The E3 ubiquitin ligase SlATL2 suppresses tomato immunity by promoting SlCSN5a degradation during Pseudomonas syringae pv. tomato DC3000 infection

Abstract

Plant immunity involves complex regulatory mechanisms that mediate the activation of defense responses against pathogens. Protein degradation via ubiquitination plays a crucial role in modulating these defenses, with E3 ubiquitin ligases functioning as central regulators. This study investigates the role of SlATL2, an ARABIDOPSIS TÓXICOS EN LEVADURA (ATL)-type E3 ubiquitin ligase localized in the plasma membrane, in the immune response of tomato plants against Pseudomonas syringae pv. tomato (Pst) DC3000. Our findings demonstrate that SlATL2 expression is induced upon Pst DC3000 infection and treatment with defense hormones salicylic acid and jasmonic acid. Functionally, SlATL2 negatively regulates immune responses, impairing resistance to Pst DC3000 and suppressing flg22-triggered immunity. In addition, SlATL2 limits pathogen-induced reactive oxygen species and callose accumulation by targeting the COP9 signalosome subunit 5a (SlCSN5a), a key positive regulator of tomato defense responses against Pst DC3000. This interaction, which occurs via the N-terminal residue of SlATL2, results in the ubiquitination and 26S proteasomal degradation of SlCSN5a, thereby suppressing SA-dependent expression of defense response genes associated and limiting reactive oxygen species production. This work sheds light on the molecular mechanism through which the E3 ubiquitin ligase SlATL2 attenuates tomato immune responses by targeting a COP9 signalosome subunit for degradation. These discoveries deepen our insights into the post-translational mechanisms governing plant immune responses and provide fresh opportunities to bolster crop resistance against bacterial pathogens.

Acknowledgements

This research was supported by the National Natural Science Foundation of China under grant no. 31972415 and the Zhejiang Provincial Natural Science Foundation of China under grant no. LY21C140003.

Author contributions

D.L., F.S., and Y.D. conceived the study and designed the experiments; Y.D., X.L., Y.H., L.Z., and Y.B. conducted the research; D.L., Y.D., and F.S. analyzed the data; D.L. and F.S wrote, edited and revised the manuscript. All authors read and approved of its content.

Data availability

All study data are incorporated in the submitted article.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Supplementary Data

Supplementary data is available at Horticulture Research online.

References

[1] Zhou JM, Zhang Y. Plant immunity: danger perception and signaling. Cell. 2020; 181:978-89

[2] Jones JD, Dangl JL. The plant immune system. Nature. 2006; 444: 323-9

[3] Saijo Y, Loo EP, Yasuda S.Pattern recognition receptors and signaling in plant-microbe interactions. Plant J. 2018; 93: 592-613

[4] Couto D, Zipfel C. Regulation of pattern recognition receptor signalling in plants. Nat Rev Immunol. 2016; 16:537-52

[5] Wang W, Feng B, Zhou JM. et al. Plant immune signaling: advanc-ing on two frontiers. J Integr Plant Biol. 2020; 62:2-24

[6] Tang D, Wang G, Zhou JM. Receptor kinases in plant-pathogen interactions: more than pattern recognition. Plant Cell. 2017; 29: 618-37

[7] Monteiro F, Nishimura MT. Structural, functional, and genomic diversity of plant NLR proteins: an evolved resource for rational engineering of plant immunity. Annu Rev Phytopathol. 2018; 56: 243-67

[8] Cesari S. Multiple strategies for pathogen perception by plant immune receptors. New Phytol. 2018; 219:17-24

[9] Cui H, Tsuda K, Parker JE. Effector-triggered immunity: from pathogen perception to robust defense. Annu Rev Plant Biol. 2015; 66:487-511

[10] Yuan M, Ngou BPM, Ding P. et al. PTI-ETI crosstalk: an integrative view of plant immunity. Curr Opin Plant Biol. 2021; 62:102030

[11] Yuan M, Jiang Z, Bi G. et al. Pattern-recognition receptors are required for NLR-mediated plant immunity. Nature. 2021; 592: 105-9

[12] Ngou BPM, Ahn HK, Ding P. et al. Mutual potentiation of plant immunity by cell-surface and intracellular receptors. Nature. 2021; 592:110-5

[13] Ciechanover A. The unravelling of the ubiquitin system. Nat Rev Mol Cell Biol. 2015; 16:322-4

[14] Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem. 1998; 67:425-79

[15] Smalle J, Vierstra RD. The ubiquitin 26S proteasome proteolytic pathway. Annu Rev Plant Biol. 2004; 55:555-90

[16] Kim D, Jeon SJ, Hong JK. et al. The auto-regulation of ATL 2 E3 ubiquitin ligase plays an important role in the immune response against Alternaria brassicicola in Arabidopsis thaliana. Int J Mol Sci. 2024; 25:2388

[17] Serrano M, Guzman P. Isolation and gene expression analysis of Arabidopsis thaliana mutants with constitutive expression of ATL2, an early elicitor-response RING-H2 zinc-finger gene. Genetics. 2004; 167:919-29

[18] Deng F, Guo T, Lefebvre M. et al. Expression and regulation of ATL9, an E3 ubiquitin ligase involved in plant defense. PLoS One. 2017; 12:e0188458

[19] Kong F, Guo T, Ramonell KM. Arabidopsis Toxicos en Levadura 12 (ATL12): a gene involved in chitin-induced, hormone-related and NADPH oxidase-mediated defense responses. J Fungi (Basel). 2021; 7:7

[20] Maekawa S, Inada N, Yasuda S. et al. The carbon/nitrogen regu-lator ARABIDOPSIS TOXICOS EN LEVADURA31 controls papilla formation in response to powdery mildew fungi penetration by interacting with SYNTAXIN OF PLANTS121 in Arabidopsis. Plant Physiol. 2014; 164:879-87

[21] Maekawa S, Sato T, Asada Y. et al. The Arabidopsis ubiquitin ligases ATL31 and ATL6 control the defense response as well as the carbon/nitrogen response. Plant Mol Biol. 2012; 79:217-27

[22] LiuX, ZhouY, DuM. et al. The calcium-dependent protein kinase CPK28 is targeted by the ubiquitin ligases ATL31 and ATL6 for proteasome-mediated degradation to fine-tune immune signal-ing in Arabidopsis. Plant Cell. 2022; 34:679-97

[23] Takai R, Matsuda N, Nakano A. et al. EL5, a rice N-acetylchitooligosaccharide elicitor-responsive RING-H2 finger protein, is a ubiquitin ligase which functions in vitro in co-operation with an elicitor-responsive ubiquitin-conjugating enzyme, OsUBC5b. Plant J. 2002; 30:447-55

[24] Ariani P, Regaiolo A, Lovato A. et al. Genome-wide character-isation and expression profile of the grapevine ATL ubiquitin ligase family reveal biotic and abiotic stress-responsive and development-related members. Sci Rep. 2016; 6:38260

[25] Lin L, Chen Q, Yuan K. et al. PbrATL18, an E3 ubiquitin ligase identified by genome-wide identification, is a positive factor in pear resistance to drought and Colletotrichum fructicola infection. Hortic Plant J. 2023; 10:698-712

[26] Zhong C, Ren Y, Qi Y. et al. PAMP-responsive ATL gene StRFP1 and its orthologue NbATL60 positively regulate Phytophthora infestans resistance in potato and Nicotiana benthamiana. Plant Sci. 2018; 270:47-57

[27] Takahashi H, Hase S, Kanayama Y. et al. Identification of a protein that interacts with LeATL6 ubiquitin-protein ligase E3 upregulated in tomato treated with elicitin-like cell wall pro-teins of Pythium oligandrum. J Phytopathol. 2010; 158:132-6

[28] Hondo D, Hase S, Kanayama Y. et al. The LeATL6-associated ubiquitin/proteasome system may contribute to fungal elicitor-activated defense response via the jasmonic acid-dependent signaling pathway in tomato. Mol Plant-Microbe Interact. 2007; 20: 72-81

[29] Qin N, Xu D, Li J. et al. COP9 signalosome: discovery, conservation, activity, and function. J Integr Plant Biol. 2020; 62:90-103

[30] Stratmann JW, Gusmaroli G. Many jobs for one good COP - the COP9 signalosome guards development and defense. Plant Sci. 2012;185-186:50-64

[31] Hua Z, Vierstra RD. The cullin-RING ubiquitin-protein ligases. Annu Rev Plant Biol. 2011; 62:299-334

[32] Cope GA, Deshaies RJ. COP9 signalosome: a multifunctional regulator of SCF and other cullin-based ubiquitin ligases. Cell. 2003; 114:663-71

[33] Jin D, Li B, Deng XW. et al. Plant COP9 signalosome subunit 5, CSN5. Plant Sci. 2014; 224:54-61

[34] Stuttmann J, Lechner E, Guerois R. et al. COP9 signalosome-and 26S proteasome-dependent regulation of SCFTIR1 accumulation in Arabidopsis. JBiolChem. 2009; 284:7920-30

[35] Wang X, Feng S, Nakayama N. et al. The COP9 signalosome interacts with SCF UFO and participates in Arabidopsis flower development. Plant Cell. 2003; 15:1071-82

[36] Lozano-Duran R, Rosas-Diaz T, Gusmaroli G. et al. Geminiviruses subvert ubiquitination by altering CSN-mediated derubylation of SCF E3 ligase complexes and inhibit jasmonate signaling in Arabidopsis thaliana. Plant Cell. 2011; 23:1014-32

[37] Dohmann EM, Nill C, Schwechheimer C. DELLA proteins restrain germination and elongation growth in Arabidopsis thaliana COP9 signalosome mutants. Eur J Cell Biol. 2010; 89:163-8

[38] Feng S, Ma L, Wang X. et al. The COP9 signalosome interacts physically with SCF COI1 and modulates jasmonate responses. Plant Cell. 2003; 15:1083-94

[39] Franciosini A, Lombardi B, Iafrate S. et al. The Arabidopsis COP9 SIGNALOSOME INTERACTING F-BOX KELCH 1 protein forms an SCF ubiquitin ligase and regulates hypocotyl elongation. Mol Plant. 2013; 6:1616-29

[40] Zhang Y, Feng S, Chen F. et al. Arabidopsis DDB1-CUL4 ASSOCI-ATED FACTOR1 forms a nuclear E3 ubiquitin ligase with DDB1 and CUL 4 that is involved in multiple plant developmental processes. Plant Cell. 2008; 20:1437-55

[41] Gusmaroli G, Figueroa P, Serino G. et al. Role of the MPN subunits in COP9 signalosome assembly and activity, and their regulatory interaction with Arabidopsis Cullin3-based E3 ligases. Plant Cell. 2007; 19:564-81

[42] Dubiel D, Rockel B, Naumann M. et al. DiversityofCOP9sig-nalosome structures and functional consequences. FEBS Lett. 2015; 589:2507-13

[43] Korczeniewska J, Barnes BJ. 5 protein stabil-ity. Mol Cell Biol. 2013; 33:1124-38

[44] Kwok SF, Solano R, Tsuge T. et al. Arabidopsis homologs of a c-Jun coactivator are present both in monomeric form and in the COP9 complex, and their abundance is differentially affected by the pleiotropic COP/det/fus mutations. Plant Cell. 1998; 10:1779-90

[45] Dohmann EM, Kuhnle C, Schwechheimer C. Loss of the CONSTI-TUTIVE PHOTOMORPHOGENIC9 signalosome subunit5 is suffi-cient to cause the cop/det/fus mutant phenotype in Arabidopsis. Plant Cell. 2005; 17:1967-78

[46] Gusmaroli G, Feng S, Deng XW. The Arabidopsis CSN5A and CSN5B subunits are present in distinct COP9 signalosome com-plexes, and mutations in their JAMM domains exhibit differen-tial dominant negative effects on development. Plant Cell. 2004; 16:2984-3001

[47] Mukhtar MS, Carvunis AR, Dreze M. et al. Independently evolved virulence effectors converge onto hubs in a plant immune sys-tem network. Science. 2011; 333:596-601

[48] Bournaud C, Gillet FX, Murad AM. et al. Meloidogyne incognita PASSE-MURAILLE (MiPM) gene encodes a cell-penetrating protein that interacts with the CSN5 subunit of the COP9 signalosome. Front Plant Sci. 2018; 9:904

[49] Zhang H, Wang X, Giroux MJ. et al. Awheat COP9 subunit 5-like gene is negatively involved in host response to leaf rust. Mol Plant Pathol. 2017; 18:125-33

[50] Hind SR, Pulliam SE, Veronese P. et al. The COP9 signalosome controls jasmonic acid synthesis and plant responses to her-bivory and pathogens. Plant J. 2011; 65:480-91

[51] Yang L, Miao M, Lyu H. et al. Genome-wide identification, evo-lution, and expression analysis of RING finger gene family in Solanum lycopersicum. Int J Mol Sci. 2019; 20:4864

[52] Nelson BK, Cai X, Nebenfuhr A. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 2007; 51:1126-36

[53] Veronese P, Nakagami H, Bluhm B. et al. The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens. Plant Cell. 2006; 18:257-73

[54] Zhang Y, Li D, Zhang H. et al. Tomato histone H2B monoubiq-uitination enzymes SlHUB1 and SlHUB2 contribute to disease resistance against Botrytis cinerea through modulating the bal-ance between SA-and JA/ET-mediated signaling pathways. BMC Plant Biol. 2015; 15:252

[55] Gao C, Tang D, Wang W. The role of ubiquitination in plant immunity: fine-tuning immune signaling and beyond. Plant Cell Physiol. 2022; 63:1405-13

[56] Cho SK, Ryu MY, Kim JH. et al. RING E3 ligases: key regulatory elements are involved in abiotic stress responses in plants. BMB Rep. 2017; 50:393-400

[57] Morreale FE, Walden H. Types of ubiquitin ligases. Cell. 2016; 165: 248-248.e1

[58] Wei S, Li X, Gruber MY. et al. COP9 signalosome subunit 5A affects phenylpropanoid metabolism, trichome formation and transcription of key genes of a regulatory tri-protein complex in Arabidopsis. BMC Plant Biol. 2018; 18:134

[59] Schwechheimer C, Deng XW. COP9 signalosome revisited: a novel mediator of protein degradation. Trends Cell Biol. 2001; 11: 420-6

[60] Zhou Y, Li XH, Guo QH. et al. Salt responsive alternative splicing of a RING finger E3 ligase modulates the salt stress tolerance by fine-tuning the balance of COP9 signalosome subunit 5A. PLoS Genet. 2021; 17:e1009898

[61] Lu J, Yu J, Liu P. et al. Ubiquitin-mediated degradation of SlPsbS regulates low night temperature tolerance in tomatoes. Cell Rep. 2024; 43:114757

[62] Wu M, Musazade E, Yang X. et al. ATL protein family: novel regulators in plant response to environmental stresses. J Agric Food Chem. 2023; 71:20419-40

[63] Fei W, Liu Y. Biotrophic fungal pathogens: a critical overview. Appl Biochem Biotechnol. 2023; 195:1-16

[64] Mapuranga J, Zhang N, Zhang L. et al. Infection strategies and pathogenicity of biotrophic plant fungal pathogens. Front Micro-biol. 2022; 13:799396

[65] Mengiste T. Plant immunity to necrotrophs. Annu Rev Phy-topathol. 2012; 50:267-94

[66] Liu C, Liu Q, Mou Z. Redox signaling and oxidative stress in systemic acquired resistance. JExp Bo.t 2024; 75: 4535-48

[67] Singh Y, Nair AM, Verma PK. Surviving the odds: from percep-tion to survival of fungal phytopathogens under host-generated oxidative burst. Plant Commun. 2021; 2:100142

[68] Xin XF, He SY. Pseudomonas syringae pv. Tomato DC3000: a model pathogen for probing disease susceptibility and hormone signal-ing in plants. Annu Rev Phytopathol. 2013; 51:473-98

[69] Wang Y, Li X, Fan B. et al. Regulation and function of defense-related callose deposition in plants. Int J Mol Sci. 2021; 22:2393

[70] Dhawan R, Luo H, Foerster AM. et al. HISTONE MONOUBIQUI-TINATION1 interacts with a subunit of the mediator complex and regulates defense against necrotrophic fungal pathogens in Arabidopsis. Plant Cell. 2009; 21:1000-19

[71] Lee D, Lal NK, Lin ZD. et al. Regulation of reactive oxygen species during plant immunity through phosphorylation and ubiquitination of RBOHD. Nat Commun. 2020; 11:1838

[72] Nie WF, Wang MM, Xia XJ. et al. Silencing of tomato RBOH1 and MPK2 abolishes brassinosteroid-induced H2O2 generation and stress tolerance. Plant Cell Environ. 2013; 36:789-803

[73] Sato T, Maekawa S, Yasuda S. et al. Identification of 14-3-3 proteins as a target of ATL31 ubiquitin ligase, a regulator of the C/N response in Arabidopsis. Plant J. 2011; 68:137-46

[74] Yan Y, Wang H, Bi Y. et al. OsATL32 ubiquitinates the reactive oxygen species-producing OsRac5-OsRbohB module to suppress rice immunity. J Integr Plant Biol. 2024; 66:1459-80

[75] Schulze-Niemand E, Naumann M. The COP9 signalosome: a versatile regulatory hub of Cullin-RING ligases. Trends Biochem Sci. 2023; 48:82-95

[76] Xiaoxia L, Zhang J, Jinkai S. et al. The Salix SmSPR1 involved in light-regulated cell expansion by modulating microtubule arrangement. Front Cell Dev Biol. 2019; 7:309

[77] Wang J, Zhang C, Li H. et al. OsJAB1 positively regulates ascor-bate biosynthesis and negatively regulates salt tolerance due to inhibiting early-stage salt-induced ROS accumulation in rice. Plants (Basel). 2023; 12:12

[78] Fan J, Bai P, Ning Y. et al. The monocot-specific receptor-like kinase SDS2 controls cell death and immunity in Rice. Cell Host Microbe. 2018; 23:498-510.e5

[79] Liu J, Park CH, He F. et al. The RhoGAP SPIN6 associates with SPL11 and OsRac1 and negatively regulates programmed cell death and innate immunity in rice. PLoS Pathog. 2015; 11: e1004629

[80] Li JZ, Gu YL, Zhang W. et al. Pseudomonas syringae lytic trans-glycosylase HrpH interacts with host ubiquitin ligase ATL 2 to modulate plant immunity. Cell Rep. 2025; 44:115145

[81] Shang Y, Wang K, Sun S. et al. COP9 signalosome CSN4 and CSN5 subunits are involved in jasmonate-dependent defense against root-knot nematode in tomato. Front Plant Sci. 2019; 10:1223

[82] Bai X, Huang X, Tian S. et al. RNAi-mediated stable silencing of TaCSN5 confers broad-spectrum resistance to Puccinia striiformis f. sp. tritici. Mol Plant Pathol. 2021; 22:410-21

[83] Zhang C, Fang L, He F. et al. Ubiquitination of OsCSN5 by OsPUB45 activates immunity by modulating the OsCUL3a-OsNPR1 module. Sci Adv. 2025;11:eadr2441

[84] Cui K-C, Liu M, Ke G-H. et al. Transient silencing of VvCSN5 enhances powdery mildew resistance in grapevine (Vitis vinifera). Plant Cell Tissue Organ Cult. 2021; 146:621-33

[85] Yang W, Zhai H, Wu F. et al. Peptide REF1 is a local wound signal promoting plant regeneration. Cell. 2024; 187:3024-3038.e14

[86] Xu S, Liao CJ, Jaiswal N. et al. Tomato PEPR1 ORTHOLOG RECEPTOR-LIKE KINASE1 regulates responses to systemin, necrotrophic fungi, and insect herbivory. Plant Cell. 2018; 30: 2214-29

[87] Liu S, Wang J, Jiang S. et al. Tomato SlSAP3, a member of the stress-associated protein family, is a positive regulator of immu-nity against pseudomonas syringae pv. Tomato DC3000. Mol Plant Pathol. 2019; 20:815-30

[88] Hu Z, Fang H, Zhu C. et al. Ubiquitylation of PHYTOSULFOKINE RECEPTOR 1 modulates the defense response in tomato. Plant Physiol. 2023; 192:2507-22

[89] Howe GA, Lightner J, Browse J. et al. An octadecanoid pathway mutant (JL5) of tomato is compromised in signaling for defense against insect attack. Plant Cell. 1996; 8:2067-77

[90] Liu Y, Schiff M, Dinesh-Kumar SP. Virus-induced gene silencing in tomato. Plant J. 2002; 31:777-86

[91] Ai Y, Li Q, Li C. et al. Tomato LysM receptor kinase 4 mediates chitin-elicited fungal resistance in both leaves and fruit. Hortic Res. 2023;10:uhad082

[92] Luo Q, Wang J, Wang P. et al. Transcriptomic and genetic approaches reveal that low-light-induced disease susceptibil-ity is related to cellular oxidative stress in tomato. Hortic Res. 2023;10:uhad173

[93] Xu J, Wang X, Zu H. et al. Molecular dissection of rice phyto-hormone signaling involved in resistance to a piercing-sucking herbivore. New Phytol. 2021; 230:1639-52

[94] Chakrabarty R, Banerjee R, Chung SM. et al. PSITE vec-tors for stable integration or transient expression of aut-ofluorescent protein fusions in plants: probing Nicotiana ben-thamiana-virus interactions. Mol Plant-Microbe Interact. 2007; 20: 740-50

[95] Zhu Y, Schluttenhoffer CM, Wang P. et al. CYCLIN-DEPENDENT KINASE8 differentially regulates plant immunity to fungal pathogens through KINASE-dependent and -independent func-tions in Arabidopsis. Plant Cell. 2014; 26:4149-70

[96] Guo M, Li Z, Wang L. et al. BAG8 positively regulates cold stress tolerance by modulating photosystem, antioxidant system and protein protection in Solanum lycopersicum. Plant Physiol Biochem. 2023; 206:108267

[97] Wang H, Ye X, Bi Y. et al. Eukaryotic translation elongation factor OseEF1A negatively regulates rice immunity against blast disease. Plant Physiol. 2024; 195:1796-801

[98] Yuan Y, Fan Y, Huang L. et al. The SINA1-BSD1 module regulates vegetative growth involving gibberellin biosynthesis in tomato. Adv Sci (Weinh). 2024; 11:e2400995

[99] Wang X, Ding Y, Li Z. et al. PUB25 and PUB26 promote plant freez-ing tolerance by degrading the cold signaling negative regulator MYB15. Dev Cell. 2019; 51:222-235.e5