The AL1 protein of tomato golden mosaic virus (TGMV), a member of the 
geminivirus family, is essential for viral replication in plants. Its N terminus 
contains three conserved motifs that mediate origin recognition and DNA cleavage 
during the initiation of rolling-circle replication. We used the N-terminal 
domain of TGMV AL1 as bait in a yeast two-hybrid screen of a random peptide 
aptamer library constrained in the active site of the thioredoxin A (TrxA) gene. 
The screen selected 88 TrxA peptides that also bind to the full-length TGMV AL1 
protein. Plant expression cassettes corresponding to the TrxA peptides and a 
TGMV A replicon encoding AL1 were cotransfected into tobacco protoplasts, and 
viral DNA replication was monitored by semiquantitative PCR. In these assays, 31 
TrxA peptides negatively impacted TGMV DNA accumulation, reducing viral DNA 
levels to 13 to 64% of those of the wild type. All of the interfering aptamers 
also bound to the AL1 protein of cabbage leaf curl virus. A comparison of the 
20-mer peptides revealed that their sequences are not random. The alignments 
detected seven potential binding motifs, five of which are more highly 
represented among the interfering peptides. One motif was present in 18 
peptides, suggesting that these peptides interact with a hot spot in the AL1 N 
terminus. The peptide aptamers characterized in these studies represent new 
tools for studying AL1 function and can serve as the basis for the development 
of crops with broad-based resistance to single-stranded DNA viruses.