Geminiviruses are single-stranded DNA viruses that infect important crops worldwide, often occurring as mixed infections in the field. In Africa, they cause significant losses in cassava, maize, tomato, cotton and bean crops. Efforts to generate geminivirus-resistant plants based on conventional breeding and pathogen-derived transgenic approaches have been of limited success, often resulting in reduced susceptibility or tolerance to a narrow range of viruses and with the resistant plants serving as reservoirs for viral evolution. Hence, it is essential to develop an alternative resistance strategy that confers broad-based, stable immunity to geminivirus infection. To address this need, we used a combinatorial chemistry approach to identify peptide aptamers that interfere with the geminivirus replication protein (Rep). Rep is highly conserved among geminiviruses and other eukaryotic single-stranded DNA viruses. Its N-terminus contains several conserved motifs that are required for viral replication, making them excellent targets for an interfering peptide strategy. We screened a constrained random peptide library in yeast two-hybrid assays to identify peptides that bind to tomato golden mosaic virus (TGMV) Rep. The library plasmids contained a LexA DNA binding domain fused to the bacterial thioredoxin (Trx) coding sequence with a random 60-mer sequence inserted into the active site. Using the first 130 amino acids of Rep fused to a synthetic activation domain as bait, we isolated 597 positive candidates in a screen of 2 x 107 yeast colonies. Interaction with Rep was confirmed for 287 colonies, and prey plasmids were recovered and sequenced from 130 colonies. We selected 88 unique sequences for further analysis in transient replication interference assays. In these assays, tobacco protoplasts were cotransfected with a wild-type TGMV A replicon and a plant expression cassette corresponding to the Trx-peptide fusion, and viral DNA replication was measured after a 36-hour culture period. To facilitate high throughput screening, we developed a semi-quantitative polymerase chain reaction (PCR) protocol to measure viral DNA accumulation. These experiments established that 28 of the 88 selected Trx-peptide fusions interfere with geminivirus replication. Sequence comparison of the interfering peptides revealed that they contain homologous amino acids with conserved spacing. To date, we have identified three different sequence groups that are represented among the interfering peptides. We also used yeast growth assays to show that the Rep protein from the cabbage leaf curl virus (CaLCuV) binds to 77 of the 88 selected peptides, including the 28 peptides that display replication interference activity. This result is particularly promising because TGMV and CaLCuV belong to different begomovirus clades with distinct Rep proteins. We are currently examining the peptide binding properties of Rep proteins from the three major geminivirus genera. Breadth and strength of interaction and replication interference will be used to select Trx-peptides for expression in transgenic plants and testing in geminivirus infectivity assays.