Interaction with host SGS3 is required for suppression of RNA silencing by tomato yellow leaf curl virus V2 protein.

The V2 protein of tomato yellow leaf curl geminivirus (TYLCV) functions as an RNA-silencing suppressor that counteracts the innate immune response of the host plant. The host-cell target of V2, however, remains unknown. Here we show that V2 interacts directly with SlSGS3, the tomato homolog of the Arabidopsis SGS3 protein (AtSGS3), which is known to be involved in the RNA-silencing pathway. SlSGS3 genetically complemented an AtSGS3 mutation and restored RNA silencing, indicating that SlSGS3 is indeed a functional homolog of AtSGS3. A point mutant of V2 that is unable to bind SlSGS3 also lost its ability to suppress RNA silencing, suggesting a correlation between the V2-SlSGS3 interaction in planta and the suppressor activity of V2.

Suppressor of RNA silencing encoded by Tomato yellow leaf curl virus-Israel.

The Israeli isolate of Tomato yellow leaf curl geminivirus (TYLCV-Is) is a major tomato pathogen, causing extensive crop losses both in the New and Old World. Surprisingly, however, little is known about the molecular mechanisms of TYLCV-Is interactions with tomato cells. Here, we have identified a TYLCV-Is protein, V2, which acts as a suppressor of RNA silencing and which is unrelated to presently known viral suppressors. Specifically, V2, but not other proteins of TYLCV-Is, inhibited RNA silencing of a reporter transgene, GFP. This inhibition elevated the cellular levels of the GFP transcript and the GFP protein, but it had no apparent effect on the accumulation of GFP-specific short interfering RNAs (siRNAs), suggesting that TYLCV-Is V2 targets a step in the RNA silencing pathway which is subsequent to the Dicer-mediated cleavage of dsRNA. Visualization of the sub-cellular localization of TYLCV-Is V2 in plant protoplasts and tissues showed that this protein is associated with cytoplasmic strands and inclusion bodies in the cortical regions of the cell.

Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta.

Bimolecular fluorescence complementation (BiFC) represents one of the most advanced and powerful tools for studying and visualizing protein-protein interactions in living cells. In this method, putative interacting protein partners are fused to complementary non-fluorescent fragments of an autofluorescent protein, such as the yellow spectral variant of the green fluorescent protein. Interaction of the test proteins may result in reconstruction of fluorescence if the two portions of yellow spectral variant of the green fluorescent protein are brought together in such a way that they can fold properly. BiFC provides an assay for detection of protein-protein interactions, and for the subcellular localization of the interacting protein partners. To facilitate the application of BiFC to plant research, we designed a series of vectors for easy construction of N-terminal and C-terminal fusions of the target protein to the yellow spectral variant of the green fluorescent protein fragments. These vectors carry constitutive expression cassettes with an expanded multi-cloning site. In addition, these vectors facilitate the assembly of BiFC expression cassettes into Agrobacterium multi-gene expression binary plasmids for co-expression of interacting partners and additional autofluorescent proteins that may serve as internal transformation controls and markers of subcellular compartments. We demonstrate the utility of these vectors for the analysis of specific protein-protein interactions in various cellular compartments, including the nucleus, plasmodesmata, and chloroplasts of different plant species and cell types.