Characterization of the C4 protein encoded by parsley yellow leaf curl virus

Abstract

Geminiviruses encode small, rapidly evolving proteins that reprogram host functions to facilitate infection. Among these, C4/AC4 proteins frequently act as determinants of pathogenicity and symptom expression. Parsley yellow leaf curl virus (PYLCV; Pylecuvirus petroselini), a recently described monopartite geminivirus associated with yellowing and leaf curling in parsley, encodes a C4 protein whose biological activities and cellular distribution remain uncharacterized. Here, we combined comparative sequence analysis, confocal microscopy, site-directed mutagenesis, heterologous expression, and RNA silencing suppression assays to characterize PYLCV C4. In silico analysis of the 85-amino-acid C4 protein identified several candidate targeting features, including a predicted N-terminal myristoylation motif, a putative palmitoylation site, nuclear trafficking-related motifs, and a chloroplast transit peptide. Confocal imaging showed that C4–GFP localizes to the plasma membrane and also accumulates in the nucleus and chloroplasts. Substitution of Gly2 within the predicted N-myristoylation motif strongly reduced plasma membrane association and increased the chloroplast-associated signal of C4–GFP, supporting an important role for the Gly2-containing N-terminal region in membrane targeting and suggesting that chloroplast-associated targeting becomes more prominent when Gly2-dependent cell-peripheral association is disrupted. Expression of PYLCV C4 from a potato virus X (PVX; Potexvirus ecspotati)-based vector in Nicotiana benthamiana induced severe developmental alterations, including mosaic, yellowing, leaf curling, stem deformation, and plant death, without affecting PVX RNA accumulation. PYLCV C4 did not behave as a strong suppressor of local or systemic post-transcriptional gene silencing (PTGS) under the conditions tested, although a possible delay in the spread of GFP silencing was observed in N. benthamiana 16c plants. Together, these findings provide the first experimental characterization of PYLCV C4 and establish a framework for future mechanistic studies addressing how this protein may contribute to host developmental perturbation and PYLCV–plant interactions.