Abstract
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.
Highlights
Pepino mosaic virus (PepMV; genus Potexvirus) is a mechanically-transmitted virus, which has evolved in only a few years from an emerging to important endemic pathogen in several tomato (Solanum lycopersicum)-growing areas of the world [1]
The four related protein groups (GRX, protein disulphide isomerases (PDIs), glutathione peroxidases (GPs), glutathione S-transferases (GSTs)) in the TRX superfamily (Figure 2A) were included, together with sequences from phosducin-like proteins 3 (PLP3) from A. thaliana, S. lycopersicum and N. benthamiana, as these shared the highest degree of amino acid identity with SlTXND9 in the TAIR database, using a 1 × 10−5 E-value as a threshold
Initial experimentation showed that the SlTXND9/PepMV TGBp1 complex can be detected in the cytoplasm of agroinfiltrated N. benthamiana epidermal cells and, as for the catalase 1 (CAT1)/PepMV TGBp1 protein complex [18], the transcriptome and fractionated protein samples from PepMV-infected leaves did not contain elevated levels of NbTXND9 mRNA or protein, when compared to those of healthy plants
Summary
Pepino mosaic virus (PepMV; genus Potexvirus) is a mechanically-transmitted virus, which has evolved in only a few years from an emerging to important endemic pathogen in several tomato (Solanum lycopersicum)-growing areas of the world [1]. PepMV CP binds the heat-shock cognate protein 70 (Hsc70) isoform 3 to facilitate PepMV infection [16,17], whereas TGBp1 interacts with tomato catalase 1 (CAT1) to elevate its hydrogen peroxide scavenging efficiency, most likely to regulate plant defense mechanisms and facilitate virus accumulation [18]. TRXs have been shown to participate in (and affect) multiple processes including gene expression, signal transduction, proliferation and apoptosis, and in particular tolerance of oxidative stress. In the latter, TRXs may play an active role as scavengers of reactive oxygen species (ROS) or participate in signaling within the antioxidant network [24]. The manner of action of TXND9 in PepMV-infected plants remains to be addressed
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