Evaluating the strength of RNA silencing suppressor proteins encoded by two geminiviruses using assay based on reversal of GFP silencing

Virus Induced Gene Silencing (VIGS) is conserved and dynamic gene regulation machinery, which plays an active role in shielding plants against viral infections. In counter-defense, viruses encode proteins that can suppress RNA silencing to ensure successful systemic invasion. The AC2 protein of Tomato Leaf Curl New Delhi Virus (ToLCV-AC2) and Mungbean Yellow Mosaic India Virus (MYMIV-AC2) are well-characterized suppressors of RNA silencing. In this study we have compared the structure and suppressor strength of ToLCV-AC2 and MYMIV-AC2, to gain insight on the conservation of the RNA silencing suppression activities. The relative suppressor strength was measured using an assay based on the reversal of GFP silencing and quantitated in terms of the amount of reversal of GFP expression and the sustenance of the reversal activity. The phenotypic observations in the reversal of silencing assay were further corroborated with molecular analysis. Our results indicate that ToLCV-AC2 exhibited weaker RNA silencing suppression than MYMIV-AC2. The suppression activity of ToLCV-AC2 is supplemented by related, though weak, activities encoded by ToLCV-AC4 and ToLCV-AV2 proteins. Collectively the three ToLCV proteins showed suppressor strength comparable to MYMIV-AC2. These results indicate that in all probability the ToLCV deploys more than one RNA silencing suppressor protein to synergistically counteract the host RNA silencing mechanism.