Viral tug-of-war: palmitoylation switch governs geminivirus infection in plants

A single fatty acid, attached to and removed from one cysteine residue on a viral protein, may determine whether a devastating plant virus runs rampant through a crop or stalls almost before it begins. That is the central finding of a new study on tomato yellow leaf curl Chuxiong virus (TYLCCxV), a monopartite begomovirus that causes severe leaf curling, crumpling, chlorosis and stunting in infected plants. Researchers report that the viral C4 protein, a well-established symptom determinant and immune suppressor, undergoes reversible S-palmitoylation at cysteine residue 4, and that two opposing host enzymes in Nicotiana benthamiana fight for control of this modification. The work, published in Stress Biology, provides the first full characterization of an S-acylation cycle during a plant-geminivirus interaction and proposes a new model of host-pathogen conflict fought at the level of post-translational chemistry.

S-palmitoylation, also called S-acylation, is the covalent attachment of a saturated 16-carbon palmitate to a cysteine residue through a thioester bond. Unlike other lipid modifications, the thioester linkage is intrinsically unstable, which makes the modification reversible and dynamically regulated by two antagonistic enzyme families: palmitoyl acyltransferases (PATs), which carry a conserved DHHC catalytic motif and add palmitoyl groups, and depalmitoylases, which hydrolyze the thioester bond and strip acyl groups away. In humans, 23 PATs have been identified and many are implicated in disease, but the enzymology of S-acylation in plants remains comparatively underexplored. Arabidopsis encodes 24 PATs with distinct subcellular localizations, and S-palmitoylated proteins have been catalogued in rice, maize and soybean, yet validated enzyme-substrate pairs in plant-virus systems have been scarce, limiting mechanistic insight into how this modification shapes infection.

The research team, led by Yan Xie and Xueping Zhou of Zhejiang University together with colleagues, began by establishing where TYLCCxV C4 resides inside plant cells. When C4 fused to green fluorescent protein was expressed in N. benthamiana leaf cells, fluorescence accumulated predominantly at the cytoplasm and plasma membrane, with weak nuclear signal. Subcellular fractionation experiments reinforced the picture: C4 co-sedimented with a plasma membrane marker in the membrane-enriched pellet fraction, while free GFP stayed in the soluble supernatant. Bioinformatic prediction tools suggested that C4 lacks a transmembrane domain but carries two candidate lipid modifications, N-myristoylation at glycine 2 and S-acylation at cysteine 4, hinting that a fatty anchor rather than a hydrophobic helix might explain the membrane association.

To test that hypothesis directly, the researchers deployed the biotin-switch assay, a technique in which hydroxylamine selectively cleaves thioester bonds and the newly freed cysteines are tagged with biotin for detection. A strong S-acylation signal appeared in hydroxylamine-treated samples of C4 but not in untreated controls. Quantitative mass spectrometry using Q-Exactive liquid chromatography-tandem mass spectrometry then pinpointed the modification to cysteine 4. When the team substituted that cysteine with serine, generating a mutant called C4(C4S), the S-acylation signal vanished entirely. The consequences were striking: the mutant redistributed to the cytoplasm and nucleus, and its protein accumulation collapsed to roughly a tenth of wild-type levels, even though semi-quantitative RT-PCR showed that transcript abundance was unchanged. Treating plants with 2-bromopalmitate, a chemical inhibitor of palmitoylation, produced the same dual effect, driving C4 out of the membrane and reducing its steady-state abundance without touching its mRNA.

The functional stakes became clear in infection experiments. When C4 or the palmitoylation-deficient C4(C4S) was expressed from a potato virus X vector, plants receiving wild-type C4 developed systemic upward leaf curling and stem elongation by 14 days post-inoculation, while plants receiving the mutant showed only mild chlorotic symptoms indistinguishable from the empty-vector control. Western blots confirmed that C4 protein levels and PVX coat protein accumulation were comparable across treatments, ruling out differences in expression or vector fitness. More importantly, the team engineered an infectious TYLCCxV clone carrying the same C4S mutation. Plants inoculated with the mutant virus developed dramatically milder symptoms than those infected with wild-type TYLCCxV, and Southern blot analysis revealed weak viral DNA signals at 15 days that diminished further by 30 days. S-acylation of a single cysteine, in other words, is a major determinant of geminiviral pathogenicity and accumulation.

Having established that the modification matters, the researchers went hunting for the enzymes that control it. Screening four candidate palmitoyl acyltransferases from N. benthamiana with a yeast split-ubiquitin assay, they found that NbPAT4 and NbPAT6 interacted with C4. They focused on NbPAT4 and confirmed the interaction in plant cells using bimolecular fluorescence complementation, which reconstituted yellow fluorescent protein signal at the cytomembrane, and co-immunoprecipitation, which pulled down NbPAT4 specifically with C4 but not with free GFP. Genetic evidence followed: in CRISPR-generated NbPAT4 knockout plants, TYLCCxV induced only mild leaf curling and accumulated less viral DNA, whereas plants overexpressing NbPAT4 developed more severe symptoms, including vein swelling and leaf clustering, and carried higher viral loads. Biotin-switch measurements placed the enzyme at the chemistry itself: C4 S-acylation levels fell to about half of wild type in knockout plants and rose to nearly double in overexpression lines.

Palmitoylation is a two-way street, so the team also screened 19 candidate depalmitoylases, including four acyl-protein thioesterases and fifteen alpha/beta hydrolase domain-containing proteins, for interactors of C4. Only one emerged: NbABHD6, a 274-amino-acid enzyme containing a conserved alpha/beta hydrolase domain with a catalytic triad of serine 147, aspartate 212 and histidine 241, mirroring the architecture of the mammalian ABHD17 family. Yeast split-ubiquitin, bimolecular fluorescence complementation and co-immunoprecipitation all confirmed a specific interaction with C4. Functionally, co-expression of NbABHD6 reduced C4 S-acylation levels to roughly 0.6 of control values and cut C4 protein accumulation by half, again without altering transcript levels. When the three catalytic residues were simultaneously mutated to alanine, the resulting NbABHD6(mSDH) mutant still bound C4 but lost all ability to reduce its S-acylation or abundance, demonstrating that the depalmitoylase activity, not mere physical association, drives the effect.

The fate of depalmitoylated C4 proved to be destruction by the ubiquitin-proteasome system. Treatment with the proteasome inhibitor MG132 increased C4 accumulation, while inhibitors of autophagy, E64d and 3-methyladenine, had no effect, indicating that C4 is normally turned over through the 26S proteasome. When NbABHD6 was co-expressed, C4 levels dropped sharply in vehicle-treated leaves, but MG132 partially rescued the protein, and the catalytically dead mutant had no such effect. The authors note that even with the proteasome blocked, NbABHD6 still reduced C4 below control levels, hinting at additional regulatory mechanisms consistent with C4’s multifunctional nature. They also observed that NbABHD6 did not markedly change C4’s membrane localization, likely because the myristoylation site at glycine 2 provides a second, independent lipid anchor, a reminder that subcellular localization alone cannot reliably report S-acylation status.

The findings sit within a growing appreciation that S-acylation is a battleground in plant-microbe encounters. Rice stripe virus disrupts the S-palmitoylation of host Remorin 1 and shunts it into autophagy; potato mop-top virus hijacks the acylated stress sensor HIPP26 for long-distance movement; and the C4 protein of beet severe curly top virus uses its own S-acylation to engage the receptor kinase CLAVATA 1. In mammals, an elegant palmitoylation cycle on STAT3, catalyzed by DHHC7 and reversed by APT2, governs inflammatory signaling, illustrating how dynamic acylation can act as a molecular switch. The new study extends this logic to plant antiviral immunity and, notably, identifies only the second defined plant depalmitoylase-viral substrate pair, after the recent report that Arabidopsis ABAPT3 targets the C4 protein of beet severe curly top virus.

The authors frame their results as a host-pathogen enzymatic tug-of-war aligned with the recently proposed concept of apoplastic interactive balance. Early in infection, TYLCCxV exploits the host’s own S-acylation machinery: NbPAT4 palmitoylates C4, anchoring it at the plasma membrane, stabilizing the protein and amplifying viral accumulation. The plant counters by deploying NbABHD6, whose depalmitoylase activity destabilizes C4 and channels it into proteasomal degradation, suppressing infection. The equilibrium between these opposing enzymes sets the stoichiometry of C4 acylation and, with it, the trajectory of disease. Open questions remain, including whether C4 recruits specific E3 ubiquitin ligases and how S-acylation crosstalks with myristoylation, phosphorylation and ubiquitination. But the identification of NbPAT4 and NbABHD6 as antagonistic regulators offers a concrete enzymatic axis, and potentially a target for engineering crop resistance to one of agriculture’s most damaging virus families.

Subject of Research: Reversible S-palmitoylation of the geminiviral C4 protein and its regulation by host palmitoylation enzymes during plant-virus interaction

Article Title: Reversible S-palmitoylation of C4 protein encoded by TYLCCxV orchestrates geminiviral pathogenesis

Article References: Xie, Y., Zhao, M., Liu, X., Yan, J., Yang, W., Chen, Y., Yang, M., Wang, X., Fu, S., & Zhou, X. (2026). Reversible S-palmitoylation of C4 protein encoded by TYLCCxV orchestrates geminiviral pathogenesis. Stress Biology, 6(1), Article 35. https://doi.org/10.1007/s44154-026-00308-2

Image Credits: AI Generated

DOI: 10.1007/s44154-026-00308-2

Keywords: S-palmitoylation, geminivirus, C4 protein, TYLCCxV, palmitoyl acyltransferase, depalmitoylase, NbPAT4, NbABHD6, Nicotiana benthamiana, plant-virus interaction, 26S proteasome, post-translational modification