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.

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).

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.