‘Candidatus Liberibacter Asiaticus’ Effector SECP8 Subverts Salicylic Acid‐Mediated Citrus Immunity via a Two‐Pronged Impairment of CsTCP15 Dimerization

ABSTRACT

Citrus Huanglongbing (HLB), a devastating disease caused by the unculturable bacterium ‘Candidatus Liberibacter asiaticus’ (CLas), poses a severe threat to global citrus production. CLas secretes effectors to suppress host immune responses and facilitate its colonisation. Previously, the CLas effector SECP8 (CLIBASIA_05330) has been identified as an immune inhibitor. However, its molecular mechanisms on host immune suppression remain unclear. This study identifies the citrus transcription factor CsTCP15 as a target of SECP8. Transgenic citrus plants overexpressing CsTCP15 enhanced resistance to CLas, whereas CsTCP15‐RNAi interference plants became more susceptible, confirming its role as a positive immune regulator. Meanwhile, CsTCP15 was demonstrated to directly bind to cis‐elements of salicylic acid (SA)‐responsive genes CsPR5 and CsWRKY22, and overexpression of either gene strengthened citrus hairy roots' resistance against CLas. However, SECP8 directly interacts with CsTCP15 and inhibits its homodimerization. Concurrently, mSECP8 facilitates CsBRG3‐mediated degradation and further prevents the dimerization of CsTCP15. This two‐pronged interference eventually impairs the transcriptional activation of CsPR5 and CsWRKY22, thereby compromising salicylic acid‐mediated immunity and promoting CLas infection. Our findings reveal a virulence strategy whereby a CLas effector manipulates a key host immune regulator to establish pathogenesis.

1. Introduction

Citrus Huanglongbing (HLB), a devastating disease caused by Candidatus Liberibacter species, is primarily associated with three pathogenic variants: ‘Candidatus Liberibacter africanus’ (CLaf), ‘Candidatus Liberibacter asiaticus’ (CLas), and ‘Candidatus Liberibacter americanus’ (CLam) (Jagoueix et al. 1994). Among these, CLas exhibits the broadest geographic distribution and is predominantly transmitted by the Asian citrus psyllid ( Diaphorina citri ), posing a severe threat to global citrus production. CLas infection induces characteristic symptoms, including asymmetrical leaf chlorosis (blotchy mottle), leaf thickening, stunted growth, fruit abscission, and, in severe cases, plant mortality. Since the release of the CLas genome in 2009 (Duan et al. 2009), significant progress has been made in CLas research. Despite reports of successful cultivation and the preliminary fulfilment of Koch's postulates, challenges remain in achieving consistent high titers in culture and efficient inoculation (Zheng et al. 2024).

Plants utilise a two‐tiered innate immune system, including pattern‐triggered immunity (PTI) and effector‐triggered immunity (ETI), to defend against pathogen invasion. To counteract these defenses, pathogens secrete effector proteins that function as virulence factors, suppressing or evading host immune responses (Mukhtar et al. 2011). Numerous CLas effectors have been identified as key players in modulating citrus immunity by interacting with host proteins, thereby facilitating bacterial colonisation. For instance, the effector SDE1 (CLIBASIA_05315) inhibits the activity of citrus papain‐like cysteine proteases (PLCPs), promoting CLas proliferation (Clark et al. 2018). Similarly, SDE15 (CLIBASIA_04025) targets CsACD2 to suppress plant immunity and enhance CLas survival (Pang et al. 2020). SDE3 (CLIBASIA_00420) directly interacts with citrus glyceraldehyde‐3‐phosphate dehydrogenase GAPC to specifically inhibit the ATG8‐mediated immune response (Shi et al. 2023). SDE5 (CLIBASIA_02470) enhances the PUB21‐MYC2 interaction, thereby suppressing JA‐mediated defence and promoting CLas infection (Zhao et al. 2025). Despite these advances, the molecular mechanisms of many CLas effectors largely remain elusive. For instance, SECP8 has been strongly implicated in immunosuppression and virulence, yet its host targets remain unidentified (Shen et al. 2022). Given the devastating agricultural impact of HLB, deciphering the pathogenic strategies of CLas carries profound economic and societal implications.

The TCP gene family derives its name from three founding members: TEOSINTE BRANCHED1 (TB1) in maize ( Zea mays ), CYCLOIDEA (CYC) in snapdragon ( Antirrhinum majus ), and PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR (PCF) in rice ( Oryza sativa ) (Cubas et al. 2002). TCP transcription factors serve as central regulators in plant developmental and stress‐responsive pathways, governing processes such as cell proliferation (Kieffer et al. 2011; Luo et al. 1996; Sarvepalli and Nath 2018), phytohormone biosynthesis (Steiner et al. 2012), immune responses, and circadian rhythm modulation (Fu et al. 2025). Phylogenetically, the TCP family is divided into two classes: class I (PCF subfamily) and class II (further subdivided into CIN and CYC/TB1 subfamilies) (Martin‐Trillo and Cubas 2010). Class I members bind to the distinct cis‐element GGNCCCAC, while class II members bind to GTGGNCCC to modulate downstream gene expression (Kosugi and Ohashi 2002; Aggarwal et al. 2010). Notably, TCPs play pivotal roles in plant responses to both abiotic and biotic stresses (Lopez et al. 2015).

TCP transcription factors have been confirmed to directly or indirectly participate in plant defence responses, and they are also one of the important targets of pathogen action. Pathogens frequently exploit TCP transcription factors to subvert host immunity. For example, the oomycete effector CRN12_997 targets tomato SlTCP14‐2 (a homologue of Arabidopsis AtTCP14), enhancing host susceptibility (Stam et al. 2021). Similarly, the tospovirus NSs protein suppresses jasmonic acid (JA), auxin (AUX), and strigolactone (SL) signalling by interacting with TCP21, thereby dampening immune activation (Chen, Zhao, et al. 2022). Phytoplasma effectors, such as SAP11, destabilise Arabidopsis AtTCP2/4/7/13, attenuating LOX2 activity and JA‐mediated defences (Mackey et al. 2019; Sugio et al. 2011). The Pseudomonas syringae effector HopBB1 binds AtTCP14 and AtJAZ3, hijacking JA‐responsive genes to promote infection (Yang et al. 2017). Additionally, the phytoplasma effector Zaofeng6 disrupts SL signalling by inhibiting ZjTCP7 at the transcriptional and translational levels (Chen, Chen, et al. 2022), while SWP1 degrades BRC1 (TCP18) to facilitate pathogen proliferation (Wang et al. 2018). Despite these insights, the role of TCPs in citrus resistance to CLas remains unexplored.

Protein ubiquitination is a common post‐translational modification that participates in the regulation of various protein substrates in different cellular pathways through a three‐enzyme cascade reaction, while the 26S proteasome degrades the ubiquitinated substrates (Craig et al. 2009). In this process, the successive involvement of ubiquitin‐activating enzyme E1, ubiquitin‐conjugating enzyme E2, and ubiquitin ligase E3 triggers the degradation of substrates mediated by the 26S proteasome, among which E3 ligases are most extensively studied in plant immune signalling. For instance, E3 ligase PUB22 regulates JA signalling through the degradation of JAZ (Wu et al. 2024). BRG3 is an E3 ligase containing a highly conserved RING motif. MdBRG3 has been confirmed to mediate the ubiquitination and degradation of nitrate response factor MdLBD36 (Guo et al. 2025). Moreover, BRG3 can weaken the inhibition of tomato fruit ripening by degrading WRKY71 (Sun et al. 2023). However, the role of CsBRG3 in the resistance of citrus to CLas infection has not been elucidated.

It was previously shown that SECP8 significantly suppresses salicylic acid (SA)‐responsive gene expression (Shen et al. 2022). However, the molecular mechanisms for SA pathway modulation in CLas‐infected citrus remained unresolved. Here, we demonstrate that the mature form of SECP8 (mSECP8) disrupts CsTCP15 dimerization through both direct and indirect mechanisms, ultimately compromising its transcriptional activity. Importantly, we demonstrate that CsTCP15 serves as a positive regulator of citrus resistance to CLas by directly activating the expression of defence‐related genes CsPR5 and CsWRKY22. These findings establish CsTCP15 as a central hub in the SA‐mediated immunity network and highlight its potential as a molecular target for HLB control strategies.

2. Results

2.1. mSECP8 Physically Interacts With Citrus CsTCP15

To elucidate the molecular mechanism underlying mSECP8‐mediated suppression of systemic acquired resistance (SAR) signalling, we conducted a yeast two‐hybrid (Y2H) screen using mSECP8 as bait to identify its targets against the citrus library. CsTCP15 was identified (Figure S1), and its physical interaction was confirmed through Y2H assays (Figure 1a). In Y2H assays, the yeast carried both pGADT7 (AD)‐CsTCP15 and pGBKT7 (BD)‐mSECP8 constructs grew on SD‐LTHA (with X‐α‐gal) medium, whereas yeast harbouring the control plasmids failed to grow (Figure 1a). To further validate their interaction, a Split luciferase complementation assay (LCA), Bimolecular fluorescence complementation (BiFC), Co‐immunoprecipitation (Co‐IP), and GST pull‐down assays were carried out. The mSECP8‐CsTCP15 interaction was reinforced with LCA assays, showing a strong luminescence signal upon co‐expression of mSECP8 and CsTCP15, rather than the controls (Figure 1b). The BiFC analysis confirmed that mSECP8 interacts with CsTCP15 in the nucleus, with a fluorescence signal observed (Figure 1c). Co‐IP assays showed that the FLAG‐mSECP8‐GFP was specifically co‐immunoprecipitated by HA‐CsTCP15, but not by the negative control HA‐GUS (Figure 1d). Additionally, GST pull‐down assays further substantiated the direct interaction between mSECP8 and CsTCP15 in vitro, with His‐SUMO‐mSECP8 specifically pulled down by GST‐CsTCP15, but not by GST (Figure 1e).

FIGURE 1.

mSECP8 interacts with CsTCP15 in vivo and in vitro. (a) mSECP8 interacts with CsTCP15 in Y2H. Y2H gold yeast cells co‐transformed with AD‐CsTCP15 and BD‐mSECP8 plated on SD‐LT (lacking leucine and tryptophan) and SD‐LTHA (lacking leucine, tryptophan, histidine and adenine with X‐α‐gal) plates. BD‐53/AD‐T7 was used as the positive control. BD‐Lam/AD‐T7, BD/AD‐CsTCP15 and BD‐mSECP8/AD were used as the negative control. (b) LCA analysis of the interaction between mSECP8 and CsTCP15. CsTCP15‐nLuc+cLuc‐GUS, GUS‐nLuc+cLuc‐GUS, GUS‐nLuc+cLuc‐mSECP8 were used as the negative control. (c) mSECP8 interacts with CsTCP15 in BiFC assay. Yn/CsTCP15:Yc and Yn:mSECP8/Yc were used as the negative control. Scale bar = 100 μm. (d) Co‐IP assays showing mSECP8 and CsTCP15 interaction. Total proteins were extracted from the N. benthamiana leaves of HA‐CsTCP15/HA‐GUS and FLAG‐mSECP8‐GFP co‐infiltrated and immunoprecipitated with HA‐Trap. Immunoblot analysis was detected by anti‐HA and anti‐FLAG antibodies, respectively. (e) The GST pull‐down assay confirms the interaction between mSECP8 and CsTCP15. All experiments were repeated at least three times with similar results.

To understand their subcellular localization, mSECP8‐BFP and CsTCP15‐GFP were individually or paired transiently expressed in Nicotiana benthamiana (N. benthamiana) leaves. In separate localization studies, mSECP8 was observed to localise to both the nucleus and cytoplasm, while CsTCP15 exclusively distributes to the nucleus. Co‐expression of mSECP8 and CsTCP15 found them to overlap in the nuclear compartment (Figure S2a). Moreover, when mSECP8‐GFP was transiently expressed in citrus leaves, it exclusively localised to the nucleus (Figure S2b). Collectively, these results demonstrated that mSECP8 physically interacts with CsTCP15 primarily in the nuclear compartment.

2.2. CsTCP15 Positively Regulates SA‐Mediated Immunity

To elucidate the biological function of CsTCP15 in the immunity of citrus, CsTCP15‐overexpressing (OE‐CsTCP15) and RNA interference (RNAi‐CsTCP15) transgenic citrus lines were generated (Figure 2a,b, Figure S3a,d). Three OE‐CsTCP15 (OE‐1, OE‐2 and OE‐3) and three RNAi‐CsTCP15 (R‐1, R‐2 and R‐3) independent lines were obtained and verified via GFP fluorescence observation (Figure 2a,b), PCR and western blot (Figure S3b,c,e). RT‐qPCR assay indicates that CsTCP15 is significantly overexpressed in OE‐CsTCP15 and significantly downregulated in RNAi‐CsTCP15 when compared with the wild‐type (WT) citrus (Figure 2c,d).

FIGURE 2.

Susceptibility assay of OE‐CsTCP15 and RNAi‐CsTCP15 transgenic citrus plants against CLas challenge. (a, b) Green fluorescent signal visualised by UV light in three independent OE‐CsTCP15 and RNAi‐CsTCP15 transgenic lines, respectively. (c, d) The relative transcript levels of CsTCP15, CsPR5 and CsWRKY22 in the OE‐CsTCP15 and RNAi‐CsTCP15 transgenic citrus plants. (e) The transcript levels of CsPR5 and CsWRKY22 in transcriptome profiling from RNAi‐CsTCP15 transgenic citrus plants. (f) The SA content was evaluated in WT, OE‐CsTCP15, RNAi‐CsTCP15 and mSECP8 transgenic citrus. (g) Quantitative analysis of CLas population at 2, 4, and 6 mpi. CLas population densities (CLas cells μg⁻¹ of citrus DNA) were quantified by qPCR analysis. (h) CLas‐infected plants and newly flushing and mature leaves with symptoms were captured at 6 mpi. The leaves exhibiting HLB symptoms are shown in the most right panel. Scale bar = 1 cm. Statistical analysis was performed using the Student's t‐test, *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001, n = 3 technical replicates in (c, d), n = 3 biologically independent transgenic lines in (f, g). All experiments were repeated at least three times with similar results.

To systematically investigate the regulatory roles of CsTCP15 in citrus, high‐throughput RNA sequencing was conducted on R‐2 due to its more efficient suppression of CsTCP15 among the three transgenic lines (Figure 2d). A total of 1271 differentially expressed genes (DEGs) were identified in R‐2 compared to the WT (Figure S4a). Functional enrichment analysis was conducted on the downregulated genes. The results revealed that transcriptional regulation processes, such as protein dimerization activity, transcription regulator activity, and DNA‐binding transcription factor activity, were significantly enriched (Figure S4b). KEGG enrichment analysis revealed that these downregulated genes are involved in peroxisome, phagosome, and efferocytosis pathways (Figure S4c). Notably, the SA‐responsive genes CsPR5 and CsWRKY22 downregulated in mSECP8 transgenic plants (Shen et al. 2022) also showed reduced expression in the R‐2 line (Figure S4d and Figure 2e). Consistent with this, RT‐qPCR analysis confirmed that both CsPR5 and CsWRKY22 were strongly induced in OE‐CsTCP15 plants but suppressed in RNAi‐CsTCP15 citrus (Figure 2c,d). Furthermore, the accumulation level of SA contents was significantly higher in OE‐CsTCP15 citrus, whereas there was no significant change in the RNAi‐CsTCP15 and mSECP8 transgenic citrus (Figure 2f). RNAi‐CsTCP15 citrus did not show a significant reduction in SA content, potentially due to functional redundancy among TCP family members (Danisman et al. 2013; Zhao et al. 2024). Collectively, these findings demonstrate that CsTCP15 overexpression activates SA‐mediated immunity, enhancing the expression of key SAR‐related genes.

2.3. CsTCP15 Enhances Citrus Resistance Against CLas Infection

To evaluate the biological role of CsTCP15 in CLas colonisation, the OE‐CsTCP15 and RNAi‐CsTCP15 citrus were graft‐inoculated with CLas‐infected buds for susceptibility assessment. Time‐lapsed CLas detection revealed distinct colonisation patterns. At 2 months post‐inoculation (2 mpi), the CLas population in R‐1, R‐2, and R‐3 was significantly greater than that in WT citrus, whereas no CLas was detected in OE‐1, OE‐2, and OE‐3 citrus (Figure 2g). At 4 mpi, CLas was detected in OE‐1, OE‐2, and OE‐3 transgenic lines with a significantly lower CLas population than that in WT citrus plants (Figure 2g). Notably, CLas load in OE‐CsTCP15 transgenic lines had reached levels comparable to those in WT plants by 6 mpi (Figure 2g). With HLB progressing, CLas‐infected plants exhibited typical HLB characteristics, including leaf mottling, yellowing, curling, and small leaves, particularly within RNAi‐CsTCP15 citrus plants (Figure 2h). These findings indicate that CsTCP15 acts as a positive regulator in the citrus host defence response, limiting CLas proliferation at the early infection stage.

2.4. mSECP8 Inhibits the Dimerization of CsTCP15

CsTCP15 has been identified as a transcriptional activator involved in SA‐mediated immune defence responses, whereas mSECP8 suppressed SA‐mediated pathways (Shen et al. 2022). We hypothesised that mSECP8 suppresses SAR signalling by inhibiting the transcriptional function of CsTCP15. In previous studies, it has been confirmed that transcription factors exert transcriptional activity by forming homodimers (Aggarwal et al. 2010; Zhang et al. 2023). Then the LCA, BiFC, and GST pull‐down assays were employed to confirm the dimerization of CsTCP15. The LCA demonstrated robust signals for the CsTCP15‐nLuc+cLuc‐CsTCP15 pairing (Figure S5a). BiFC assays revealed that the dimerization of CsTCP15 occurred within the nuclear compartment (Figure S5b). GST pull‐down assays showed His‐SUMO‐CsTCP15 recruitment by GST‐CsTCP15 but not by GST (Figure S5c).

To investigate the effect of mSECP8 on CsTCP15 homodimerization, the yeast three‐hybrid (Y3H) assays were utilised. The Y3H results showed that the presence of mSECP8 inhibits the formation of CsTCP15 homodimers (Figure 3a). Additionally, LCA assays showed that the relative integral optical density (IOD) of CsTCP15‐nLuc+cLuc‐CsTCP15 + mSECP8 was significantly decreased compared to that of CsTCP15‐nLuc+cLuc‐CsTCP15 and CsTCP15‐nLuc+cLuc‐CsTCP15 + EV (Figure 3b). The His pull‐down competition assay results indicated that the protein abundance of GST‐CsTCP15, pulled down by His‐SUMO‐CsTCP15, and gradually reduced with increasing abundance of MBP‐mSECP8‐GFP (Figure 3c). These results indicate that mSECP8 directly interferes with the dimerization of CsTCP15.

FIGURE 3.

mSECP8 disrupts the dimerization of CsTCP15. (a) The effect of mSECP8 on CsTCP15 dimerization was assayed with Y3H. Y2H gold yeast cells co‐transformed with pBridge‐CsTCP15‐MBP/pBridge‐CsTCP15‐mSECP8 and AD‐CsTCP15 plated on SD‐LT (lacking leucine and tryptophan) and SD‐MHLT (lacking methionine, histidine, leucine and tryptophan) plate. (b) LCA assay results showing the mSECP8 disrupts the dimerization of CsTCP15. CsTCP15‐nLuc+cLuc‐CsTCP15, CsTCP15‐nLuc+cLu‐CsTCP15 + EV and CsTCP15‐nLuc+cLuc‐CsTCP15 + mSECP6 were used as the negative control. (c) mSECP8 disrupts the dimerization of CsTCP15 in GST pull‐down competition assay. The GST‐CsTCP15 proteins were pulled down by His‐SUMO‐CsTCP15, and the abundance of GST‐CsTCP15 decreased with increasing amounts of MBP‐mSECP8‐GFP (using MBP as the control). Immunoblot analysis was detected by anti‐GST, anti‐His and anti‐MBP antibodies. Statistical analysis was performed using the Student's t‐test, *p < 0.05, n = 3 biologically independent samples. All experiments were repeated at least three times with similar results.

To further identify the critical domain of CsTCP15 by which mSECP8 inhibits its dimerization, CsTCP15 was truncated into three truncates on the basis of its conserved domain (Figure S6a). However, LCA assays demonstrated that all three truncates of CsTCP15 are capable of forming dimers, particularly with the TCP domain appearing to be the most critical for this interaction (Figure S6e). Similarly, a further LCA assay found that mSECP8 interacts with all three truncates of CsTCP15 (Figure S6f).

2.5. mSECP8 Mediates CsTCP15 Proteolytic Degradation

To investigate the effect of mSECP8 on CsTCP15 protein stability, FLAG‐mSECP8‐GFP or FLAG‐GFP was co‐expressed with HA‐CsTCP15 in N. benthamiana leaves. Western blot assay revealed that the protein abundance of CsTCP15 was significantly reduced when co‐expressing with FLAG‐mSECP8‐GFP compared to co‐expressing with FLAG‐GFP (Figure 4a). Furthermore, CsTCP15 protein abundance recovered significantly upon MG132 treatment compared to dimethyl sulfoxide (DMSO) treatment (Figure 4b). Subsequently, cell‐free degradation assays were conducted by incubating total protein extracts from OE‐CsTCP15 transgenic citrus leaves with those from mSECP8 transgenic leaves (with WT serving as the control) at 28°C. The results showed a progressive reduction in CsTCP15 protein abundance as the amount of total protein from mSECP8 transgenic citrus increased, whereas CsTCP15 abundance remained relatively stable in WT extracts (Figure 4c). Additionally, the degradation of CsTCP15 in mSECP8 transgenic citrus extracts was inhibited upon MG132 treatment (Figure 4c). Collectively, these findings indicate that mSECP8 promotes the degradation of CsTCP15 via the 26S proteasome pathway.

FIGURE 4.

mSECP8 facilitates the degradation of CsTCP15 via the 26S proteasome pathway. (a, b) The effect of mSECP8 on CsTCP15 degradation assay without and with 26S proteasome inhibitor MG132 treatment. Total proteins were extracted from the N. benthamiana leaves at 48 hpi and detected with anti‐HA, anti‐FLAG and anti‐Actin antibodies, respectively. N. benthamiana actin protein was used as the internal control. (c) Cell‐free degradation assays. Total proteins were extracted from the OE‐CsTCP15, WT, and mSECP8‐transgenic citrus leaves. The protein extracts from OE‐CsTCP15 plants were co‐incubated with those from mSECP8‐transgenic or WT citrus, respectively. The target proteins were detected using anti‐FLAG and anti‐Actin antibodies. Citrus Actin protein was used as the internal control. (d) mSECP8 enhances the degradation of CsTCP15. Total proteins were extracted from the N. benthamiana leaves of FLAG‐mSECP8‐GFP/FLAG‐GFP, HA‐CsTCP15 and MYC‐CsBRG3 co‐infiltrated and target proteins were detected by corresponding antibodies. (e) mSECP8 enhances the interaction between CsTCP15 and CsBRG3 in LCA. CsBRG3‐nLuc+cLuc‐CsTCP15 + buffer, CsBRG3‐nLuc+cLuc‐CsTCP15 + EV were used as the negative control. (f) mSECP8 promote the interaction of CsTCP15 and CsBRG3 in Y3H. Y2H gold yeast cells co‐transformed with pBridge‐CsTCP15‐MBP/pBridge‐CsTCP15‐mSECP8 and AD‐CsBRG3 plated on SD‐LT (lacking leucine and tryptophan) and SD‐MHLT (lacking methionine, histidine, leucine and tryptophan) plate. (g) Ubiquitination of GST‐CsTCP15 mediated by His‐SUMO‐CsBRG3 in an in vitro ubiquitination assay. (h) mSECP8 enhances the ubiquitination of GST‐CsTCP15 mediated by His‐SUMO‐CsBRG3 in an in vitro ubiquitination assay. Target proteins were detected by anti‐Ub, anti‐His and anti‐GST antibodies. (i) CsBRG3 disrupts the dimerization of CsTCP15 in LCA assay. CsTCP15‐nLuc+cLuc‐CsTCP15 + EV were used as the negative control. (j) mSECP8 promotes CsBRG3‐induced disruption of CsTCP15 dimerization in the LCA assay. CsTCP15‐nLuc+cLuc‐CsTCP15 + CsBRG3 + EV were used as the negative control. Statistical analysis was performed using the Student's t‐test, **p < 0.005, ****p < 0.0001, n = 3 biologically independent samples. All experiments were repeated at least three times with similar results.

2.6. mSECP8 Enhances the Interaction Between CsBRG3 and CsTCP15 to Promote the Degradation of CsTCP15

mSECP8 promotes CsTCP15 degradation via the 26S proteasome pathway, likely involving an E3 ubiquitin ligase. To elucidate the molecular mechanism underlying mSECP8‐mediated CsTCP15 degradation, the E3 ubiquitin ligase CsBRG3 was screened by Y2H, and its expression was markedly upregulated in mSECP8‐transgenic citrus (Figure S7a), suggesting a potential regulatory interplay among these molecular components.

To confirm the CsTCP15‐CsBRG3 interaction, Y2H, LCA, BiFC, and His pull‐down assays were conducted. In Y2H assays, yeast co‐transformed with AD‐CsTCP15 and BD‐CsBRG3 recombinants grew on SD‐LTHA medium, while control plasmid‐bearing yeast failed to proliferate (Figure S7b). LCA revealed clear signals for the CsBRG3‐nLuc+cLuc‐CsTCP15 pairing, with no activity observed in other combinations (Figure S7c). BiFC assays demonstrated CsTCP15‐CsBRG3 interaction in the nucleus (Figure S7d). His pull‐down assays showed GST‐CsTCP15 recruitment by His‐SUMO‐CsBRG3 but not by His‐FLAG‐GFP (Figure S7e). Notably, CsBRG3 interacts specifically with CsTCP15 but not mSECP8 (Figure S7f,g). These evidences confirmed the CsTCP15‐CsBRG3 interaction both in vivo and in vitro.

To investigate whether CsBRG3 mediates CsTCP15 degradation, MYC‐CsBRG3 and HA‐CsTCP15 were co‐expressed in N. benthamiana leaves, with MYC‐GUS/HA‐CsTCP15 serving as negative controls. CsBRG3 overexpression significantly reduced CsTCP15 protein abundance (Figure S7h), and the reduction was reversed by MG132 treatment (Figure S7i), implicating the 26S proteasome pathway in this regulatory mechanism. Moreover, when the HA‐CsTCP15, MYC‐CsBRG3, and FLAG‐mSECP8‐GFP proteins were co‐expressed in N. benthamiana leaves, the protein abundance of CsTCP15 was remarkably reduced in the presence of mSECP8 than in the control (HA‐CsTCP15, MYC‐CsBRG3, and FLAG‐GFP co‐expression) (Figure 4d). To understand whether mSECP8 affects CsBRG3‐CsTCP15 interaction, LCA and Y3H were conducted and revealed that mSECP8 expression enhanced their interaction (Figure 4e,f). Furthermore, the impact of mSECP8 on the degradation of CsTCP15 mediated by CsBRG3 was investigated using an in vitro ubiquitination assay. The polyubiquitinated bands were observed in lanes 1 and 6 (anti‐Ub), while they were absent in other lanes (Figure 4g). The polyubiquitinated bands in lane 6 (anti‐Ub) indicated that CsBRG3 exhibits E3 ubiquitin ligase activity (Figure 4g). Additionally, the intensity of polyubiquitination in lane 1 is greater than that in lane 6, suggesting that CsTCP15 is ubiquitinated by CsBRG3 (Figure 4g). Meanwhile, the addition of mSECP8 enhances the ubiquitination of CsTCP15 by CsBRG3 compared to the control (Figure 4h). In summary, the CLas effector mSECP8 facilitates CsTCP15 degradation by enhancing CsBRG3‐CsTCP15 interaction.

Based on the observed dual functions of mSECP8‐inhibiting CsTCP15 dimerization while promoting its CsBRG3‐mediated degradation, we investigated whether these processes are functionally connected with a competitive LCA assay. The results revealed that CsBRG3‐mediated degradation of CsTCP15 reduces the formation of CsTCP15 dimers (Figure 4i). Moreover, the presence of mSECP8 strengthened the inhibitory effect of CsBRG3 on CsTCP15 dimerization (Figure 4j). These results suggest that mSECP8 indirectly disrupts CsTCP15 dimer formation by accelerating CsBRG3‐dependent degradation, which was achieved with enhanced CsBRG3‐CsTCP15 interaction. In summary, mSECP8 suppresses the transcriptional activity of CsTCP15 through a dual mechanism: direct inhibition of homodimerization and enhancement of CsBRG3‐mediated ubiquitination and degradation, which further disrupts dimer formation.

2.7. mSECP8 Affects SA‐Mediated Immunity to Enhance Plant Disease Susceptibility in a CsTCP15‐Dependent Manner

The expression levels of defence‐related genes CsPR5 and CsWRKY22 were consistently upregulated in OE‐CsTCP15 lines and downregulated in RNAi‐CsTCP15 lines, respectively (Figure 2c,d). These observations led us to hypothesise that CsTCP15 directly regulates the transcription of these downstream target genes. To test this hypothesis, their promoter sequences (~−2000 bp) were extracted from the citrus database (http://citrus.hzau.edu.cn/). The promoter sequences of CsPR5 and CsWRKY22 contain a GGATCCAC cis‐element (−606 ~ −599 bp) and a GTTCCCAC cis‐element (−1388 ~ −1381 bp), respectively, indicating CsPR5 and CsWRKY22 as potential direct targets of CsTCP15. Then, dual‐luciferase reporter assay system, chromatin immunoprecipitation qPCR (ChIP‐qPCR), and electrophoretic mobility shift assays (EMSAs) were performed. The results demonstrated that CsTCP15 significantly activates the expression of the dual‐luciferase reporter gene driven by the promoters of CsPR5 and CsWRKY22 (Figure 5a–c). ChIP‐qPCR revealed significant enrichment of CsPR5 and CsWRKY22 promoter fragments containing cis‐elements (Figure 5d,e). To further investigate the molecular mechanism underlying this regulation, we conducted EMSAs to examine the DNA‐binding capacity of CsTCP15. The results confirmed that CsTCP15 specifically binds to the promoters of CsPR5 and CsWRKY22 (Figure 5f,g), whereas the mutated probes did not bind to CsTCP15, and the cold probe significantly abolished the binding of CsTCP15 to the labelled WT probe (Figure 5f,g). Therefore, CsTCP15 directly regulates CsPR5 and CsWRKY22 expression via binding to their promoters.

FIGURE 5.

mSECP8 suppresses the transcriptional activation of CsTCP15 on CsPR5 and CsWRKY22. (a) Schematic representations of the effector and reporter constructs employed in dual‐luciferase assays. (b, c) The LUC‐effectors were co‐infected with EV or CsTCP15 in N. benthamiana leaves. (d, e) ChIP‐qPCR was utilised to detect the binding of CsTCP15 to the CsPR5/CsWRKY22 promoter with OE‐3 transgenic lines. IgG was used as a negative control. (f, g) The EMSA assay of His‐SUMO‐CsTCP15 binding to the promoters of CsPR5 and CsWRKY22. His‐FLAG‐GFP were used as the negative control. The 10× and 50× unlabeled probes of CsPR5/CsWRKY22 were used as competitors. (h, i) The dual‐luciferase‐effectors and CsTCP15 were co‐infected with EV or mSECP8 in N. benthamiana leaves. (j) mSECP8 did not affect the binding between CsTCP15 and CsPR5/CsWRKY22 in EMSA assays. His‐FLAG‐GFP was used as the negative control. The 10× and 50× His‐SUMO‐mSECP8 were used as competitors. The DNA‐protein shift did not change with the addition of His‐SUMO‐mSECP8. Statistical analysis was performed using the Student's t‐test, **p < 0.005, ***p < 0.001, ****p < 0.0001, n = 3 biologically independent samples in (b, c) and (h, i), n = 3 technical replicates in (d, e). All experiments were repeated at least three times with similar results.

However, when mSECP8 was co‐expressed, it remarkably attenuated CsTCP15‐mediated transactivation of these promoters compared to the control (Figure 5h,i). This finding is tightly correlated with the observed downregulation of CsPR5 and CsWRKY22 in mSECP8‐transgenic citrus (Shen et al. 2022). Of particular interest, in vitro EMSA assay revealed that the presence of mSECP8 did not impair CsTCP15 binding capacity to their promoter sequences (Figure 5j), indicating that mSECP8 suppresses CsTCP15 transcriptional activity by interfering with its dimerization rather than its DNA‐binding capacity. Together, our findings demonstrate that mSECP8 modulates SA‐mediated immunity and enhances disease susceptibility in citrus by inhibiting CsTCP15 dimerization.

To further elucidate the functional role of CsTCP15 in immunity, OE‐CsPR5 and OE‐CsWRKY22 transgenic citrus hairy roots were generated with healthy Citrus trifoliate stems. Successful transformation was confirmed by GFP fluorescence observation and PCR (Figure 6a–c). RT‐qPCR revealed significantly higher expression levels of CsPR5 and CsWRKY22 in the transgenic hairy roots compared to the control (pNmGFPer:00‐infiltrated hairy roots) (Figure 6d,e). For susceptibility assessment, three independent OE‐CsPR5 (OE‐CsPR5‐1/‐2/‐3) and OE‐CsWRKY22 (OE‐CsWRKY22‐1/‐2/‐3) hairy roots were graft‐inoculated with CLas‐infected buds. At 20 days post‐inoculation (20 dpi), CLas titers were quantified, and the results showed their population in both OE‐CsPR5 and OE‐CsWRKY22 hairy roots was significantly lower than that in the pNmGFPer:00 control (Figure 6f,g). Taken together, these results suggest that CsTCP15 enhanced citrus plant resistance against CLas by activating SA‐mediated gene expression of CsPR5 and CsWRKY22.

FIGURE 6.

mSECP8 suppresses CsTCP15‐mediated citrus immunity by inhibiting CsWRKY22 and CsPR5 expression. (a) Green fluorescent signal visualised by UV light in citrus hairy roots. Scale bar = 1 cm. (b, c) PCR‐diagnostic image of CsPR5 and CsWRKY22, respectively (P: Plasmid). (d, e) The relative transcript levels of CsPR5 and CsWRKY22 in OE‐CsPR5 and OE‐CsWRKY22 citrus hairy roots, respectively. (f, g) CLas population (CLas cells μg⁻¹ of citrus DNA) were quantified through qPCR analysis. (h) The working model of mSECP8 manipulating CsTCP15 to suppress immunity. Left: Under non‐infected conditions, CsTCP15 homodimers activates the transcription of SA‐mediated genes (CsPR5 and CsWRKY22) through dimerization, thereby positively regulating plant immunity. Meanwhile, the host E3 ligase CsBRG3 negatively regulates both the protein stability and dimerization of CsTCP15 via the ubiquitin‐mediated degradation pathway, thus maintaining immune homeostasis. Right: Upon CLas infection, the effector protein mSECP8 directly interacts with CsTCP15 to inhibit dimer formation, while simultaneously enhancing CsBRG3‐mediated ubiquitination and degradation of CsTCP15. This synergistic suppression of SA‐mediated immune responses ultimately facilitates pathogen infection. Statistical analysis was performed using the Student's t‐test, *p < 0.05, **p < 0.005, ****p < 0.0001, n = 3 technical replicates in (d, e), n = 3 biologically independent samples in (f, g). All experiments were repeated at least three times with similar results.

3. Discussion

Pathogenic bacteria commonly employ virulence strategies that involve secreting effectors into the host to suppress plant immunity and promote colonisation. In a previous study, the CLas effector mSECP8 has been identified to suppress citrus SAR and PTI, thereby promoting CLas proliferation. However, the mechanism by which mSECP8 hijacks or disrupts host factors to suppress SAR remains unclear (Shen et al. 2022). Here, we revealed the molecular mechanism that mSECP8 inhibits citrus SA‐mediated immunity by impairing the CsTCP15 dimerization (Figure 6h).

Transcription factor TCP15 plays a multifaceted role in plant growth and development, orchestrating processes ranging from seed germination to floral transition through its transcriptional regulation of cell cycle‐associated genes and hormone response pathways (Kieffer et al. 2011; Li et al. 2012; Lucero et al. 2015; Resentini et al. 2021). Recent evidence further suggests that TCP factors are key regulators of phloem architecture, as demonstrated by the role of poplar PtoTCP20 in vascular differentiation via the miR396d‐PtoGRF15 axis (Wang et al. 2023). Beyond its developmental functions, TCP15 also contributes to plant immunity (Li et al. 2018; Zhang et al. 2017). Our findings evidenced that CsTCP15 acts as a positive regulator of citrus immunity against CLas proliferation. CLas, a notorious phloem‐restricted bacterium, colonises citrus phloem tissues (Huang et al. 2020; Merfa et al. 2019), yet how it modulates host vascular development for colonisation remains unclear. Considering that CLas infection requires host vascular remodelling, it is worth investigating whether CsTCP15 might coordinate citrus phloem development by transcriptionally regulating downstream genes. Future studies integrating single‐cell RNA sequencing of vascular tissues and CRISPR‐mediated CsTCP15 knockout could dissect its role in coordinating phloem differentiation and SA‐responsive immunity, thereby illuminating CLas colonisation strategies. Additionally, for phloem‐infecting pathogens relying on insect vectors, secreted effectors often evolve specialised functions to manipulate host physiology for vector fitness. A paradigm case is the phytoplasma effector SAP11, which destabilises Arabidopsis CIN‐TCP transcription factors to suppress jasmonic acid (JA)‐mediated defences, thereby enhancing fecundity of its leafhopper vector (Sugio et al. 2011). This highlights a conserved strategy where phloem pathogens exploit TCP‐mediated regulatory networks to subvert host immunity. Whether CLas similarly modulates TCP pathways to simultaneously suppress citrus immunity and enhance psyllid attraction remains an open question—one that could be tested through psyllid behavioural assays using genetically modified citrus plants.

Transcription factors, including TCP proteins, often depend on dimerization to regulate gene expression (Kosugi and Ohashi 2002; Martin‐Trillo and Cubas 2010). Pathogens have evolved effectors that target this dimer‐dependent regulatory system. For instance, the oomycete effector Pi22798 enhances virulence by promoting homodimerization of StKNOX3 (Zhou et al. 2022), while the TSWV NSs protein disrupts TCP17 homodimerization to suppress immunity and facilitate infection (Zhao et al. 2024). Similarly, we found that mSECP8 inhibits CsTCP15 homodimerization, thereby impairing the transcriptional activation of downstream immune genes. Notably, mSECP8 acts through a dual mechanism: directly blocking dimerization and indirectly promoting CsBRG3‐mediated degradation via the 26S proteasome. This dual strategy is also employed by another CLas effector, SDE5, which acts by disrupting MYC2 function (Zhao et al. 2025). Interestingly, like NSs on TCP17, mSECP8 does not affect the DNA‐binding ability of CsTCP15. Despite our efforts to map the critical motifs governing CsTCP15 dimerization and SECP8 binding, all three truncated variants retained both capabilities, suggesting multiple regions are involved. The structural complexity of TCP factors has obscured the precise link between mSECP8‐induced degradation and dimerization disruption. Future work employing cryo‐EM and mutagenesis should therefore focus on elucidating the structural basis of TCP dimerization to reveal how pathogens exploit these interfaces for immune suppression.

In summary, our work reveals a virulence strategy whereby the CLas effector mSECP8 suppresses plant immunity by directly and indirectly disrupting CsTCP15 dimerization (Figure 6h). This finding not only identifies the immune components CsTCP15, CsPR5, and CsWRKY22 as prime targets for breeding resistant citrus but also opens a path for therapeutic intervention. Future efforts should focus on developing transgenic lines and inhibitory peptides targeting the CsBRG3 ubiquitination pathway, which would offer both a sustainable management strategy and fundamental insights into ubiquitin‐mediated immune coordination.

4. Materials and Methods

4.1. Plant Materials and Plant Growth Conditions

N. benthamiana plants were grown in a growth chamber maintained at 25°C with a 16‐h light/8‐h dark photoperiod and 65% relative humidity. N. benthamiana aged 3–4 weeks old were used for experiments. The Wanjincheng orange ( Citrus sinensis ) was grown in the plant greenhouse of the Germplasm Creation Science Center in western Chongqing, China.

4.2. Generation of Transgenic Citrus

The full‐length coding sequence (CDS) of CsTCP15 (Cs_ont_7g026320), CsPR5 (Cs_ont_1g028270), and CsWRKY22 (Cs_ont_5g048610) was successfully amplified from Wanjincheng orange, and CsTCP15 was fused with FLAG‐tag. Subsequently, CsTCP15‐FLAG, CsPR5, and CsWRKY22 were cloned into the pNmGFPer vector to generate pNmGFPer:CsTCP15‐FLAG, pNmGFPer:CsPR5, and pNmGFPer:CsWRKY22 constructs. Meanwhile, the CsTCP15 interference (235–581 bp) vector (CsTCP15‐RNAi) was constructed following the protocol established by Zou et al. (2021). The pNmGFPer:CsTCP15‐FLAG and CsTCP15‐RNAi constructs were transformed into Agrobacterium tumefaciens ( A. tumefaciens ) EHA105 to obtain transgenic citrus. Additionally, pNmGFPer:CsPR5 and pNmGFPer:CsWRKY22 constructs were introduced into A. tumefaciens K599 to generate OE‐CsPR5 and OE‐CsWRKY22 citrus hairy roots, with pNmGFPer:00 serving as the negative control. Finally, transgenic lines were robustly validated through GFP fluorescence observation, construct detection, and RT‐qPCR assays. Young leaves from both the R‐2 and WT citrus were collected for transcriptome sequencing, with the WT serving as the control group. The transcriptome sequencing was constructed by Novogene Co. Ltd. Primers for constructs and RT‐qPCR detection are detailed in Table S1.

4.3. The Susceptibility Evaluations of Transgenic Citrus to Huanglongbing

The tolerance of transgenic plants to CLas was evaluated using the grafting‐based method described by Zou et al. (2017). To ensure a consistent initial CLas population in the buds used for transformation, we selected those with CLas population densities (CLas cells μg⁻¹ of citrus DNA) falling within 6.34 ± 0.5, which corresponds to the average density measured in the grafted buds (Figure S8a). CLas‐infected buds were graft‐inoculated approximately 2 cm away from the transgenic scion graft union on transgenic (OE‐CsTCP15 and RNAi‐CsTCP15) and WT citrus plants (Figure S8b), with 3 ~ 5 biological replicates per line. To monitor CLas proliferation, midribs from 3 ~ 5 leaves from newly flushing and mature leaves with suspect symptoms were collected from each plant and pooled for DNA isolation at bimonthly intervals. Meanwhile, the OE‐CsPR5, OE‐CsWRKY22, and pNmGFPer:00 citrus hairy roots were graft‐inoculated with CLas‐infected buds on the aerial parts. The selection criteria for CLas‐infected buds were similar to those applied to the transgenic citrus plants (Figure S8c) that CLas population densities (CLas cells μg⁻¹ of citrus DNA) falling within 6.56 ± 0.5, with 3 ~ 5 biological replicates/lines per gene (Figure S8d). Thereafter, 20 dpi, CLas had successfully colonised the branch and was translocated basipetally, resulting in substantial accumulation of the bacterium in the root tissues. Genomic DNA was extracted from the citrus hairy roots. The bacterial titers were quantified using the citrus 18S rRNA gene as an internal reference, following the qPCR‐based protocol of Zou et al. (2017) to assess the resistance conferred by CsPR5 and CsWRKY22 overexpression. The primers used are listed in Table S1.

4.4. Expression and Purification of Recombinant Proteins

The coding sequences of CsTCP15 were fused with GST and cloned into the pGEX‐6p vector. The coding sequences of CsTCP15, mSECP8, CsBRG3 (Cs_ont_5g032190), and FLAG‐GFP were cloned into the pET‐28a vector with a His tag to obtain His‐SUMO‐CsTCP15, His‐SUMO‐mSECP8, His‐SUMO‐CsBRG3, and His‐FLAG‐GFP. The coding sequences of mSECP8 were cloned into the pMAL‐C5X vector with MPB and GFP tag to obtain MBP‐mSECP8‐GFP. Subsequently, these constructs were transformed into the Escherichia coli Rosetta (DE3) strain for protein expression. These cells proliferate to a density of OD₆₀₀ = 0.4–0.5 at 37°C and are induced with 0.1 mM isopropyl β‐D‐thiogalactoside (IPTG) at 16°C for 16 h. Cells were harvested and resuspended in 1 × PBS buffer, decomposed by ultrasound, and then centrifuged at 12,000 rpm for 10 min. Finally, the recombinant proteins were purified using a GST or His Purification Kit.

4.5. Split Luciferase Complementation Assay (LCA)

The full‐length coding sequences of CsTCP15, mSECP8, and CsBRG3 were cloned into the LCA vector pCAMBIA‐1300 to obtain CsTCP15‐nLuc, cLuc‐CsTCP15, cLuc‐mSECP8, and CsBRG3‐nLuc. These constructs were then separately transformed into A. tumefaciens GV3101 (pSoup). The A. tumefaciens harbouring nLuc and cLuc were mixed in a 1:1 ratio at OD₆₀₀ = 0.6. Two days post infiltration, the infiltrated N. benthamiana leaves were sprayed with 100 mM D‐luciferin and then kept in the dark for 5 min for observation. The signals were detected and analysed using the Tanon‐5200 imaging system and ImageJ software.

4.6. BiFC Assay and Subcellular Localization Assays

For BiFC assays, the full‐length coding sequences of CsTCP15, mSECP8, and CsBRG3 were cloned to construct pCV‐CsTCP15‐nYFP, pCV‐CsTCP15‐cYFP, pCV‐mSECP8‐nYFP, and pCV‐CsBRG3‐nYFP. Meanwhile, the pCV‐CsTCP15‐GFP, pCV‐mSECP8‐GFP, and pCV‐mSECP8‐BFP were constructed for subcellular localization assays. These constructs were then separately transformed into A. tumefaciens GV3101 (pSoup). The BiFC and subcellular localization assay injection protocol is consistent with the LCA experimental protocol. After 2 days of infiltration, fluorescence images of the epidermal cells in infiltrated N. benthamiana leaves were captured using an Olympus FV3000 confocal microscope. The subcellular localization assay in Wanjincheng orange leaves was conducted 3 days after infiltration. Epidermal cells along the citrus leaves midribs were gently scraped with a razor blade and examined using an Olympus FV3000 confocal microscope. The YFP fluorescence signal was detected under a 514 nm excitation wavelength; the GFP was detected under a 552 nm excitation wavelength; BFP was detected under a 400 nm excitation wavelength with an Olympus FV3000 confocal microscope.

4.7. Co‐Immunoprecipitation (Co‐IP) Assay

The full‐length coding sequences of CsTCP15 and mSECP8 were cloned into the pCAMBIA‐1300 vector with HA tag and FLAG‐GFP tag. These constructs were then separately transformed into A. tumefaciens GV3101 (pSoup). The A. tumefaciens containing HA‐CsTCP15 and FLAG‐mSECP8‐GFP were co‐expressed in N. benthamiana plants with the LCA experimental protocol. Total proteins were extracted using an extraction buffer (Cat No. bc3720, Solarbio, Beijing Solarbio Science and Technology) and then incubated at 4°C for 2 h with gentle agitation in the presence of HA‐tagged beads. The mixture was then washed 5 times with PBS buffer and detected by immunoblotting using anti‐HA antibody and anti‐FLAG antibody. The bands were detected using the Tanon‐5200 imaging system.

4.8. Pull‐Down Assay

To verify the interaction between CsTCP15 and mSECP8 (or CsTCP15), GST‐CsTCP15/GST was incubated with gentle agitation at 4°C for 2 h in the presence of GST‐tagged beads. The mixture was subsequently washed five times with 1× PBS buffer. His‐SUMO‐mSECP8 (or His‐SUMO‐CsTCP15) was then added to the mixture and incubated at 4°C for another 2 h. Following this, the mixture was washed five times with 1× PBS buffer and detected by immunoblot analysis using anti‐GST or anti‐His antibodies.

Pull‐down competition assay was performed by incubating five equal aliquots of His‐SUMO‐CsTCP15 with His‐tagged magnetic beads at 4°C for 2 h with gentle agitation. After five washes with 1× PBS buffer, equal amounts of GST‐CsTCP15 were added to the mixtures (Groups 1–4). Increasing amounts of MBP‐mSECP8‐GFP (1×, 2×, and 3×) to groups 2–3, and MBP to group 1 were followed by addition. The samples were then incubated for an additional 2 h at 4°C. Protein interactions were finally analysed by immunoblotting using anti‐GST, anti‐His, and anti‐MBP antibodies.

The His pull‐down assay was performed to verify the interaction between CsTCP15 and CsBRG3. The His‐SUMO‐CsBRG3/His‐FLAG‐GFP was incubated with gentle agitation at 4°C for 2 h in the presence of His‐tagged beads. After five washes with 1× PBS buffer, the GST‐CsTCP15 was then added to the mixture and incubated at 4°C for another 2 h. Following this, the mixture was washed five times with 1× PBS buffer and detected by immunoblot analysis using anti‐GST or anti‐His antibodies.

4.9. ChIP‐qPCR Analysis

ChIP‐qPCR assays were performed with the EpiQuik Plant ChIP Kit (Cat No. P‐2014, EpigenTek Group Inc) in accordance with the manufacturer's instructions. Leaves from OE‐3 citrus plants were harvested and immediately fixed under vacuum with 1% formaldehyde for 15 min, followed by quenching with glycine buffer. After crosslinking, the chromatin complexes were fragmented by sonication to an average size of approximately 500–1000 bp. Immunoprecipitation was conducted using an anti‐FLAG antibody, with IgG as the control. The enrichment of target DNA fragments was evaluated by qPCR and normalised to the input DNA. All primers used for ChIP‐qPCR are listed in Table S1.

4.10. Cell‐Free Protein Degradation Assay

Total proteins were extracted from leaves of mSECP8 and OE‐CsTCP15 transgenic citrus, along with WT citrus as controls. Eight equal aliquots of total protein from OE‐CsTCP15 transgenic citrus leaves were separately mixed with 1×, 2×, 3×, and 3× (+MG132) mSECP8/WT citrus leaf proteins, followed by co‐incubation at 28°C for 2 h. Protein abundance of CsTCP15 was finally analysed by immunoblotting using anti‐FLAG and anti‐Actin antibodies.

4.11. In Vitro Ubiquitination Assay

The ubiquitination reaction mixture (30 μL) containing ubiquitination reaction buffer (50 mM Tris–HCl (pH 7.5), 5 mM MgCl₂, 2 mM DTT, and 2 mM ATP), E1 (Cat No. E‐305, R&D Systems), E2 (Cat No. E2‐627, R&D Systems), and ubiquitin (Cat No. U‐110, R&D Systems) was prepared. After incubation at 30°C for 3 h, the proteins were separated using 10% SDS‐PAGE gels and analysed by immunoblotting using anti‐ubiquitin, anti‐His, and anti‐GST antibodies.

4.12. Yeast Two‐Hybrid (Y2H) Assay

The full‐length coding sequences of mSECP8 and CsBRG3 were cloned into the pGBKT7 vector to fuse with the GAL4 binding domain, and CsTCP15 was cloned into the pGADT7 vector. The AD‐prey and BD‐bait were transformed into Y2H gold yeast cells and grown at 30°C on the SD‐LT plates (lacking leucine and tryptophan) and SD‐LTHA (with X‐α‐gal or not) plates (lacking leucine, tryptophan, histidine, and adenine).

4.13. Yeast Three‐Hybrid (Y3H) Assay

For the Y3H assay, the full‐length coding sequences of CsTCP15 and mSECP8 were cloned to construct the pBridge‐CsTCP15‐MBP and pBridge‐CsTCP15‐mSECP8. The AD‐CsTCP15 plasmid was co‐transformed with pBridge‐CsTCP15‐MBP and pBridge‐CsTCP15‐mSECP8 into the yeast Y2H gold yeast cells and cultured at 30°C on SD‐LT plates (lacking leucine and tryptophan) and SD‐MHLT plates (lacking methionine, histidine, leucine, and tryptophan), respectively.

4.14. Dual‐Luciferase Reporter Assay System

The promoter sequences (~−2000 bp) of CsPR5 and CsWRKY22 were cloned into the pGreenII 0800 LUC vector. These constructs were then separately transformed into A. tumefaciens GV3101 (pSoup). The A. tumefaciens harbouring pCAMBIA‐1300:HA‐CsTCP15 and 0800‐CsPR5 or 0800‐CsWRKY22 were mixed in a 1:1 ratio at OD₆₀₀ = 0.6. After 2 days of infiltration, the infiltrated N. benthamiana leaves were sprayed with 100 mM D‐luciferin and then kept in the dark for 5 min. The signals were detected and analysed using the Tanon‐5200 imaging system and ImageJ software. A Dualucif Firefly & Renilla Assay Kit (Cat No. F6075, UElandy, Suzhou, China) was used to detect luciferase activity according to the manufacturer's instructions.

4.15. Electrophoretic Mobility Shift Assay (EMSA)

Synthesised 5′‐terminal biotin‐labelled forward primers were annealed with reverse single‐stranded oligonucleotides to generate double‐stranded probes at 95°C for 5 min. The EMSA was conducted using the Chemiluminescent EMSA Kit (Cat No. MH104, Coolaber, Beijing, China) following the manufacturer's protocol. Each reaction contained 5 pmol of protein and 20 fmol of biotin‐labelled double‐stranded probe. Unlabelled competitive probes were included at a 200‐fold molar excess (10 μM) relative to the biotin‐labelled probes. Additionally, mutated versions of the competitive probes were tested at the same concentration (10 μM) to assess binding specificity.

4.16. Determination of Phytohormone Levels

To determine SA levels, leaf samples (0.2 g) from OE‐CsTCP15, RNAi‐CsTCP15, mSECP8 transgenic, and WT citrus were processed using the Plant Salicylic Acid (SA) ELISA Detection Kit (Cat No. YX‐190100P, Sinobestbio, Shanghai, China) in accordance with the manufacturer's protocol. Three biological replicates were included for statistical reliability.

4.17. RNA Extraction and Quantitative RT‐PCR

Total RNA was extracted from citrus leaves using the EASYspin Plant RNA Extraction Kit (Cat No. RN3801, Aidlab). First‐strand cDNA was synthesised using the abm All‐In‐One 5× RT MasterMix (Cat No. G592, ABM). Quantitative real‐time PCR was performed with BlasTaq 2× qPCR MasterMix (Cat No. G891, ABM) following the manufacturer's protocols. All primers used for RT‐qPCR are listed in Table S1.

4.18. Transcriptome Sequencing

For high‐throughput RNA sequencing, fully expanded young leaves from both R‐2 and WT plants were collected, with WT serving as the control group. The R‐2 and WT groups each contained multiple leaf samples, which were subsequently ground into powder. To minimise technical variability during sequencing, three aliquots (~0.3 g each) per biological replicate were processed for RNA library construction. The resulting libraries were subjected to high‐throughput sequencing on the Illumina HiSeq 2500 platform.

Author Contributions

Mingyue Qin analysed the data, and wrote the manuscript. Mingyue Qin and Rongyu Pan carried out the experiment. Yaqian Shi, Yuanjian Qiu, and Xuejin Cui constructed the transgenic citrus vector. Pan Shen provided the mSECP8 transgenic citrus material. Shimin Fu, Changyong Zhou, and Xuefeng Wang revised it critically for important intellectual content. Shimin Fu and Changyong Zhou supervised the research. All authors read and approved the final version of the manuscript.

Funding

This work was supported by the National Natural Sciences Foundation of China (32402312, U23A20196), Natural Science Foundation of Chongqing, China (CSTB2024NSCQ‐MSX1133), Special Fund for Youth Team of Southwest University (SWU‐XJLJ202310), Innovation Research 2035 Pilot Plan of Southwest University (SWU‐XDZD22002), and the National Key Research and Development Program of China (2021YFD1400800).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: The identification of CsTCP15 in citrus.

Phylogenetic tree in TCP families from Arabidopsis thaliana and Citrus sinensis .

Figure S2: Subcellular localization of CsTCP15 and mSECP8. (a) The subcellular localization of CsTCP15 and mSECP8 proteins when individually expressed or co‐expressed in N. benthamiana leaves. Scale bar = 100 μm. (b) The subcellular localization of mSECP8 proteins when individually expressed in Wanjincheng orange leaves. Scale bar = 100 μm. All experiments were repeated at least three times with similar results.

Figure S3: OE‐CsTCP15 and RNAi‐CsTCP15 transgenic plant construction and identification. (a) Schematic diagrams of the vector used for constructing the OE‐CsTCP15 transgenic citrus. (b) PCR‐diagnostic image of CsTCP15 in OE‐CsTCP15 transgenic citrus (P: Plasmid). (c) The accumulation of CsTCP15 protein in OE‐CsTCP15 plants, as determined by western blotting. Total proteins were extracted from the leaves of OE‐1, OE‐2, and OE‐3, and immunoblot analysis was performed using anti‐FLAG and anti‐Actin antibodies, respectively. Actin protein served as the internal control. (d) Schematic diagrams of the vector used for constructing the RNAi‐CsTCP15 transgenic citrus. (e) PCR‐diagnostic image of CsTCP15 in RNAi‐CsTCP15 transgenic citrus (P: Plasmid). All experiments were repeated at least three times with similar results.

Figure S4: Transcriptome analysis of R‐2 transgenic lines. (a) Volcano plot of DEGs between R‐2 transgenic lines and WT. (b) GO enrichment analysis of downregulated genes. (c) KEGG enrichment analysis of downregulated genes. (d) Heatmap of SA‐related genes in RNA sequencing of R‐2 transgenic lines.

Figure S5: CsTCP15 forms homodimeric complexes. (a) LCA assay of the homodimeric complexes of CsTCP15. CsTCP15‐nLuc+cLuc‐GUS, GUS‐nLuc+cLuc‐GUS, GUS‐nLuc+cLuc‐CsTCP15 were used as the negative control. (b) CsTCP15 has the ability to form homodimeric complexes in BiFC assay. Yn:CsTCP15/Yc were used as the negative control. Scale bar = 100 μm. (c) The GST pull‐down assay confirms the homodimeric complexes of CsTCP15. All experiments were repeated at least three times with similar results.

Figure S6: The interaction between CsTCP15 and its truncates with mSECP8. (a) Schematic representations of the truncated sequence of CsTCP15. (b‐e) The interaction between CsTCP15 and the truncated sequence in the LCA assay. (f) LCA assay of the interaction between mSECP8 and the truncates. Statistical analysis was performed using the Student's t‐test, **p < 0.005, n = 3 biologically independent samples. All experiments were repeated at least three times with similar results.

Figure S7: E3 ligase CsBRG3 facilitates the degradation of CsTCP15 via the 26S proteasome pathway. (a) The mRNA expression of CsBRG3 in mSECP8‐transgenic citrus. (b) CsBRG3 interacts with CsTCP15 in Y2H. Y2H gold yeast cells co‐transformed with AD‐CsTCP15 and BD‐CsBRG3 plated on SD‐LT (lacking leucine and tryptophan) and SD‐LTHA (lacking leucine, tryptophan, histidine and adenine) plates. BD‐53/AD‐T7 was used as the positive control. BD‐Lam/AD‐T7, BD/CsTCP15 and BD‐CsBRG3/AD were used as the negative control. (c) LCA assay of the interaction between CsBRG3 and CsTCP15. CsBRG3‐nLuc+cLuc‐GUS, GUS‐nLuc+cLuc‐GUS, GUS‐nLuc+cLuc‐CsTCP15 were used as the negative control. (d) CsBRG3 interacts with CsTCP15 in BiFC assay. Yn:CsBRG3/Yc were used as the negative control. Scale bar = 100 μm. (e) The His pull‐down assay confirms the interaction between CsBRG3 and CsTCP15. (f) CsBRG3 does not interact with mSECP8 in BiFC assay. Scale bar = 100 μm. (g) CsBRG3 does not interact with mSECP8 in Co‐IP assay. Total proteins were extracted from the N. benthamiana leaves of FLAG‐mSECP8‐GFP/FLAG‐GFP and MYC‐CsBRG3 co‐infiltrated and immunoprecipitated with FLAG‐Trap. Protein bands were detected by anti‐FLAG and anti‐MYC antibodies, respectively. (h‐i) The effect of CsBRG3 on CsTCP15 degradation assay without and with 26S proteasome inhibitor MG132 treatment. Total proteins were extracted from the N. benthamiana leaves at 48 hpi and detected by anti‐MYC, anti‐HA and anti‐Actin antibodies, respectively. N. benthamiana actin protein was used as the internal control. Statistical analysis was performed using the Student's t‐test, ***p < 0.001, ****p < 0.0001, n = 3 technical replicates. All experiments were repeated at least three times with similar results.

Figure S8: Schematic diagram of CLas grafting inoculation sites and parts sampled for CLas detection. (a) CLas population densities (CLas cells μg⁻¹ of citrus DNA) of buds grafted on the OE‐CsTCP15, WT and RNAi‐CsTCP15 transgenic citrus plants were quantified by qPCR analysis. The average (6.34) represents that the average CLas population densities (CLas cells μg⁻¹ of citrus DNA) were measured in the grafted buds. (b) Schematic diagram of CLas grafting and inoculation of OE‐CsTCP15, WT and RNAi‐CsTCP15. (c) CLas population densities (CLas cells μg⁻¹ of citrus DNA) of buds grafted on the pNmGFPer: 00, OE‐CsPR5 and OE‐CsWRKY22 citrus hairy roots were quantified by qPCR analysis. The average (6.56) represents that the average CLas population densities (CLas cells μg⁻¹ of citrus DNA) were measured in the grafted buds. (d) Schematic diagram of CLas grafting and inoculation of pNmGFPer: 00, OE‐CsPR5 and OE‐CsWRKY22 citrus hairy roots. Statistical analysis was performed using the Student's t‐test, n = 3 technical replicates. All experiments were repeated at least three times with similar results.

Table S1: Primers used in this study.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.