Cucurbit chlorotic yellows virus p22 suppressor of RNA silencing binds single-, double-stranded long and short interfering RNA molecules in vitro.

Cucurbit chlorotic yellows virus (CCYV) is a new member of the genus Crinivirus (family Closteroviridae) with a bi-partite genome. CCYV RNA 1-encoded p22 has recently been reported to be a weak local suppressor of RNA silencing for which an interaction with cucumber SKP1LB1 through an F-box-like motif was demonstrated to be essential. Using a bacterially expressed maltose-binding protein (MBP) fusion of CCYV p22 in electrophoretic mobility shift assays (EMSA), we have examined in vitro its ability to bind different RNA templates. Our experiments showed that CCYV p22 is able to bind to ss and ds long RNAs, in addition to ss and ds small interfering (si) RNA molecules. CCYV p22 deletion mutants (MBP_CCYV DEL1-4) were produced that covered the entire protein, with MBP_CCYV DEL2 corresponding to the F-box motif and its flanking sequences. None of these deletions abolished the capacity of CCYV p22 to bind ss- and dsRNA molecules. However, deletions affecting the C-terminal half of the protein resulted in decreased binding efficiency for either ss- or dsRNA molecules indicating that essential elements for these interactions are located in this region. Taken together, our data add to current knowledge of the mode of action of suppressors of RNA silencing encoded by genes sited at the 3'-terminus of crinivirus genomic RNA 1, and shed light on the involvement of CCYV p22 in the suppression of RNA silencing and/or in another role in the virus life cycle via RNA binding.

A Thioredoxin Domain-Containing Protein Interacts with Pepino mosaic virus Triple Gene Block Protein 1.

Pepino mosaic virus (PepMV) is a mechanically-transmitted tomato pathogen of importance worldwide. Interactions between the PepMV coat protein and triple gene block protein (TGBp1) with the host heat shock cognate protein 70 and catalase 1 (CAT1), respectively, have been previously reported by our lab. In this study, a novel tomato interactor (SlTXND9) was shown to bind the PepMV TGBp1 in yeast-two-hybrid screening, in vitro pull-down and bimolecular fluorescent complementation (BiFC) assays. SlTXND9 possesses part of the conserved thioredoxin (TRX) active site sequence (W__PC vs. WCXPC), and TXND9 orthologues cluster within the TRX phylogenetic superfamily closest to phosducin-like protein-3. In PepMV-infected and healthy Nicotiana benthamiana plants, NbTXND9 mRNA levels were comparable, and expression levels remained stable in both local and systemic leaves for 10 days post inoculation (dpi), as was also the case for catalase 1 (CAT1). To localize the TXND9 in plant cells, a polyclonal antiserum was produced. Purified α-SlTXND9 immunoglobulin (IgG) consistently detected a set of three protein bands in the range of 27⁻35 kDa, in the 1000 and 30,000 g pellets, and the soluble fraction of extracts of healthy and PepMV-infected N. benthamiana leaves, but not in the cell wall. These bands likely consist of the homologous protein NbTXND9 and its post-translationally modified derivatives. On electron microscopy, immuno-gold labelling of ultrathin sections of PepMV-infected N. benthamiana leaves using α-SlTXND9 IgG revealed particle accumulation close to plasmodesmata, suggesting a role in virus movement. Taken together, this study highlights a novel tomato-PepMV protein interaction and provides data on its localization in planta. Currently, studies focusing on the biological function of this interaction during PepMV infection are in progress.