A New Strategy For Plant Resistance Against Geminivirus Disease

Geminiviruses constitute a large family of single-stranded DNA viruses that cause significant losses in economically important crops worldwide. They often exist in disease complexes and undergo high levels of recombination and mutation, allowing them to adapt rapidly to new hosts and new environments. Thus, an effective resistance strategy must be general in character and able to target multiple viruses. The geminivirus Rep protein is a good target for broad-based disease control because it is highly conserved and required for viral replication. In an earlier study, we identified a set of peptide aptamers that bind to Rep and reduce viral replication in cultured plant cells. In the studies reported here, we selected 16 of the peptide aptamers for further analysis in yeast two-hybrid assays. These experiments showed that all 16 peptide aptamers interact with the Rep proteins from 9 geminiviruses representing the three major Geminiviridae genera and identified two peptide aptamers (A22 and A64) that strongly interact with different regions in the Rep N-terminus. We developed a protocol for Agrobacterium mediated transformation of A22 and A64 into tomato (Solanum lycopersicum ). We optimized conditions for tomato growth in our laboratory chambers and developed a protocol for Tomato yellow leaf curl (TYLCV) and Tomato mottle virus (ToMoV) agroinoculation of tomato. Transgenic tomato lines expressing A22 or A64 inoculated with TYLCV accumulated significantly reduced levels of viral DNA. Strikingly, the effect on symptoms was stronger, with many of the plants showing no symptoms or strongly attenuated symptoms. To establish the efficacy of the peptide aptamer technology for broad-based geminivirus disease resistance, the A22 and A64 lines displaying the strongest resistance to TYLCV are currently being challenged with ToMoV. Together, these results established that efficacy of using Rep-binding peptide aptamers to develop crops that are resistant to geminiviruses.;Rep is an excellent candidate to develop broad-based resistance strategies, and it is imperative to continue characterizing the Rep protein in an effort to increase our understanding of essential viral functions that are conserved across geminiviruses. We highlighted a conserved sequence found in the Rep proteins of all dicotyledonous infecting geminiviruses except for members of the Squash leaf curl (SLC) clade. Some amino acids in this conserved sequence are necessary for viral infection but are missing in SLC clade Rep proteins. Even though more studies are needed to fully understand the functional differences between these Rep proteins, the results from our functional assays suggest that CaLCuV Rep domain organization is similar to TGMV Rep and that both the DNA binding and oligomerization domains are required for its dsDNA binding activity.