The research, conducted by Hasan Zeitooni and Masoud Shams-Bakhsh at Tarbiat Modares University in Tehran together with Rosa Lozano-Durán of Eberhard Karls University Tübingen, the Max Planck Institute for Plant Breeding Research, and the GreenRobust Cluster of Excellence, was published in Virology Journal. Because PYLCV was only recently identified and assigned to the genus Pylecuvirus as the species Pylecuvirus petroselini, essentially nothing was known about how its individual proteins behave inside plant cells. The team set out to fill that gap by focusing on C4, a protein of just 85 amino acids whose biological activities and cellular distribution had remained entirely uncharacterized.

The investigation began with comparative sequence and bioinformatic analysis of the PYLCV C4 protein. Even at only 85 residues, the protein carries an impressive collection of candidate targeting features. The computational screen identified a predicted N-terminal myristoylation motif—a lipid attachment signal that in many viral and cellular proteins anchors them to membranes—a putative palmitoylation site that could reinforce membrane association, motifs potentially related to nuclear trafficking including nuclear localization signals and a nuclear export signal, and a chloroplast transit peptide that could direct the protein into plastids. Such a combination of targeting determinants in a protein this small hints at a multifaceted role during infection, consistent with the pleiotropic effects that C4 proteins from related geminiviruses exert on their hosts.

To test these predictions experimentally, the researchers turned to confocal microscopy. They fused the C4 protein to green fluorescent protein and expressed the construct in plant tissue, allowing them to track exactly where the protein accumulates within the cell. The imaging revealed that PYLCV C4–GFP localizes prominently to the plasma membrane, the boundary between the cell interior and its surroundings, and also accumulates in two additional compartments: the nucleus, where many geminivirus proteins carry out their manipulations of host gene expression and cell cycle control, and the chloroplasts, the photosynthetic organelles that are frequent targets of viral effectors.

A key experiment followed from the predicted myristoylation motif. N-myristoylation is a covalent modification in which a myristoyl lipid group is attached to a glycine residue at position two of the protein, a process that typically requires the removal of the initiator methionine and is essential for membrane anchoring. By substituting the glycine at position 2, the team created a mutant form of C4 and examined its localization. The result was striking: loss of Gly2 strongly reduced the plasma membrane association of the C4–GFP fusion and, at the same time, increased the chloroplast-associated signal. This finding supports an important role for the Gly2-containing N-terminal region in directing the protein to the cell periphery, and it suggests a competitive relationship between targeting pathways—when Gly2-dependent membrane association is disrupted, chloroplast targeting becomes more prominent. In other words, the same short protein can be routed to different destinations depending on whether its lipid modification site is intact.

Localization studies alone cannot establish pathogenicity, so the researchers next asked what happens when the protein is produced in plants at physiological relevance. They expressed PYLCV C4 from a potato virus X–based vector in Nicotiana benthamiana, a widely used experimental host in plant virology. PVX is an RNA virus whose genome can be engineered to carry additional sequences, providing a convenient system to test whether a heterologous protein induces symptoms. The outcome was dramatic. Plants expressing PYLCV C4 developed severe developmental alterations, including mosaic patterns, yellowing, leaf curling, stem deformation, and in the most extreme cases, plant death. Critically, these symptoms arose without any change in PVX RNA accumulation, demonstrating that the damage was caused by the C4 protein itself rather than by an alteration in the vector virus’s replication. This separation of protein effect from viral accumulation is a hallmark of a genuine pathogenicity determinant.

Given that many geminivirus C4 proteins function as suppressors of RNA silencing—a cornerstone of the plant antiviral immune response—the team also examined whether PYLCV C4 interferes with post-transcriptional gene silencing, or PTGS. They employed standard silencing suppression assays, monitoring both local and systemic silencing of a reporter transgene in N. benthamiana, including the well-known 16c line that carries a green fluorescent protein transgene used to visualize silencing spread. Under the conditions tested, PYLCV C4 did not behave as a strong suppressor of local or systemic PTGS. However, the researchers observed a possible delay in the spread of GFP silencing in 16c plants, leaving open the possibility that the protein has a subtle or context-dependent effect on silencing mobility rather than a robust suppressor activity of the kind seen with classic viral silencing suppressors.

The combination of results paints a picture of a compact but multifunctional protein. PYLCV C4 is a membrane-associated factor whose N-terminal lipidation motif governs its subcellular distribution, with an apparent routing switch between the plasma membrane and the chloroplasts controlled by a single glycine residue. When expressed in plants, it is sufficient to cause severe developmental perturbation on its own, echoing the symptoms associated with the natural disease. At the same time, its weak apparent activity as a silencing suppressor distinguishes it from some of its homologs in other geminivirus genera, suggesting that different C4 proteins may achieve pathogenicity through overlapping but non-identical mechanisms.

These findings carry broader implications for understanding geminivirus evolution and host manipulation. The family Geminiviridae encompasses many genera, and their C4/AC4 proteins have diversified while retaining core functions as symptom determinants. Comparative analysis across these genera, as undertaken here with a dataset of C4/AC4 and C3 protein homologs from twelve genera, helps place PYLCV within that evolutionary landscape. The discovery that a single point of lipidation—the glycine at position two—acts as a switch between membrane and chloroplast targeting offers a mechanistic handle for future studies dissecting how subcellular localization translates into developmental reprogramming. It also raises questions about which host proteins C4 engages at each destination, whether chloroplast-associated C4 contributes to the yellowing symptoms typical of the disease, and whether nuclear accumulation supports interactions with cell cycle regulators.

As the first experimental characterization of any PYLCV protein, this work establishes a framework for future mechanistic studies of how a recently emerged geminivirus interacts with its hosts. The methodology—combining comparative sequence analysis, confocal microscopy, site-directed mutagenesis, heterologous expression, and silencing assays—provides a template that can now be extended to the virus’s remaining proteins and to PYLCV’s interactions with parsley and experimental model hosts. For a pathogen family whose members continue to emerge and recombine at alarming rates, understanding the molecular toolkit of each new virus is an essential step toward anticipating and managing the diseases they cause.

Subject of Research: Functional characterization of the C4 pathogenicity protein of parsley yellow leaf curl virus

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

Article References: Zeitooni, H., Lozano-Durán, R., & Shams-Bakhsh, M. (2026). Characterization of the C4 protein encoded by parsley yellow leaf curl virus. Virology Journal. https://doi.org/10.1186/s12985-026-03309-9

Image Credits: AI Generated

DOI: 10.1186/s12985-026-03309-9

Keywords: geminivirus, PYLCV, C4 protein, myristoylation, plasma membrane, chloroplast, Nicotiana benthamiana, potato virus X, pathogenicity, gene silencing, symptom determinants, plant virology