No abstract

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.

We have developed a simple genetic assay to detect active nuclear localization (NLS) and export signals (NES) on the basis of their function within yeast cells. The bacterial LexA protein was modified (mLexA) to abolish its intrinsic NLS and fused to the activation domain of the yeast Gal4p (Gal4AD) with or without the SV40 large T-antigen NLS. In the import assay, if a tested protein fused to mLexA-Gal4AD contains a functional NLS, it will enter the cell nucleus and activate the reporter gene expression. In the export assay, if a tested protein fused to mLexA-SV40 NLS-Gal4AD contains a functional NES, it will exit into the cytoplasm, decreasing the reporter gene expression. We tested this system with known NLS and NES and then used it to demonstrate a NES activity of the capsid protein of a plant geminivirus. This approach may help to identify, analyze, and select for proteins containing functional NLS and NES.

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.

Cell-to-cell tobacco mosaic virus movement protein (TMV MP) mediates viral spread between the host cells through plasmodesmata. Although several host factors have been shown to interact with TMV MP, none of them coresides with TMV MP within plasmodesmata. We used affinity purification to isolate a tobacco protein that binds TMV MP and identified it as calreticulin. The interaction between TMV MP and calreticulin was confirmed in vivo and in vitro, and both proteins were shown to share a similar pattern of subcellular localization to plasmodesmata. Elevation of the intracellular levels of calreticulin severely interfered with plasmodesmal targeting of TMV MP, which, instead, was redirected to the microtubular network. Furthermore, in TMV-infected plant tissues overexpressing calreticulin, the inability of TMV MP to reach plasmodesmata substantially impaired cell-to-cell movement of the virus. Collectively, these observations suggest a functional relationship between calreticulin, TMV MP, and viral cell-to-cell movement.Following initial infection (usually by mechanical inoculation), many plant viruses, such as tobacco mosaic virus (TMV), spread from cell to cell through plasmodesmata (for review, see Heinlein, 2002;Waigmann et al., 2004), presumably utilizing the existing cellular pathways of plasmodesmal transport. In the case of TMV, this virus encodes a specific cell-to-cell movement protein (MP), which mediates the transport of the viral genomic RNA through plasmodesmata (Deom et al., 1987). To date, MP has been shown to bind TMV RNA (Citovsky et al., 1990(Citovsky et al., , 1992, associate with the cytoskeleton (Heinlein et al., 1995;McLean et al., 1995;Boyko et al., 2000) and endoplasmic reticulum (ER; Heinlein et al., 1998;Reichel and Beachy, 1999), target to plasmodesmata within plant cell walls (Tomenius et al., 1987;Ding et al., 1992a), increase plasmodesmal permeability (Wolf et al., 1989;Waigmann et al., 1994), and undergo negative regulation by phosphorylation (Citovsky et al., 1993;Waigmann et al., 2000;Trutnyeva et al., 2005). To perform many of these functions, TMV MP most likely cooperates with different cellular factors, some of which, such as cell wall pectin methylesterases (PME; Dorokhov et al., 1999;Chen et al., 2000), microtubules (MTs) and actin filaments (Heinlein et al., 1995;McLean et al., 1995;Boyko et al., 2000), and a RIO protein kinase (Yoshioka et al., 2004), are known, while others remain to be discovered. It would be especially interesting to determine whether cellular proteins exist that not only recognize TMV MP but also coreside with it within plasmodesmata.Using TMV MP as specific ligand in a biochemical purification protocol, we showed that it interacts with plant calreticulin. Calreticulin is a Ca 21 -sequestering protein that is highly conserved between different species, including plants ( Nelson et al., 1997; Borisjuk et al., 1998, and refs. therein;Michalak et al., 1998). Functionally, calreticulin is involved in Ca 21 storage and signaling, chaperone activity, cell ad...