Reversible S-palmitoylation of C4 protein encoded by TYLCCxV orchestrates geminiviral pathogenesis.

Cysteine palmitoylation (S-palmitoylation or S-acylation) is a reversible post-translational modification dynamically controlled by opposing enzymes: palmitoyl acyltransferases (PATs) and depalmitoylases. Despite its established roles in other systems, the mechanistic details of S-acylation in plants, particularly its spatiotemporal regulation during plant-virus interactions, remain poorly understood, largely due to the lack of validated enzyme-substrate pairs. Using the geminivirus tomato yellow leaf curl Chuxiong virus (TYLCCxV) as a model, we show that the viral effector C4 undergoes S-palmitoylation at Cys-4, a modification essential for its plasma membrane anchorage and subsequent mediation of viral pathogenesis. NbPAT4, a palmitoyl acyltransferase from Nicotiana benthamiana, catalyzes C4 S-palmitoylation, promoting its membrane localization, protein stability, and viral infection. Conversely, the C4S substitution (C4C4S) abolishes S-palmitoylation, leading to cytoplasmic redistribution, protein destabilization, and impaired viral pathogenicity. We further identify NbABHD6 as a depalmitoylase that interacts with C4 and catalyzes its S-depalmitoylation, triggering C4 degradation via the 26S proteasome pathway. This study reveals a regulatory axis in plant-geminivirus interactions, identifying NbPAT4 and NbABHD6 as antagonistic enzymes that dynamically regulate the stoichiometry of C4 S-acylation. These findings support a host-pathogen enzymatic tug-of-war model in which competitive S-palmitoylation homeostasis governs viral subcellular trafficking and pathogenicity.