothesis that the mild symptoms in plants inoculated with A2 and B1 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, as shown by the mild strain mutant A2-CRB1, 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 (17), TGMV (13, 25), and beet curly top virus (BCTV) (4, 5, 32).

Of the four amino acid differences in the N-terminal region (amino acids 1 to 110) between the Rep genes of A1 and A2, we chose to mutate Ile9 and Asn10 because of their proximity to motif I (FLTYPKC), a conserved element found in all the initiator proteins that replicate via a rolling circle mechanism (18). Replication assays in protoplasts and in plants obtained with the mild strain mutants A2RepM1/CRA1 and A2-RepM1/CRB1 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-Nde has not been biochemically determined. We used site-directed mutagenesis to determine whether the 13-nucleotide sequence identified in the ori interacts in a functional way with the Rep protein. The severe strain mutants A1-CRM1 and A1-CRM2, which contain single nucleotide deletions in the 13-mer sequence, failed to accumulate viral DNA, 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 (26).

The mutant A2-RepM2/CRM3, which contains an Asp10-to-Asn mutation in the Rep protein and corresponding changes in the potential binding site sequence, GGCGTCTGGCGTC to GGTGTCTGGAGTC (identical to the severe strain), restored the replication efficiency of A2 DNA. These results indicated that Asn10 may differentiate between A1 and A2 strains and determine the specificity in recognizing ori sequences. In addition, the fact that replication of B1 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. We did not study the role of the other two amino acids, Lys40 and Glu54, that are different between the two Rep proteins and therefore do not conclude that Asn10 is solely responsible for strain specificity.

Since the iteron sequences in the binding sites of A1 and A2 are different with respect to the 3rd and 10th nucleotides, we examined the significance of these differences in context of Asn10 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 Asn10 may recognize the third base pair in the 5′ iteron GGTGTC. It is possible that Asn10 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 (25). The inability of the Cal/Logan strain to replicate the Worland or the CFH strain of BCTV (32) was correlated to the differences in the third 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 (17) whose Rep proteins share more than 76% amino acid identity but are not interchangeable and between TGMV and BGMV (12), probably because of differences in their iteron sequences, supporting the importance of specific interaction between the Rep protein and its recognition sequence.

The iteron sequences of A1 and B1 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 A1, suggesting that both iterons are required for replication. Similarly, differential contributions of the two iterons have been reported for TGMV (11) and BCTV (5). Unlike the reports of Fontes et al. (11) and Choi and Stenger (5), 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′ than the 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.

Our 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 position 10 (Asp to Asn) in the Rep protein with a concomitant change in the third nucleotide of the putative binding site (A2-RepM2/CRM3) resulted in increased levels of virus accumulation. Similarly, the mutant, A1-RepM4, containing a change of Asn10 to Asp in Rep protein without any change in its binding site accumulated very low levels of virus DNA. Further, the change of Asn10 to Asp accompanied by a change in the 5′ iteron of the binding site (mutant A1-RepM4/CRM4) restored virus replication, suggesting that the correlation between the amino acid at position 10 and the third nucleotide of the iteron may indeed be related to specificity of replication.

The observed specificity of the Rep protein with sequences in the ori accounts for selective replication of A1 and A2 strains. While earlier work (4, 5, 17, 22) 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 Asn10 of Rep protein in case of the two strains of ToLCV-Nde. Since Asn10 is very closely associated with the conserved motif I sequence, FLTYPKC (18), in Rep protein, we suggest that it may function as a part of motif I to mediate specific replication of the cognate genomes. Experiments are in progress to determine if changes at Asn10 in Rep protein affects its capacity to bind DNA.