Functional Modulation of the Geminivirus AL2 Transcription Factor and Silencing Suppressor by Self-Interaction

Abstract

The DNA genomes of geminiviruses have a limited coding capacity that is compensated for by the production of small multifunctional proteins. The AL2 protein encoded by members of the genus Begomovirus (e.g., Tomato golden mosaic virus) is a transcriptional activator, a silencing suppressor, and a suppressor of a basal defense. The related L2 protein of Beet curly top virus (genus Curtovirus) shares the pathogenicity functions of AL2 but lacks transcriptional activation activity. It is known that AL2 and L2 can suppress local silencing by interacting with adenosine kinase (ADK) and can suppress basal defense by interacting with SNF1 kinase. However, how the activities of these viral proteins are regulated remains an unanswered question. Here, we provide some answers by demonstrating that AL2, but not L2, interacts with itself. The zinc finger-like motif (CCHC) is required but is not sufficient for AL2 self-interaction. Alanine substitutions for the invariant cysteine residues that comprise the motif abolish self-interaction or cause aberrant subnuclear localization but do not abolish interaction with ADK and SNF1. Using bimolecular fluorescence complementation, we show that AL2:AL2 complexes accumulate primarily in the nucleus, whereas AL2:ADK and L2:ADK complexes accumulate mainly in the cytoplasm. Further, the cysteine residue mutations impair the ability of AL2 to activate the coat protein promoter but do not affect local silencing suppression. Thus, AL2 self-interaction correlates with nuclear localization and efficient activation of transcription, whereas AL2 and L2 monomers can suppress local silencing by interacting with ADK in the cytoplasm.

Tomato SlSnRK1 Protein Interacts with and Phosphorylates βC1, a Pathogenesis Protein Encoded by a Geminivirus β-Satellite

“…BiFC experiments were performed as described previously (Yang et al, 2007). YFP fluorescence was observed and photographed by confocal microscopy (Leica TCS SP5) at 48 to72 h after infiltration.…”

“…The full-length coding sequence of tomato SlSnRK1 was amplified using the primers listed in Supplemental Table S2 and cloned into the PacI-AscI site of p2YC (Yang et al, 2007) as a fusion with the C-terminal fragment of YFP, resulting in pSlSnRK1-YFP C .…”

“…For the production of BiFC vectors, the full-length coding sequence of bC1 was amplified using the primers listed in Supplemental Table S2 and cloned into the PacI-AscI site of p2YN (Yang et al, 2007) as a fusion with the N-terminal fragment of YFP, resulting in pbC1-YFP N . The full-length coding sequence of tomato SlSnRK1 was amplified using the primers listed in Supplemental Table S2 and cloned into the PacI-AscI site of p2YC (Yang et al, 2007) as a fusion with the C-terminal fragment of YFP, resulting in pSlSnRK1-YFP C .…”

P0 proteins of European beet-infecting poleroviruses display variable RNA silencing suppression activity

“…The RSSs characterized for sugar beet viruses are: L2 of beet curly top virus (genus Curtovirus; Yang et al, 2007), P14 of beet necrotic yellow vein virus (BNYVV, genus Benyvirus; Dunoyer et al, 2002), P21 of beet yellows virus (genus Closterovirus; Reed et al, 2003) and P0 of turnip yellows virus (TuYV, genus Polerovirus; Pfeffer et al, 2002). TuYV [syn.…”

C2 from Beet curly top virus meddles with the cell cycle

“…However, the fact that C2 from TYLCSV seems to affect hormone signaling in a similar manner 25 and yet this related pathogenicity factor does not have a promoting effect on geminivirus replication or the plant cell cycle 10,25 argues against this hypothesis. D) Even though C2 from BCTV is considered not to have transcriptional activation activity, because it lacks a transcriptional activation domain present in C2 from begomoviruses 26,27 and it does not cause transcriptional activation in yeast, 28 the possibility that it may be acting as a transcriptional factor itself cannot be definitively ruled out. E) Finally, C2 may be interacting with cellular components other than BCTV infection, transgenic expression of C2 from BCTV in Arabidopsis plants leads to changes in the expression of genes involved in a diverse array of cellular processes ( Fig.…”