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

Abstract

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 (C4^C4S) 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.

Keywords

S-palmitoylation / Palmitoyl acyltransferases / Depalmitoylases / Geminivirus / Pathogenicity / Plant-virus interaction

References

[1] Batistic O. Genomics and localization of the Arabidopsis DHHC-cysteine-rich domain S-acyltransferase protein family. Plant Physiol, 2012, 160(3): 1597-1612

[2] Bi HP, Fan WJ, Zhang P. C4 protein of Sweet Potato Leaf Curl Virus regulates brassinosteroid signaling pathway through interaction with AtBIN2 and affects male fertility in Arabidopsis. Front Plant Sci, 2017, 8 Article ID: 1689

[3] Bürger M, Willige BC, Chory J. A hydrophobic anchor mechanism defines a deacetylase family that suppresses host response against YopJ effectors. Nat Commun, 2017, 8 Article ID: 2201

[4] Carluccio AV, Prigigallo MI, Rosas-Diaz T, Lozano-Duran R, Stavolone L. S-acylation mediates mungbean yellow mosaic virus AC4 localization to the plasma membrane and in turns gene silencing suppression. PLoS Pathog, 2018, 14(8): Article ID: e1007207

[5] Chen DQ, Hao FS, Mu HQ, Ahsan N, Thelen JJ, Stacey G. S-acylation of P2K1 mediates extracellular ATP-induced immune signaling in Arabidopsis. Nat Commun, 2021, 12(1): Article ID: 2750

[6] Cowan GH, Roberts AG, Jones S, Kumar P, Kalyandurg PB, Gil JF, Savenkov EI, Hemsley PA, Torrance L. Potato mop-top virus co-opts the stress sensor HIPP26 for long-distance movement. Plant Physiol, 2018, 176(3): 2052-2070

[7] De I, Sadhukhan S. Emerging roles of DHHC-mediated protein S-palmitoylation in physiological and pathophysiological context. Eur J Cell Biol, 2018, 97(5): 319-338

[8] Fu S, Xu Y, Li CY, Li Y, Wu JX, Zhou XP. Rice stripe virus interferes with S-acylation of remorin and induces its autophagic degradation to facilitate virus infection. Mol Plant, 2018, 11(2): 269-287

[9] Gao J, Huang G, Chen X, Zhu YX. Protein S-acyl transferase 13/16 modulate disease resistance by S-acylation of the nucleotide binding, leucine-rich repeat protein R5L1 in Arabidopsis. J Integr Plant Biol, 2022, 64(9): 1789-1802

[10] Hemsley PA. S-acylation in plants: an expanding field. Biochem Soc Trans, 2020, 48: 529-536

[11] Hemsley PA, Kemp AC, Grierson CS. The TIP GROWTH DEFECTIVE1 s-acyl transferase regulates plant cell growth in Arabidopsis. Plant Cell, 2005, 17: 2554-2563

[12] Hemsley PA, Taylor L, Grierson CS. Assaying protein palmitoylation in plants. Plant Methods, 2008, 4 Article ID: 2

[13] Hurst C, Turnbull D, Xhelilaj K, Myles S, Pflughaupt R, Kopischke M, Davies P, Jones S, Robatzek S, Zipfel C, et al.. S-acylation stabilizes ligandinduced receptor kinase complex formation during plant pattern-triggered immune signaling. Curr Biol, 2023, 33(8): 1588-1596

[14] Ismayil A, Haxim Y, Wang YJ, Li HG, Qian LC, Han T, Chen TY, Jia Q, Liu AY, Zhu SB, et al.. Cotton leaf curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase. PLoS Pathog, 2018, 14(8): Article ID: e1007282

[15] Lai JB, Chen H, Teng KL, Zhao QZ, Zhang ZH, Li Y, Liang LM, Xia R, Wu YR, Guo HS, et al.. RKP, a RING finger E3 ligase induced by BSCTV C4 protein, affects geminivirus infection by regulation of the plant cell cycle. Plant J, 2009, 57: 905-917

[16] Lennicke C, Cochemé HM. Redox metabolism: ROS as specific molecular regulators of cell signaling and function. Mol Cell, 2021, 81: 3691-3707

[17] Li YX, Lin JZ, Li L, Peng YC, Wang WW, Zhou YB, Tang DY, Zhao XY, Yu F, Liu XM. DHHC cysteine-rich domain S-acyltransferase protein family in rice: organization, phylogenetic relationship and expression pattern during development and stress. Plant Syst Evol, 2016, 302(10): 1405-1417

[18] Li HY, Zeng RX, Chen Z, Liu XS, Cao ZD, Xie Q, Yang CW, Lai JB. S-acylation of a geminivirus C4 protein is essential for regulating the CLAVATA pathway in symptom determination. J Exp Bot, 2018, 69(18): 4459-4468

[19] Li FF, Zhang MZ, Zhang CW, Zhou XP. Nuclear autophagy degrades a geminivirus nuclear protein to restrict viral infection in solanaceous plants. New Phytol, 2020, 225(4): 1746-1761

[20] Li XQ, Zhao M, Yang WY, Zhou XP, Xie Y. The C4 protein of TbLCYnV promotes SnRK1 β2 degradation via the autophagy pathway to enhance viral infection in N. benthamiana. Viruses, 2024, 16(2): 234

[21] Linder ME, Deschenes RJ. Palmitoylation: policing protein stability and traffic. Nat Rev Mol Cell Biol, 2007, 8: 74-84

[22] Liu XS, Li M, Li Y, Chen ZA, Zhuge C, Ouyang YW, Zhao YW, Lin YX, Xie Q, Yang CW, et al.. An ABHD17-like hydrolase screening system to identify de-S-acylation enzymes of protein substrates in plant cells. Plant Cell, 2021, 33(10): 3235-3249

[23] Liu XS, Chen Z, Huang LT, Ouyang YW, Wang ZY, Wu S, Ye WX, Yu BY, Zhang YH, Yang CW, et al.. Salicylic acid attenuates brassinosteroid signaling via protein de-S-acylation. EMBO J, 2023, 42(13): e112998

[24] Liu JZ, Xu QY, Li WD, Ding XH. Equilibrium in the apoplast: the molecular tug-of-war between plant and pathogen. Mol Plant, 2026, 19: 217-220

[25] Magee T, Seabra MC. Fatty acylation and prenylation of proteins: what’s hot in fat. Curr Opin Cell Biol, 2005, 17(2): 190-196

[26] Martin K, Kopperud K, Chakrabarty R, Banerjee R, Brooks R, Goodin MM. Transient expression in Nicotiana benthamiana fluorescent marker lines provides enhanced definition of protein localization, movement and interactions in planta. Plant J, 2009, 59: 150-162

[27] Medina-Puche L, Tan H, Dogra V, Wu MS, Rosas-Diaz T, Wang LP, Ding X, Zhang D, Fu X, Kim C, et al.. A defense pathway linking plasma membrane and chloroplasts and co-opted by pathogens. Cell, 2020, 182(5): 1109-1124

[28] Mei YZ, Wang YQ, Hu T, Yang XL, Lozano-Duran R, Sunter G, Zhou XP. Nucleocytoplasmic shuttling of Geminivirus C4 protein mediated by phosphorylation and myristoylation is critical for viral pathogenicity. Mol Plant, 2018, 11(12): 1466-1481

[29] Mills-Lujan K, Andrews DL, Chou CW, Deom CM. The roles of phosphorylation and SHAGGY-like protein kinases in Geminivirus C4 protein induced hyperplasia. PLoS ONE, 2015, 10(3): Article ID: e0122356

[30] Mitchell DA, Vasudevan A, Linder ME, Deschenes RJ. Protein palmitoylation by a family of DHHC protein S-acyltransferases. J Lipid Res, 2006, 47: 1118-1127

[31] Porcellato E, González-Sánchez JC, Ahlmann-Eltze C, Elsakka MA, Shapira I, Fritsch J, Navarro JA, Anders S, Russell RB, Wieland FT, et al.. The S-palmitoylome and DHHC-PAT interactome of Drosophila melanogaster S2R⁺ cells indicate a high degree of conservation to mammalian palmitoylomes. PLoS ONE, 2022, 17(8): Article ID: e0261543

[32] Rojas MR, Hagen C, Lucas WJ, Gilbertson RL. Exploiting chinks in the plant’s armor: evolution and emergence of geminiviruses. Annu Rev Phytopathol, 2005, 43: 361-394

[33] Rosas-Diaz T, Zhang D, Fan PF, Wang LP, Ding X, Jiang YL, Jimenez-Gongora T, Medina-Puche L, Zhao XY, Feng ZY, et al.. A virus-targeted plant receptor-like kinase promotes cell-to-cell spread of RNAi. Proc Natl Acad Sci U S A, 2018, 115(6): 1388-1393

[34] Tao XR, Zhou XP, Li GX, Yu JL. The pathogenicity on legumes of cucumber mosaic virus was determined by 243 nucleotides on 2a polymerase gene of viral RNA2. Chin Sci Bull, 2002, 47: 748-750

[35] Varma A, Malathi VG. Emerging geminivirus problem: a serious threat to crop production. Ann Appl Biol, 2003, 142: 145-164

[36] Wei YP, Li JT, Li J, Liu C, Guo XZ, Liu ZR, Zhang LY, Bao SL, Wu XH, Su WH, et al.. Dietary long-chain fatty acids promote colitis by regulating palmitoylation of STAT3 through CD36-mediated endocytosis. Cell Death Dis, 2024, 15(1): Article ID: 60

[37] Won SJ, Cheung See Kit M, Martin BR. Protein depalmitoylases. Crit Rev Biochem Mol Biol, 2018, 53: 83-98

[38] Xie Y, Liu XN, Luo CH, Hu QQ, Che X, Zhao LL, Zhao M, Wu LQ, Ding M. Distinct tomato yellow leaf curl Chuxiong virus isolated from whiteflies and plants in China and its symptom determinant and suppressor of post-transcriptional gene silencing. Virology, 2024, 594 Article ID: 110040

[39] Yang J, Brown MS, Liang G, Grishin NV, Goldstein JL. Identification of the acyltransferase that octanoylates ghrelin, an appetite—stimulating peptide hormone. Cell, 2008, 132(3): 387-396

[40] Yang Y, Hsu JM, Sun LL, Chan LC, Li CW, Hsu JL, Wei YK, Xia WY, Hou JW, Qiu YF, et al.. Palmitoylation stabilizes Pd-L1 to promote breast tumor growth. Cell Res, 2019, 29(1): 83-86

[41] Yang M, Ismayil A, Gao T, Ye ZH, Yue N, Wu J, Zheng XY, Li YQ, Wang Y, Hong YG, et al.. Cotton leaf curl Multan virus C4 protein suppresses autophagy to facilitate viral infection. Plant Physiol, 2023, 193(1): 708-720

[42] Yao H, Lan J, Li CS, Shi HB, Brosseau JP, Wang HB, Lu HJ, Fang CY, Zhang Y, Liang LX, et al.. Author correction: inhibiting Pd-L1 palmitoylation enhances T-cell immune responses against tumours. Nat Biomed Eng, 2019, 3(5): 414

[43] Ye QY, Zheng LH, Liu P, Liu QW, Ji T, Liu JL, Gao YJ, Liu L, Dong JL, Wang T. The S-acylation cycle of transcription factor MtNAC80 influences cold stress response in Medicago truncatula. Plant Cell, 2024, 36: 2629-2651

[44] Yokoi N, Fukata Y, Sekiya A, Murakami T, Kobayashi K, Fukata M. Identification of PSD-95 depalmitoylating enzymes. J Neurosci, 2016, 36(24): 6431-6444

[45] Yuan XW, Zhang SZ, Sun MH, Liu SY, Qi BX, Li XZ. Putative DHHC-cysteine-rich domain S-acyltransferase in plants. PLoS ONE, 2013, 8(10): Article ID: e75985

[46] Yue N, Jiang ZH, Zhang X, Li ZG, Wang XT, Wen ZY, Gao ZY, Pi QL, Zhang YL, Wang XB, et al.. Palmitoylation of γb protein directs a dynamic switch between barley stripe mosaic virus replication and movement. EMBO J, 2022, 41(13): e110060

[47] Zaballa ME, van der Goot FG. The molecular era of protein S-acylation: spotlight on structure, mechanisms, and dynamics. Crit Rev Biochem Mol Biol, 2018, 53: 420-451

[48] Zareba-Koziol M, Bartkowiak-Kaczmarek A, Figiel I, Krzystyniak A, Wojtowicz T, Bijata M, Wlodarczyk J. Stress-induced changes in the S-palmitoylation and S-nitrosylation of synaptic proteins. Mol Cell Proteomics, 2019, 18(10): 1916-1938

[49] Zhang MM, Zhou LX, Xu YJ, Yang M, Xu YL, Komaniecki GP, Kosciuk T, Chen X, Lu X, Zou XP, et al.. A STAT3 palmitoylation cycle promotes TH17 differentiation and colitis. Nature, 2020, 586(7829): 434-439

[50] Zhao YW, Li ZG, Wang ZY, Huang LT, Li GD, Liu XS, Yuan MQ, Huang W, Ling LS, Yang CW, et al.. Improvement of plant resistance to geminiviruses via protein de-S-acylation. Stress Biol, 2024, 4: 23

Funding

National Natural Science Foundation of China(32172357)

National Natural Science Foundation of China (NSFC) International Cooperation and Exchange Program(32161143008)

RIGHTS & PERMISSIONS

The Author(s)