Tomato antiviral ubiquitin-proteasome system recognizes viral 59 kDa protein to confer tomato chlorosis virus resistance

Domain: neuroscience
Published: 2026-01-31
Editorial outcome: major_revision

Abstract
Viral diseases critically impact crop yields, necessitating insights into plant-pathogen recognition and resistance. In tomato (Solanum lycopersicum), tomato chlorosis virus (ToCV) employs 59 kDa protein (p59) to facilitate movement and degrade the host Catalase 1 (SlCAT1), disrupting redox homeostasis. Importantly, tomato has evolved an antiviral ubiquitin-proteasome system (UPS) featuring the Antiviral E2 ubiquitin-conjugating enzyme (SlAVE2) and Antiviral E3 ubiquitin ligase (SlAVE3) pair, which specifically recognizes p59 to trigger antiviral defenses. During infection, this system ubiquitinates SlWRKY6, alleviating its repression of SlAVE3 and creating a positive feedback loop that enhances SlAVE3 expression. SlAVE3 then targets p59 for ubiquitination and degradation, inhibiting viral spread. Notably, p59 co-opts this UPS to degrade SlCAT1, illustrating a counter-defense interplay. Domestication analysis reveals selection of AVE3 from S. pimpinellifolium to S. lycopersicum, with SpAVE3 conferring stronger ToCV resistance than SlAVE3 through improved p59 targeting and reduced SlCAT1 degradation. This study unveils a domesticated antiviral UPS, providing a foundation for breeding ToCV-resistant tomatoes.

Introduction
Plant disease epidemics significantly threaten local and global food security and exacerbate malnutrition1. Annual global agricultural losses due to plant viruses are estimated at $30 billion2. Tomato (Solanum lycopersicum), a major global crop with worldwide production exceeding 180 Mt, is highly susceptible to viral diseases, suffering yield losses of 70–95% that threaten supply3. Tomato-infecting viruses primarily possess RNA genomes, with Geminiviridae being the only DNA virus family among major pathogens4. A key RNA virus, tomato chlorosis virus (ToCV; Crinivirus, Closteroviridae), severely impacts global tomato production. No tomato germplasm (wild or cultivated) exhibits complete immunity to ToCV. The ToCV genome contains two single-stranded, positive-sense RNAs (RNA1/RNA2), each encapsidated in filamentous virions5. RNA1 harbors four open reading frames (ORFs) encoding proteins for virus replication (ORF1a and RNA dependent RNA polymerase, RdRp)6 and silencing suppression (22 kDa protein, p22)7. RNA2 contains nine ORFs encoding proteins putatively involved in virion encapsidation and assembly (capsid protein, CP; minor coat protein, CPm; 27 kDa protein, p27)8,9, cell-to-cell movement (7 kDa protein, p7)10, silencing suppression (CP and CPm)11, and whitefly transmission (CPm)12. Therefore, elucidating ToCV pathogenicity and host resistance requires further study of the six uncharacterized proteins, while identifying natural resistance genes from wild tomatoes is key for translating these insights into breeding applications.

Plants employ diverse defence mechanisms against viruses, including RNA silencing, immune receptor/sensor signaling, protein degradation, and defence mediated by changes of redox, hormones, and metabolism13. RNA silencing is a major antiviral pathway, often counteracted by co-evolving viral suppressors to enhance pathogenicity in susceptible hosts14. A sophisticated two-level innate immune system underpins plant antiviral defence15. The first level, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), involves surface-localized pattern recognition receptors detecting PAMPs16. Proteins like BRI1-associated kinase 1 (BAK1) and nuclear shuttle protein (NSP)-interacting kinase 1 (NIK1) initiate PTI for virus resistance17–19. The second level, effector-triggered immunity (ETI), relies on intracellular resistance (R) proteins that recognize pathogen effectors, triggering antiviral defence16. The first identified R protein, conferring resistance to Tobacco mosaic virus (TMV) in Nicotiana benthamiana, is encoded by N gene20. Subsequently, numerous R genes (Sw-5, Rx1/2, RTM1/2, RCY1, I, et al.) have been cloned and utilized in breeding21. The plant-virus arms race hinges on reciprocal recognition: the host activates immunity upon detection, and the pathogen launches virulence. Understanding these opposing recognition mechanisms remains a fundamental goal in the field.

In addition to innate immune system, another antiviral pathway that can precisely identify viral proteins is the protein degradation mediated by the ubiquitin-proteasome system (UPS) and selective autophagy pathways22,23. The ubiquitination process involves a three-step enzymatic cascade mediated by E1 ubiquitin activating enzyme (E1), E2 ubiquitin-conjugating enzyme (E2), and E3 ubiquitin ligase (E3). Substrate proteins can undergo mono-monoubiquitination (single ubiquitin at one site), multi-monoubiquitination (single ubiquitin at multiple sites), or polyubiquitination (ubiquitin chains at a single site)24. Monoubiquitination and multi-monoubiquitination typically modulate protein interactions, activation, or localization, whereas polyubiquitination often targets substrates for degradation via the UPS24. E3 determines substrate specificity and ubiquitination type24. Structurally, the E3 family is classified into four groups: three monomeric families (HECT, U-box, RING) and one multimeric family (CRLs)24. Tomato genome (SL4.0) contains approximately 1500–2100 E3s (5–6% of its total gene content), which is comparable in scale to Arabidopsis (~1400) and rice (~1300). In plant-virus interactions, the UPS performs the dual functions of viral immune suppression and host antiviral defense25,26. Virus can hijack UPS and UPS-related proteins to disrupt the host immunity27–29, preventing degradation by virus-encoded deubiquitinating enzymes30, and SUMOylation of viral replication proteins31. The UPS also plays key roles in plant antiviral defense, including mediating N gene resistance against TMV and directly degrading several viral proteins22,32. Additionally, the UPS activates other antiviral responses by recognizing viral proteins such as Rice stripe virus CP via a rice E333. Understanding how plant UPS components recognize viral proteins and transduce immune signals is key, as it underpins the mechanisms of intracellular viral recognition and host defense.

Here, we characterized a previously unstudied ToCV 59 kDa protein (p59), which functions in virion assembly, cell-to-cell movement, and pathogenicity. The p59 is targeted for degradation by tomato’s antiviral UPS, yet paradoxically exploits this system to suppress host catalase activity and generate pathogenicity-promoting oxidative stress. Notably, the E3 of this UPS underwent selection during tomato domestication, with beneficial variants from wild tomato germplasm enhancing ToCV resistance. These results demonstrate the evolutionary arms race between plants and pathogens, providing genetic resources for utilizing natural variation in biotech breeding.

Results
ToCV p59 functions in virion assembly and cell-to-cell movement
Closteroviridae genomes encode a conserved five-gene module, arranged 5’ to 3’ as follows: 6 kDa protein (p6) in RNA1, and 70 kDa heat shock protein homolog (Hsp70h), p59, CP, CPm in RNA2 (Supplementary Fig. 1a)34. In ToCV, CP and CPm are known to function in virion assembly and silencing suppression, respectively11. However, roles of p6, Hsp70h, and p59 remain undefined. To investigate their functions, we constructed ToCV mutants lacking p6 (ToCV-Δp6), Hsp70h (ToCV-ΔHsp70h), and p59 (ToCV-Δp59) (Supplementary Fig. 1b), and evaluated their pathogenicity. Here, we adopted a unified set of criteria to comprehensively assess host resistance and viral pathogenicity, including disease symptoms, disease incidence, disease index, CP transcript, and CP abundance. Inoculation of tomato plants with wild-type (WT) ToCV or the mutant variants showed that ToCV-Δp6, ToCV-ΔHsp70h, and ToCV-Δp59 all exhibited attenuated pathogenicity compared with the WT virus (Supplementary Fig. 1c–g). Among these, the reduction in pathogenicity was most pronounced in ToCV-Δp59. Furthermore, mutation of p59 led to aberrant virion morphology—mainly a reduction in virion length—suggesting its role in virion assembly (Fig. 1a and b). We then expressed p59 in tomato (PVX-p59). Compared with the empty vector control (TRV-EV), PVX-p59 not only enhanced the pathogenicity of ToCV but also restored the pathogenicity deficit in ToCV-Δp59-infected plants (Fig. 1c–e; Supplementary Fig. 1h and i). A similar effect was observed in WT and p59-overexpressing (p59-OE) N. benthamiana (Supplementary Fig. 1j–m). These results establish p59 as a key pathogenic determinant of ToCV.

The ability of a viral protein to suppress RNA silencing is a major determinant of infection severity, and most plant viruses encode at least one such suppressor. Despite this prevalence, p59 deviates from this pattern and does not function as an RNA silencing inhibitor (Supplementary Fig 2). Subcellular localization of viral pathogenic factors frequently influences viral functionality10. To determine the localization of p59, a p59-GFP fusion protein was expressed in N. benthamiana for fluorescence observation. NaCl-induced plasmolysis revealed that p59-GFP localizes to the cytoplasm and plasma membrane, exhibiting a punctate distribution pattern (Supplementary Fig. 3a). Given this pattern, we hypothesized potential localization to plasmodesmata (PD). Co-expression of p59-GFP with the PD marker TMV MP-RFP35 confirmed fluorescent co-localization, demonstrating PD targeting by p59-GFP (Fig. 1f; Supplementary Fig. 3a). As viruses commonly disturb callose deposition and utilize PD for cell-to-cell movement10,36, we investigated whether p59 modulates PD pore size and callose deposition. Upon ToCV infection, the dilation of PD apertures significantly increases as callose deposition decreases; however, mutation of p59 impair the effect of ToCV on suppressing callose deposition and dilating PD apertures (Fig. 1g and h; Supplementary Fig. 3b–d). Plant viral movement proteins target PD and facilitate rapid cytoplasmic transport to mediate intercellular viral spread37. In the cell-to-cell movement assay, free GFP (negative control) was primarily confined to one or two cells, whereas p59-GFP spread more broadly, reaching two or more cells, similar to the TMV MP-RFP positive control (Fig. 1i and j; Supplementary Fig. 4a). To confirm p59 is a movement protein, we tested its ability to complement the cell-to-cell movement deficiency in a Turnip mosaic virus mutant (TuMV-GFP-P3N-PIPO-m1)35. p59 functionally complemented the movement-deficient phenotype, mimicking the positive control P3N-PIPO by restoring expanded intercellular GFP distribution (Supplementary Fig. 4b and c). Thus, p59 functions as a viral movement protein.

Tomato antiviral UPS targets and degrades the p59 to confer ToCV resistance
To elucidate the plant recognition mechanism for p59, a yeast two-hybrid (Y2H) screen was performed using a cDNA library from ToCV-infected tomato leaves (Supplementary Table 1). Consequently, an E3 ubiquitin ligase was identified as a direct interactor with p59, suggesting that it may specifically recognize and ubiquitinate p59 for degradation, thereby potentially enhancing plant resistance to ToCV. Consequently, this gene was designated Antiviral E3 ubiquitin ligase (SlAVE3). Comparative genomic analysis reveals that SlAVE3 is the ortholog of Arabidopsis Botrytis Susceptible 1 Interactor (AtBOI), which has not been reported in plant-virus interactions.

To investigate the precise interaction sites, molecular docking analysis of SlAVE3 with p59 was conducted. The results indicated that the SlAVE3 C-terminus (Gly248 to Ser334) potentially mediates interaction with residues Lys123 to Lys517 on p59 (Supplementary Fig. 5a). Based on this docking analysis and protein domain prediction, SlAVE3 was truncated at position 245 to generate SlAVE3Δ1 (1-244 aa, containing a low-complexity region) and SlAVE3Δ2 (245-334 aa, containing coiled-coil and RING domains) (Supplementary Fig. 5b). Y2H assay confirmed that both full-length SlAVE3 and SlAVE3Δ2 interacted with p59, whereas SlAVE3Δ1 did not (Supplementary Fig. 5c). Analysis of docking data and p59 conservation within the Closteroviridae led to its truncation into three segments (p59Δ1, p59Δ2, p59Δ3) (Supplementary Fig. 5d). Y2H assay revealed that only p59Δ2, similar to full-length p59, interacted with SlAVE3 (Supplementary Fig. 5e). Within p59Δ2, three conserved amino acids Asn-Pro-Leu (NPL) were identified at positions 253-255 (Supplementary Fig. 5d). Ala substitution of all three residues (p59NPL-AAA) abolished the interaction with SlAVE3 in Y2H assay (Supplementary Fig. 5f). Further individual Ala substitutions demonstrated that only the p59L255A mutation disrupted the SlAVE3 interaction (Supplementary Fig. 5g).

The interactions were validated using co-immunoprecipitation (Co-IP), bimolecular fluorescence complementation (BiFC), and luciferase complementation imaging (LCI) assays. These assays consistently showed that the SlAVE3Δ2 region and the Leu255 residue of p59 are essential for the SlAVE3-p59 interaction (Fig. 2a and b; Supplementary Fig. 5h). Subcellular location of the SlAVE3-GFP was examined and found in both the cytoplasm and nucleus (Supplementary Fig. 6a). However, BiFC assays showed that the reconstituted YFP fluorescence from the SlAVE3-p59 interaction formed punctate aggregations specifically at the cell margins within the cytoplasm (Fig. 2b). This localization pattern was corroborated by bio-fluorescence imaging of SlAVE3-RFP co-expressed with p59-GFP (Supplementary Fig. 6b) and co-localization analysis of SlAVE3-nYFP and p59-cYFP interaction signals, which were predominantly localized to cytoplasm and PD (Supplementary Fig. 6c).

The C-terminus of SlAVE3 contains a C3-H-C5 type RING domain harboring conserved Cys and His residues (Supplementary Fig. 7a). Among these, the His residue is critical for the catalytic activity of RING-type E338. Given the interaction between SlAVE3 and p59, we hypothesized that p59 might be a ubiquitination substrate of SlAVE3. To test this, we first assessed whether SlAVE3 possesses E3 activity and whether this activity requires an intact RING domain. An E3 activity assay revealed a smear of high-molecular-weight bands when SlAVE3-Strep was incubated with ubiquitin, E1, and E2 (Supplementary Fig. 7b). In contrast, the SlAVE3H304Y-Strep mutant protein exhibited no detectable activity (Supplementary Fig. 7b). Furthermore, we performed both in vitro and in planta ubiquitination assays of p59 or its mutant p59L255A as potential substrates; p59, but not p59L255A, was ubiquitinated by SlAVE3 (Fig. 2c; Supplementary Fig. 7c).

Since ubiquitination targets proteins for 26S proteasome-dependent degradation, we next investigated whether SlAVE3 reduces p59 stability using a cell-free degradation assay. We generated two SlAVE3 loss-of-function mutants (slave3 #1 and #2) via CRISPR-Cas9-mediated knockout, employing single-guide RNAs targeting distinct exonic regions (Supplementary Fig. 8a). Additionally, we developed two SlAVE3-overexpression transgenic lines (SlAVE3-OE #1 and #2) under the CaMV 35S promoter, both exhibiting significantly elevated transcript levels (Supplementary Fig. 8b). Total protein extracts from WT and slave3 plants were incubated with purified p59-GST in an ATP-supplemented reaction mixture for 0 to 60 min. The degradation assay revealed that p59-GST degradation was delayed in slave3 extracts (Fig. 2d and e). Conversely, p59L255A-GST remained stable across all extracts, showing no differential degradation (Supplementary Fig. 7d).

Transcriptional analysis revealed predominant SlAVE3 expression in tomato stems, functional and old leaves (Supplementary Fig. 8c), which correspond to the primary sites of ToCV infection and symptom development. Furthermore, monitoring SlAVE3 expression during ToCV infection progression from 2 to 40 days post-inoculation (dpi) or across leaf positions from the basal inoculated leaf to the distal systemic leaves demonstrated an initial increase followed by a decline in transcript levels (Supplementary Fig. 8d and e). We next assessed the impact of SlAVE3-mediated p59 recognition on ToCV movement and resistance. Cell-to-cell movement assay revealed that free p59-GFP typically spread to 3–6 adjacent cells; however, co-expression with SlAVE3-Flag predominantly restricted p59-GFP to 1–2 cells (Fig. 2f and g; Supplementary Fig. 9a). Compared to p59-GST, the p59L255A-GST mutant exhibited significantly enhanced cell-to-cell movement and was unaffected by SlAVE3-Flag co-expression (Fig. 2f and g; Supplementary Fig. 9a). Analysis of systemic viral spread within tomato plants showed enhanced ToCV spread in slave3 mutants but reduced spread in SlAVE3-OE lines (Supplementary Fig. 9b and c). Similarly, the p59L255A mutation substantially increased the systemic spread rate of ToCV (Supplementary Fig. 9d and e). Compared to WT plants, slave3 mutants displayed susceptibility to ToCV, while SlAVE3-OE lines exhibited resistance phenotype (Fig. 2h–j; Supplementary Fig. 9f and g). Consistent with this, ToCV harboring the p59L255A mutation (ToCV-p59L255A) demonstrated significantly greater pathogenicity than ToCV (Supplementary Fig. 9h–l).

To identify the specific E2-E3 pair responsible for SlAVE3-mediated antiviral UPS, we performed a Y2H screen using the previous cDNA library. This screen identified an SlAVE3-interacting protein annotated as an E2. Based on subsequent functional characterization, we designated this gene Antiviral E2 ubiquitin-conjugating enzyme (SlAVE2). The SlAVE2-SlAVE3 interaction was confirmed by Y2H, LCI, and BiFC assays (Supplementary Fig. 10a–c). E3 activity assay revealed that SlAVE2 enhanced SlAVE3 activity more potently than generic E2 enzymes (Fig. 2k). We silenced SlAVE2 in both WT and slave3 backgrounds (Supplementary Fig. 10d). SlAVE2 knockdown significantly reduced ToCV resistance in WT plants but had no significant effect in slave3 mutants (Fig. 2l–n; Supplementary Fig. 10e and f). Unlike SlAVE3, transcript levels of SlAVE2 remained unchanged during ToCV infection (Supplementary Figs. 8d and 10g). Furthermore, SlAVE2 expression levels were similar across the WT, slave3, and SlAVE3-OE lines (Supplementary Fig. 10h). So, SlAVE2 functions primarily through SlAVE3 in mediating ToCV resistance.

SlAVE2-SlAVE3 UPS degrades SlWRKY6 to feedback-activate SlAVE3 transcription for ToCV resistance
During Y2H screen with SlAVE3, we additionally identified a transcription factor SlWRKY6. Two W-box motifs (canonical binding sites for WRKY) were identified in the SlAVE3 promoter (Supplementary Fig. 11a). Transcriptional profiling revealed an inverse correlation between SlAVE3 and SlWRKY6 expression during ToCV infection (Supplementary Fig. 11b and c), implying reciprocal regulation via feedback within the host-virus interaction. The interaction between SlAVE3 and SlWRKY6 was confirmed by Y2H, pull-down, and LCI assays (Fig. 3a; Supplementary Fig. 11d and e). Both in virto and in planta ubiquitination assays demonstrated that the SlAVE2-SlAVE3 pair directly ubiquitinates SlWRKY6 (Fig. 3b; Supplementary Fig. 11f). Cell-free degradation assay using extracts from WT and slave3 leaves showed that SlAVE3 promotes SlWRKY6 degradation (Fig. 3c and d).

Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR), electrophoretic mobility shift assays (EMSA), and transactivation assays were performed to elucidate the transcriptional regulation of SlAVE3 by SlWRKY6. ChIP-qPCR and EMSA assays confirmed direct binding of SlWRKY6 to SlAVE3 promoter (Fig. 3e; Supplementary Fig. 11g). Transactivation assay indicated that SlWRKY6 is a transcriptional inhibitor of SlAVE3 (Fig. 3f). Consistent with ToCV-induced SlAVE3 transcription, SlWRKY6 silencing significantly upregulated SlAVE3 transcript levels under both mock and ToCV-infected conditions, particularly under mock conditions (Supplementary Fig. 11h and i). To investigate the transcriptional network of SlWRKY6 in ToCV resistance, we performed transcriptome analysis on leaves of TRV-EV (both control and ToCV-infected conditions) and TRV-SlWRKY6 (ToCV-infected condition), forming two comparison groups: TRV-EV vs TRV-EV-ToCV and TRV-EV-ToCV vs TRV-SlWRKY6-ToCV, reflecting ToCV-infected effect and the SlWRKY6-specific effect under infection conditions. Venn diagram analysis revealed that ToCV-infected effect led to 4,936 significantly differentially expressed genes, while the SlWRKY6-specific effect resulted in 4,024 significantly differentially expressed genes, with 1,875 genes overlapping between the two effects (Supplementary Fig. 12a). KEGG pathway analysis indicated that the top three pathways in both groups were plant-pathogen interaction, MAPK signaling pathway, and plant hormone signal transduction (Supplementary Fig. 12b and c). Expression pattern analysis of the overlapping genes showed that the SlWRKY6-specific effect significantly reversed the expression changes of ToCV-infected effect (Supplementary Fig. 12d). Moreover, the top three KEGG pathways enriched in these overlapping genes remained plant-pathogen interaction, MAPK signaling pathway, and plant hormone signal transduction (Supplementary Fig. 12e). These findings demonstrate that the transcriptional regulatory function of SlWRKY6 is consistent with its observed disease-resistant phenotype.

We generated SlWRKY6-silenced lines in both WT and slave3 backgrounds (WT/TRV-EV, WT/TRV-SlWRKY6, slave3/TRV-EV, and slave3/TRV-SlWRKY6; TRV-EV served as empty vector control) to test their roles in ToCV resistance (Fig. 3g–i; Supplementary Fig. 11j and k). While SlWRKY6 silencing significantly enhanced ToCV resistance in WT background, it conferred only a marginal effect in slave3 background. Consequently, the slave3/TRV-SlWRKY6 plants exhibited a resistance profile comparable to the slave3/TRV-EV plants, not to the WT/TRV-SlWRKY6 plants. Thus, SlAVE3 is epistatic to SlWRKY6 in governing ToCV resistance. Collectively, these findings establish a feedback loop wherein degradation of SlWRKY6 by SlAVE2-SlAVE3 UPS relieves the transcriptional repression of SlAVE3, thereby promoting SlAVE3 accumulation and enhancing ToCV resistance.

p59 hijacks and degrades SlCAT1 via the SlAVE2-SlAVE3 UPS to facilitate ToCV pathogenicity
Using both p59 and SlAVE3 as bait proteins in Y2H screening, we also identified a common interactor: Catalase1 (SlCAT1). This finding implied that interactions among SlAVE3, p59, and SlCAT1 may influence redox homeostasis and ToCV resistance. We first monitored H2O2 accumulation and CAT activity during ToCV-mediated disease progression. Impaired CAT activity correlated with enhanced H2O2 accumulation and increased ToCV disease severity (Supplementary Fig. 13). This suggests that oxidative stress contributes to ToCV pathogenesis, reflecting compatibility of tomato-ToCV interaction in response to cellular reactive oxygen species (ROS).

Given the high sequence conservation among three SlCATs family members, we co-silenced SlCATs family (SlCAT1/2/3) and observed significantly reduced ToCV resistance concomitant with diminished CAT activity (Supplementary Fig. 14a–h), indicating that SlCATs function in antiviral defense. Tissue-specific expression analysis revealed that SlCAT1 transcripts were most abundant at ToCV infection sites (Supplementary Fig. 14i). We therefore focused subsequent analyses on SlCAT1. We further validated that both SlAVE3 and p59 separately interacted with SlCAT1 using Y2H, LCI, and BiFC assays (Fig. 4a; Supplementary Fig. 14j and k). Subcellular localization of SlCAT1-RFP exhibited punctate localization that co-localized with the peroxisomal marker SKL-BFP (Supplementary Fig. 15a). Notibly, BiFC assay revealed distinct subcellular patterns: SlAVE3-SlCAT1 interactions formed punctate localization, whereas p59-SlCAT1 interactions occurred diffusely throughout the cytoplasm (Fig. 4a). Consistent with this, co-localization of SlAVE3-SlCAT1 interactions were confined to peroxisomes (Supplementary Fig. 15b and c). In contrast, p59-SlCAT1 interactions showed cytoplasmic co-localization (Fig. 4a and b). Crucially, when p59 was expressed in SlCAT1-OE leaves, peroxisomal SlCAT1-GFP distributed to the cytoplasmic compartment (Fig. 4c). These findings demonstrated that p59 causes SlCAT1 to localize in the cytoplasm instead of peroxisomes, thereby subverting its biological function.

We next hypothesized that p59 functions as a viral effector to enhance SlAVE3-SlCAT1 interaction, thereby facilitating SlCAT1 degradation. MicroScale Thermophoresis (MST) quantified interaction affinities among these proteins. Dissociation constant (Kd) measurements revealed high-affinity interactions between p59 and both SlAVE3 (Kd=128.10 nM) and SlCAT1 (Kd=421.40 nM) in the nanomolar range (Supplementary Fig. 16a and b). In contrast, the SlAVE3-SlCAT1 affinity (Kd=1.89 µM) was weaker in the micromolar range (Fig. 4d). Strikingly, addition of p59 to the SlAVE3-SlCAT1 system enhanced their binding affinity 5.86-fold (Kd=322.50 nM) (Fig. 4e). Conversely, SlCAT1 addition did not significantly alter the SlAVE3-p59 interaction (Supplementary Fig. 16b and c). BiFC and Co-IP assays independently confirmed p59-mediated potentiation of SlAVE3-SlCAT1 binding (Supplementary Fig. 16d–g). Both in virto and in planta ubiquitination assays demonstrated that the SlAVE2-SlAVE3 pair directly ubiquitinates SlCAT1, with p59 significantly enhancing this function (Fig. 4f; Supplementary Fig. 16h). Cell-free degradation assay further revealed that SlAVE3 promoted the SlCAT1-GST degradation by comparing the extracts from WT and slave3 lines (Fig. 4g and h). Critically, p59 accelerated SlAVE3-mediated SlCAT1-GST degradation in all backgrounds (Fig. 4g and h). Although SlWRKY6 also interacted with SlAVE3, it did not affect the interactions between SlAVE3 and its partners, p59 or SlCAT1 (Supplementary Fig. 16i–l). These results suggested that p59 serves as a molecular scaffold that enhances SlAVE3-SlCAT1 interaction efficiency, ultimately promoting SlCAT1 ubiquitination and degradation.

Subsequently, SlCAT1 mutants (slcat1 #1 and #2) and SlCAT1-overexpression lines (SlCAT1-OE #1 and #2) were generated (Supplementary Fig. 17a–c). During ToCV infection, SlCAT1 enhanced CAT activity to reduce H2O2 accumulation, thus increasing ToCV resistance (Fig. 4i–k; Supplementary Fig. 17c–f). This is consistent with the previous observation that H2O2 homeostasis is disrupted during disease progression (Supplementary Fig. 13), confirming the function of SlCAT1 in ToCV resistance. We expressed p59 in both WT and slave3 backgrounds. p59 significantly inhibited CAT activity and increased H2O2 content in WT plants but had no effect in the slave3 mutant (Supplementary Fig. 17g and h), indicating that p59-mediated CAT inhibition depends on SlAVE3. Again, we silenced SlAVE2 in both WT and slave3 backgrounds and then expressed p59. Knockdown of SlAVE2 significantly restored CAT activity and reduced H2O2 content in WT plants, but had no significant effect in slave3 mutants (Supplementary Fig. 17i and j), suggesting that SlAVE2 primarily regulates CAT activity and H2O2 homeostasis through SlAVE3. Furthermore, we expressed SlAVE3, p59, or both in the SlCAT1-OE lines (tagged with SlCAT1-GFP). Compared to SlAVE3 alone, p59 expression more potently promoted SlCAT1 degradation, inhibited CAT activity, and elevated H2O2 levels (Supplementary Fig. 17k–m). Co-expression of SlAVE3 and p59 further enhanced these effects. Collectively, these results demonstrated that the p59 acts as an effector to induce SlAVE3-dependent degradation of SlCAT1, thereby promoting ToCV infection.

Unraveling the domestication and antiviral mechanism of the elite AVE3 haplotype
Modern cultivated big-fruited tomato (S. lycopersicum var. lycopersicum, Sll) originated through domestication of the wild relative S. pimpinellifolium (Sp) to cherry tomato (S. lycopersicum var. cerasiforme, Slc), followed by intensive breeding selection39. Intriguingly, AVE3 resides within a domestication sweep region exhibiting drastic nucleotide diversity reduction between Sp and Slc, but not between Slc and Sll (Fig. 5a). Within this region, Sp displayed a 4.1-fold higher of nucleotide diversity (π=3.7×10-3), compared with Slc (π=9.1×10-4; Supplementary Fig. 18a). We also computed Tajima’s D between Sp and Slc, and found that Slc showed a strongly negative value of Tajima’s D within the AVE3 region, further supporting the occurrence of a recent selective sweep (Supplementary Fig. 18b). Phylogenetic analysis confirmed minimal AVE3 diversity in cultivated accessions (Slc and Sll), suggesting selection during domestication (Supplementary Fig. 18c).

We identified two non-synonymous variants within the coding region of AVE3 on chromosome 5. A T-to-C substitution at 225,844 bp, changing glutamine to arginine at 199 aa; and an A-to-T variant at 226,986 bp, resulting in a serine to threonine substitution at 43 aa (Fig. 5a). We defined three AVE3 haplotypes based on these two variants, including haplotype 1 (homozygous derived), haplotype 2 (heterozygous), and haplotype 3 (reference-type) (Fig. 5b). Haplotype 1 predominated in wild Sp (64.1%, 66/103 accessions), haplotype 2 appeared exclusively in eight Slc accessions, haplotype 3 occurred in 99.8% (531/532) of cultivated accessions (Slc and Sll) (Fig. 5b). Therefore, haplotype 1 and haplotype 3 were assigned SpAVE3 and SlAVE3, respectively.

Plant domestication frequently results in diminished disease resistance40,41. To determine if AVE3 function was modified during tomato domestication, we cloned the SlAVE3 and SpAVE3 alleles from Sll and Sp accessions, respectively. We hypothesized that AVE3 sequence variation alters its substrate recognition and degradation capabilities. MST assay revealed significantly stronger binding affinity of SpAVE3 for p59 (Kd=45.82 nM; Fig. 5c) compared to SlAVE3 (Kd=421.40 nM; Supplementary Fig. 16a). Consistent with binding affinity, SpAVE3 exhibited enhanced ubiquitination and degradation of p59 relative to SlAVE3 (Fig. 5e, g and h; Supplementary Fig. 19a). Compared to SlAVE3, SpAVE3 showed reduced binding (Figs. 4d and 5d), ubiquitination (Fig. 5f; Supplementary Fig. 19b), and degradation (Fig. 5i and j) capabilities for SlCAT1. The exceptionally weak SpAVE3-SlCAT1 interaction (Kd=49.28 µM; Fig. 5d) suggests this interaction is unlikely to form physiologically, corroborated by minimal in planta ubiquitination (Supplementary Fig. 19b) and degradation (Fig. 5i and j) of SlCAT1. PVX-mediated expression of both SlAVE3 and SpAVE3 in the slave3 background (controls: PVX-EV in WT and slave3) restored ToCV resistance (Fig. 5k–m; Supplementary Fig. 19c and d). Further, SpAVE3 conferred superior ToCV resistance compared to SlAVE3. Together, SpAVE3 is an elite antiviral haplotype.

Methods
Gene accession numbers, vectors, and primers
Accession numbers of Antiviral E3 ubiquitin ligase (SlAVE3, Solyc05g005210), Antiviral E2 ubiquitin-conjugating enzyme (SlAVE2, Solyc06g070980), SlWRKY6 (Solyc02g080890), and Catalase1 (SlCAT1, Solyc12g094620) are associated with Sol Genomics Network. Accession numbers of tomato chlorosis virus (ToCV) infectious clones (RNA1, NC_007340.1; RNA2, NC_007341.1) and 59 kDa protein (p59, 3607997) are associated with National Center for Biotechnology Information. Primers and vectors are presented in the Supplementary Table 2.

Plant materials and genetic manipulations
S. lycopersicum Ailsa Craig and Nicotiana benthamiana LAB were used as wild-type (WT) plant materials.

Mutant construction. slave3 and slcat1 mutants were generated using CRISPR-Cas9. Two sgRNAs targeting the N-terminal of SlAVE3 or SlCAT1 coding sequences, respectively, were designed (CRISPOR tool; http://crispor.tefor.net/) and cloned into vector pHSE401 via homologous recombination by ClonExpress II One Step Cloning Kit (Cat# C112-01, Vazyme, China). Constructs were introduced into Agrobacterium tumefaciens LBA4404 strains for transformation of WT tomato plants. Mutants were confirmed by PCR and sequencing of genomic DNA. Homozygous were used for experiments.

Overexpression (OE) line construction. SlAVE3 and SlCAT1 coding sequences were amplified and cloned into pCAMBIA1300 under the CaMV 35S promoter, fusing a GFP tag. Constructs were transformed into WT tomato plants via LBA4404-mediated transformation. SlAVE3-OE and SlCAT1-OE lines were confirmed by PCR and gene expression analysis.

Virus-mediated gene expression. Gene expression was performed using potato virus X (PVX) vectors53. Coding sequences of target gene were amplified and cloned into PVX (PVX-target gene) under the CaMV 35S promoter with a GFP or Flag tag. PVX empty vector (PVX-EV) served as control. Reconstructed vectors in LBA4404 strains were used to infect germinated tomato seeds of specified genotypes. Seedlings need to undergo gene expression testing before all experiments.

Virus-induced gene silencing (VIGS). VIGS was performed using tobacco rattle virus (TRV) vectors TRV1 and TRV253. Target-specific sequences designed (VIGS tool; vigs.solgenomics.net) were amplified and cloned into TRV2. TRV1 and TRV2 constructs in LBA4404 strains were used to co-infect germinated seeds of specified genotypes. TRV empty vector (TRV-EV) was the control. Seedlings need to undergo gene expression testing before all experiments.

Viral materials and mutant construction
ToCV infectious clones (pCa-ToCR1 and pCa-ToCR2, Beijing isolate) were obtained from published sources54. These infectious clones were individually transformed into Agrobacterium tumefaciens GV3101 strains.

Primers were designed to generate ToCV mutants (ToCV-Δp6, ToCV-ΔHsp70h, ToCV-Δp59, ToCV-p59L255A). Sequences containing the desired mutations were amplified by PCR. Template plasmid was digested with DpnI and the product transformed into E. coli DH5α. Plasmids with confirmed mutations were introduced into GV3101 strains.

Viral infection and disease symptom assessment
One-month-old tomato plants were cultivated in a controlled greenhouse (10–13 h light and 11–14 h dark, 18–28°C, 60–70% humidity) and used for ToCV infection. The GV3101 strains carrying ToCV infectious clones were cultured overnight, pelleted, and resuspended in viral inoculation buffer (10 mM MgCl2, 100 mM MES, 200 µM acetosyringone, pH 5.2). The cultures were kept in darkness for 2 h. Cultures containing pCa-ToCR1 were mixed 1:1 with cultures containing pCa-ToCR2 (OD600 = 0.6-0.8). The mixed inoculum were inoculated into tomato stems and leaves using a modified protocol55. After inoculation, plants were maintained under the same greenhouse conditions.

At 28 days post-inoculation (dpi), disease symptoms, disease incidence, disease index, coat protein (CP) transcript, and CP abundance were evaluated to assess ToCV resistance. The second systemic leaves above the inoculation site were collected as samples. For each treatment, 20 plants were randomly selected, and the disease grade of the sample leaves was recorded based on the following scale: grade N, no noticeable symptoms; grade I, Scattered chlorotic spots (<20% of leaf area); grade II, obvious chlorotic spots (20–50% of leaf area); grade III, numerous chlorotic spots (50–80% of leaf area); grade IV: nearly complete loss of green color in mesophyll tissue (80–100% of leaf area). Representative images for each symptom grade are shown in Supplementary Fig. 13a. Disease incidence was calculated as the percentage of plants in each disease grade. The disease index was determined using the formula: Disease index = ∑(d×n)/(N×D), where d is the representative value of each disease grade, n is the number of plants in that grade, N is the total number of plants assessed, and D is the highest disease grade. Total RNA and protein were extracted from a pool of mixed sample leaves for analysis of CP transcript and CP abundance by RT-qPCR and immunoblotting assays, respectively.

Virion morphology observation and length measurement
Tomato plants were infected with ToCV or ToCV-Δp59 as previous. At 28 dpi, the second systemic leaves (100 g) were harvested for virion purification by polyethylene glycol (PEG) precipitation and differential centrifugation56. Leaf samples were homogenized in ice-cold extraction buffer (0.1 M sodium phosphate pH 7.2, 0.01 M EDTA, 0.1% β-mercaptoethanol) at a 1:2 (w/v) ratio. The homogenate was filtered through cheesecloth and centrifuged at 8000×g for 20 min (4°C). Triton X-100 was added to the supernatant (1% final concentration) and incubated on ice for 30 min. After centrifugation at 10,000×g for 15 min, PEG 6000 (6% w/v) and NaCl (1% w/v) were added to the clarified supernatant, followed by overnight incubation at 4°C. Virions were pelleted at 12,000×g for 30 min, resuspended in 0.2 M phosphate buffer (pH 8.0) containing 1% Triton X-100, and purified through 10–40% sucrose density gradients by ultracentrifugation (110,000×g, 2.5 h). Purified virions were collected in 0.05 M phosphate buffer. For virion integrity observation, samples were negatively stained with 2% uranyl acetate, loaded onto carbon-coated copper grids (230 mesh), and examined using a JEOL JEM-1400PLUS transmission electron microscope. All steps were conducted at 4°C.

Plasmodesmata observation and aperture measurement
Tomato plants were infected without (Mock) or with ToCV or ToCV-Δp59 as previous. At 28 dpi, the second systemic leaves (1–2 mm2) were harvested and fixed in 2.5% glutaraldehyde for 4 h at 4°C. After rinsing with PBS buffer, samples were post-fixed in 1% OsO4 for 2 h, dehydrated through a graded ethanol series (30–100%), and embedded in Spurr resin. Ultrathin sections were cut perpendicular to leaf veins, collected on nickel grids, and double-stained with 2% uranyl acetate (15 min) followed by lead citrate (5 min). Grids were examined using a JEOL JEM-1400PLUS transmission electron microscope operated at 120 kV. PD pore sizes were measured at aperture ends and central regions36.

Callose staining and quantification
Tomato plants were infected without (Mock) or with ToCV or ToCV-Δp59 as previous. At 28 dpi, the second systemic leaves were sectioned into 1 cm × 2 cm strips and fixed in Carnoy’s solution for 24 h. After rinses with absolute ethanol, samples were equilibrated in 50% ethanol for 30 min, then transferred to 1× PBS for 30 min. Tissues were stained with 0.01% aniline blue in 0.1 M K2HPO4 (pH 9.0) for 2 h in darkness. Following distilled water rinses, specimens were analyzed using a Zeiss LSM880 confocal laser scanning microscope (CLSM) with UV excitation (405 nm) and emission collection between 460-500 nm. Callose deposits appeared as bright blue fluorescence at cell walls and PD. Fluorescence intensity, which was quantified from confocal microscopy images using ZEISS Zen software, indicates callose levels57.

Viral movement analysis
GV3101 strains carrying p59-GFP or p59L255A-GFP were diluted to OD600 = 0.001 and infiltrated into N. benthamiana leaves without or with SlAVE3-Flag expression. GFP and TMV MP served as negative and positive controls, respectively. At 5 dpi, p59-GFP expression was analyzed using a Zeiss LSM 880 CLSM with 488 nm excitation and 510–550 nm emission detection. For complementation assay of cell-to-cell movement, p59-carrying GV3101 strains was co-infiltrated with movement-defective TuMV-GFP-P3N-PIPO-m1 (OD600 = 0.001) into N. benthamiana leaves. Co-expression of wild-type TuMV-GFP-P3N-PIPO and P3N-PIPO served as positive control. GFP expression was examined at 5 dpi under identical microscopy parameters. Cell-to-cell movement efficiency quantified from images35.

Detection of the CP in the systemic leaves was used to reflect the viral spread. WT, slave3, and SlAVE3-OE tomato plants were inoculated with ToCV or ToCV-p59L255A infectious clones. At 28 dpi, RNA was extracted from locally inoculated leaves and systemic leaves (positions 2–6 above inoculated leaf) for CP expression assay.

Real-time quantitative PCR (RT-qPCR)
Gene expression was quantified by RT-qPCR following MIQE guidelines58, using Actin as the reference gene. Total RNA was extracted using the TRIzol method. cDNA was synthesized from RNA using the Fast King RT Kit (Cat# R312-01, Vazyme, China). RT-qPCR was performed using HiScript II Q RT SuperMix for qPCR (Cat# R223-01, Vazyme, China) in 25 μL reactions containing 800 ng cDNA and 100 nM primers. Relative gene expression was calculated using the 2-ΔΔCt method59.

Total protein extraction and immunoblotting
Plant samples (50 mg) were homogenized in 150 µL extraction buffer (100 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM EDTA, 5% SDS, 20% glycerol, 20 mM DTT, 40 mM β-mercaptoethanol, 2 mM PMSF, 1× protease inhibitor cocktail, 80 µM MG132, 80 µM MG115, 1% phosphatase inhibitor cocktail, 10 mM N-ethylmaleimide). Samples were boiled (10 min), then centrifuged (16,000×g, 10 min). Supernatants were resolved by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% (w/v) non-fat milk. Target proteins were detected using specific primary antibodies followed by labeled secondary antibodies. Protein bands were visualized by chemiluminescence. For quantification, western blot bands were quantified using ImageJ according to the Protocols Handbook (Quantifications of Western Blots with ImageJ. by Hossein Davarinejad). For each protein, a single rectangular region of interest was defined around the largest band and consistently applied to all lanes. The gray mean value was measured for both bands and adjacent background areas under the set measurements option. Raw values were exported for background correction: pixel densities were inverted (255-measured value), and the inverted background was subtracted from the inverted band value to obtain the net intensity. Each target protein’s net intensity was then normalized to the corresponding loading control from the same lane to generate a relative quantification ratio60.

Yeast two-hybrid (Y2H) screening and verification
Total RNA was extracted from ToCV-infected tomato leaves. Only samples with an RNA integrity number (RIN) greater than 7 were used for subsequent experiments. A Y2H library containing 6 million primary clones was constructed in the pGADT7 (AD, prey) vector by Shanghai OE Biotech. Co., Ltd. (Shanghai, China). For library screening, the bait construct was generated using the pGBKT7 (BD, bait) vector. Y2H assay of both library screening and point-to-point protein interaction was performed using pGADT7 and pGBKT7 vectors according to the Yeast Protocols Handbook (Cat # PT3024-1, Clontech, USA). Briefly, prey (AD-library or AD-gene1) and bait (BD-gene2) vectors were constructed and co-transformed into the Y2H Gold yeast strain. Known interacting proteins (SV40 large T antigen and p53; AD-T+BD-53) and non-interacting proteins (SV40 large T antigen and Lamin C; AD-T+BD-Lam) served as positive and negative controls, respectively61. Transformants were plated on SD/-Trp-Leu medium to select for colonies containing both plasmids. Single colonies from SD/-Trp-Leu were then replica-plated onto SD/-Trp-Leu-His-Ade medium to select for interactions between prey and bait proteins.

Luciferase complementation imaging (LCI)
The genes encoding two potential interacting proteins were separately cloned into nLUC (N-terminal fragment) and cLUC (C-terminal fragment) vectors, generating gene2-nLUC and cLUC-gene1 constructs. Known non-interacting proteins (p22 and NbBAG1) served as the negative control62. These constructs were co-expressed in N. benthamiana leaves via GV3101-mediated infiltration. Luciferase activity and data quantification were detected using a IVIS Lumina In Vivo Imaging System (PerkinElmer, USA) at 2 to 3 dpi.

Bimolecular fluorescence complementation (BiFC)
The genes encoding two potential interacting proteins were separately cloned into vectors containing the N-terminal (nYFP) or C-terminal (cYFP) fragments of YFP, generating gene1-nYFP and gene2-cYFP constructs. nYFP and cYFP empty vectors served as negative controls. These constructs were co-expressed in N. benthamiana leaves via GV3101-mediated infiltration. YFP fluorescence complementation in N. benthamiana leaves was imaged at 3 dpi using a Zeiss LSM880 CLSM. YFP was excited at 514 nm and emission was captured between 525 and 585 nm. Fluorescence intensity was quantified from confocal microscopy images using ZEISS Zen software.

Co-immunoprecipitation (Co-IP)
PVX-based vectors harboring target genes were constructed. Expression of Flag-tagged or HA-tagged proteins in tomato plants was mediated by PVX as described previously. Tomato leaves were lysed in extraction buffer (10 mM Tris-HCl pH 7.6, 0.5% Nonidet P-40, 2 mM EDTA, 150 mM NaCl, 1× EDTA-free protease inhibitor cocktail [Roche]). Lysates were centrifuged and the supernatant was incubated with pre-washed anti-Flag Affinity Gel (Cat# A2220, Sigma-Aldrich, USA) for 2 h at 4°C. Following five washes, co-immunoprecipitated proteins were detected via immunoblotting with anti-Flag (Cat# AG8050, Beyotime, China) and anti-HA (Cat# AG8057, Beyotime, China) antibodies.

Pull-down
Recombinant SlAVE3-GST and SlWRKY6-His proteins were purified using BeyoGoldTM GST-tag Purification Resin (Cat# P2251, Beyotime, China) and His-tag Purification Resin (Cat# P2218, Beyotime, China), respectively. For pull-down assay, SlAVE3-GST bound to resin was incubated with SlWRKY6-His in binding buffer (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM DTT, 10 mM MgCl2) at 4°C for 2–3 h with gentle rotation. The resin was pelleted by centrifugation at 10,000×g for 5 min and washed three times with wash buffer (50 mM Tris-HCl pH 8.0, 400 mM NaCl, 1 mM EDTA, 1 mM DTT). Bound protein complexes were eluted with elution buffer (50 mM Tris-HCl pH 8.0, 20 mM reduced glutathione). Eluted proteins were detected by immunoblotting using anti-GST (Cat# AF2888, Beyotime, China) and anti-His (Cat# AF5060, Beyotime, China) antibodies.

MicroScale Thermophoresis (MST)
MST assay was performed using a Monolith NT.LabelFree instrument (NanoTemper Technologies GmbH, Germany). For each assay, 50 nM SlAVE3 or SpAVE3 was mixed with an equal volume of p59 or SlCAT1 at serial dilutions in buffer (10 mM phosphate-buffered saline pH 7.4, 137 mM NaCl). Samples were loaded into standard glass capillaries (Monolith NT.LabelFree), and thermophoresis measurements were conducted using MO. Control software (NanoTemper Technologies GmbH, Germany) with the following parameters: 40% LED power, medium MST power. For each binding experiment, three independent measurements were acquired at 360 nm. Data were processed using MO. Affinity Analysis software (NanoTemper Technologies GmbH, Germany)63.

Electrophoretic mobility shift assay (EMSA)
The coding sequence of SlWRKY6 was cloned into pGEX-6P-1 to generate an N-terminal GST fusion. Recombinant SlWRKY6-GST protein was expressed in E. coli following induction with 1 mM IPTG at 28°C for 10 h. W-box motif fragments derived from the SlAVE3 promoter were synthesized and 5’-end labeled with 5-FAM. For binding reactions, 0.08 pmol of labeled probe was incubated with 2.5 μg of purified protein in 20 μL binding buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 2 mM MgCl2, 1 mM DTT, 10% glycerol) at 25°C for 30 min. Negative controls consisted of probe alone and probe with GST (empty vector). For competition assays, unlabeled competitor DNA was added in 5- or 10-fold molar excess. Protein-DNA complexes were resolved on native 8% polyacrylamide gels in 0.5× TBE at 140 V for 90 min (4°C, dark). Gels were imaged using a Tanon 5200 Multisystem64.

Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR)
PVX-GFP (negative control) and PVX-SlWRKY6-GFP vectors were constructed and expressed in tomato plants. For ChIP assay, tomato leaves (5 g) was vacuum-infiltrated with 1% formaldehyde for 10 min, flash-frozen in liquid nitrogen, and ground to a fine powder. Chromatin was isolated, sonicated, and immunoprecipitated with anti-GFP antibody (Cat# AF2882, Beyotime, China). Purified ChIP DNA and input DNA were resuspended in 30 μL nuclease-free water. qPCR signals were normalized to input DNA, with fold enrichment calculated relative to the Actin promoter65.

Transactivation assay
The SlWRKY6 coding sequence was cloned into the pGreenII 002962-SK effector vector, and the SlAVE3 promoter was inserted into the pGreenII 0800-LUC reporter vector. Constructs were electroporated into GV3101. N. benthamiana leaves were infiltrated with transformed GV3101, and Firefly luciferase (LUC) and Renilla luciferase (REN) activities were measured at 3 dpi. Luminescence was quantified using a Synergy MX full wavelength multifunctional microplate reader (Bio Tek, USA). The LUC/REN ratio for the empty effector vector plus promoter construct was set to 1 for normalization66.

E3 ubiquitin ligase activity and ubiquitination assays
E3 ubiquitin ligase activity of SlAVE3 and its substrate ubiquitination capacity were analyzed following established methods67. Reaction mixtures contained: ubiquitin (2 μg μL−1), wheat E1-His, human E2-His (UBCH5b) or SlAVE2-His, and SlAVE3/SlAVE3H304Y/SpAVE3-Strep in reaction buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 5 mM ATP, 2 mM DTT). Following 2 h incubation at 30°C, ubiquitination was detected by immunoblotting with anti-Strep (Cat# AF2924, Beyotime, China) and anti-Ub (Cat # 3936, Cell Signaling Technology, USA) antibodies. For substrate ubiquitination, equimolar GST-tagged substrate proteins (p59, p59L255A, SlWRKY6, SlCAT1) were added to reactions prior to incubation, with ubiquitination detected by immunoblotting.

For in planta ubiquitination, target proteins with Flag tag were expressed in leaves of specified genotypes, respectively. 100 μM MG132 was infiltrated into leaves 10 h before total protein extraction. Subsequently, total proteins were extracted from specified genotypes and subjected to ubiquitination assay as previous description.

Cell-free degradation assay
Total protein was extracted from tomato leaves of specified genotypes. Equal amounts of protein extract were incubated with purified substrate proteins (recombinant GST- or His-tagged targets) and 10 mM ATP for designated time periods. MG132 (50 μM) was added to inhibit the 26S proteasome. Proteins were separated by SDS-PAGE and immunoblotted with tag-specific antibodies. Actin served as the loading control68.

Subcellular localization
The coding sequences of target proteins were inserted into the GFP vector to create 35S promoter-driven p59-GFP and SlAVE3-GFP expression constructs. Similarly, target protein coding sequences were inserted into the RFP vector to generate SlCAT1-RFP and SlAVE3-RFP expression constructs under the 35S promoter. Constructs were transformed or co-transformed into N. benthamiana leaves via GV3101-mediated infiltration. For plasmolysis, leaf tissues were incubated in 10% NaCl solution for 10 min. Epidermal fluorescence was examined using Zeiss LSM880 CLSM with the following parameters: GFP (excitation 488 nm, emission 510–550 nm) and RFP (excitation 552 nm, emission 562–632 nm). Subcellular markers included: TMV MP-RFP (PD marker)69, NbH2B-RFP (nuclear marker)70, SKL-BFP (peroxisomal marker)71.

Isolation of cytoplasmic and peroxisomal SlCAT1 proteins
Subcellular fractionation for cytoplasmic and peroxisomal isolation was adapted from established methods10,72. Fresh leaf tissues (0.5 g) of SlCAT1-OE expressed with empty vector (35S::EV) or 35S::p59 were homogenized in ice-cold buffer (50 mM Tris-HCl pH 7.5, 0.4 M sucrose, 1 mM EDTA, 1 mM DTT, protease inhibitors) and filtered through Miracloth. Sequential centrifugation was performed: 3000×g for 20 min to remove nuclear and cellular debris; the supernatant was then centrifuged at 20,000×g for 1 h to yield cytoplasmic fraction (supernatant) and a pellet containing crude peroxisomes. For peroxisome purification, the pellet was resuspended and layered onto a discontinuous sucrose gradient (35/40/45/50/60%). After ultracentrifugation (100,000×g, 3 h), peroxisomes were collected from the 40–45% sucrose interface. Fraction purity was verified by immunoblotting, with actin serving as the cytoplasmic marker. All steps were performed at 4°C. Levels of SlCAT1-GFP in different subcellular compartments were detected by immunoblotting using anti-GFP antibody (Cat# AF2882, Beyotime, China).

Hydrogen peroxide (H2O2) content and catalase (CAT) activity
Histochemical staining of H2O2 was performed using 0.1 mg/mL 3,3′-diaminobenzidine in 50 mM Tris-acetate (pH 3.8). For CAT activity, Leaf samples (0.3 g) were ground in 3 mL of ice-cold 50 mM PBS buffer (pH 7.8) with 0.2 mM ethylenediaminetetraacetic acid, 2 mM ascorbate, and 2% (w/v) polyvinylpyrrolidone, and then centrifuged at 12,000×g for 20 min at 4°C. The supernatants were used for assessing CAT activity. CAT activity was measured by the decline in absorbance at 240 nm associated with H2O2 decomposition73.

Variant calling
Illumina resequencing raw reads of 652 tomato accessions were downloaded from previous studies39,74,75. Fastp (v0.20.0) was then applied to eliminate low-quality reads76. Clean reads were aligned to the tomato reference genome Heinz 1706 version SL4.077 by BWA-mem (0.7.17-r1188)78 using parameters “-M” with readgroup header line included by adding “-R” parameter. The resulted BAM format files were sorted using SAMtools (v1.9)78 and potential PCR duplicates were marked using “MarkDuplicates” function in Genome Analysis Toolkit (GATK, Version: v4.1.4)79. SNPs and indels (<30 bp) for each sample were called by “HaplotypeCaller” in GATK, resulting in GVCF files that preserve information of both variant and reference alleles, with the parameters “--emit-ref-confidence GVCF”. “GenotypeGVCFs” in GATK was applied for joint genotyping using parameters: “--allow-old-rms-mapping-quality-annotation-data”. A set of hard filters were applied to exclude low-quality variants. Those exhibiting QD<2.0 or MQ<40.0 or FS>60.0 or SOR>3.0 or MQRankSum<−12.5 or ReadPosRankSum<−8.0 or GQ<20.0 were removed. Only be-allelic variants were retained.

Variant annotation
SnpEff (v4.3t) was applied to predict functional impact of the identified variants, in which gene upstream and downstream boundary was set to 2 kb80.

Identification of selective sweeps
SNPs with a missing rate<0.1 were used for calculating nucleotide diversity (π) by VCFtools v0.1.1781, using a sliding window approach with 100 kb windows and a 50 kb step size across the tomato reference genome. The 652 tomato individuals were classified into three groups based on their taxonomy: 69 S. pimpinellifolium (Sp), 208 S. lycopersicum var. cerasiforme (Slc) and 375 S. lycopersicum var. lycopersicum (Sll) accessions. Genomics regions displaying a significant reduction of nucleotide diversity between Sp and Slc (πSp /πSlc > the genome-wide top 5% threshold, 3.72) were defined as domestication sweeps, while regions showing a significant decrease between Slc and Sll (πSlc /πSll > the genome-wide top 5% threshold, 5.38) were considered improvement sweeps39.

Phylogeny inference
Maximum likelihood phylogenetic trees were built by RAxML (v8.2.12)82 with 100 bootstraps, using variants spanning from 2 kb upstream of SlAVE3 to 2 kb downstream. The GTRGAMMA model was employed. The tree plot was further visualized and modified at iTOL (https://itol.embl.de/) website83.

RNA sequencing (RNA-seq) assay
RNA-seq samples were extracted from leaves of normal TRV-EV, and ToCV-infected TRV-EV and TRV-SlWRKY6, respectively. Every ten samples were mixed into one sample for a single biological replicate, and a total of three biological replicates were setup in RNA-seq assay. Isolated RNA was used for cDNA library construction with the NEBNext Ultra RNA Library Prep Kit (Illumina, USA) following the manufacturer’s instructions. All libraries were subjected to paired-end 150-bp sequencing on the Illumina HiSeq 4000 platform. Illumina short paired-end reads produced previously were trimmed using Trimmomatic (v.0.36) with default parameters. The high quality reads were aligned to the tomato reference genome Heinz 1706 version SL4.077 using HISAT2 (v.2.0.4). The counts of reads aligned to each transcript were calculated with HTSeq (v.0.6.0) software, and fragments per kilobase of transcript per million mapped fragments were used to estimate the expression level of each gene. Raw data of RNA-seq have been deposited to Sequence Read Archive (SRA; https://www.ncbi.nlm.nih.gov/sra) under the accession number PRJNA1369304 with the referee access code or the clickable URL link (https://www.ncbi.nlm.nih.gov/sra/PRJNA1369304).

Statistical analyses
All experiments in this manuscript and Supplementary Information should be conducted with at least two (qualitative experiment) or three (quantitative experiment) independent biological replicates with similar results, and the sample size (biological replicate) for each independent experiment should be at least three times. The statistical graphs were generated and processed by the GraphPad Prism 9 software. All values are presented as means ± SD or box plots (median with 0.25 and 0.75 quartiles, whiskers represent values from minimum to maximum), and the statistically significant differences between the control and experimental groups were determined by t-test, one-way ANOVA, or two-way ANOVA (ns P>0.05; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Understanding how plants detect viruses, and how viruses evade this detection, is key to breeding resistant crops. Here, the\nauthors characterize a new ToCV effector, p59, and demonstrates that it is recognized by the host receptor SlAVE3. In turn,\np59 exploits this interaction to disable the host CAT1. Natural variation in AVE3, fixed during tomato domestication,\ninadvertently facilitates this viral strategy, offering a clear example of co-evolution and pointing to the use of wild SpAVE3 for\nresistance. Overall, this is a solid and systematic study that significantly advances our understanding of plant–virus\ndynamics. Only a few minor points need clarification:\nLine 119-128: The paragraph explaining the general role of ROS in plant–virus interactions is more suitable for the\nDiscussion section; please consider moving it there to keep the Introduction focused on the specific knowledge gap\naddressed in this study.\nLine 152: Please change the citation \"Fig. 1c–e\" to \"Fig. S1c–e\". In addition, Fig. S2c–d lacks photographs of N.\nbenthamiana plants infected with ToCV and ToCV-Δp59; please supply these images or revise the results accordingly.\nLine 153: Please add the citation “Fig. 1c–e” immediately after “PVX-p59” to correctly link the text to the Figures.\nLine 154-155: Please rearrange the lane order in Fig. 1c, so that PVX-EV/ToCV and PVX-p59/ToCV are adjacent; this will\nallow direct visual comparison and align with the description sequence in the text.\nLine 259: Please quantify SlAVE2 transcript levels individually for both the slave3 mutant and WT backgrounds in Fig. S12\nto allow direct comparison.\nLine 294-298: Drop-in CAT activity appears to exacerbate ToMV viral symptoms. Please briefly address whether the CAT-\nactivity decrease is a broad plant-virus response or specific to ToCV/ToMV pathogenicity in the Discussion.\nLine 334: Change the citation Fig. S21a-c to Fig. S21a-b.\nLine 370: In Fig. 5h, the SlCAT1 blot does not show an obvious reduction; please quantify the band intensities, plot a\ndegradation curve to compare the activities of SlVE3 vs SpVE3.\nLine 374: Please verify whether “restore ToCV susceptibility” is correct; if the plants actually showed reduced symptoms,\nchange it to “ToCV resistance.”\nFig. 6: Please merged the two panels into a single model that depicts AVE3-mediated co-degradation of SlCAT1 and P19\nand emphasizes the central role of AVE3 in the tomato–ToCV interaction. Additionally, show SlCAT1 being translated and\nhijacked in the cytosol (not exported from peroxisomes). For WRKY, replace the arrowhead with a blunt-end line (⊣) to\nindicate that WRKY negatively regulates ToMV progression.", "reviewer_2": "The authors revealed a p59-AVE3-CAT1 module affected the H2O2 homeostasis in tomato and a positive feedback loop of\nSlAVE2-SlAVE3-WRKY6- SlAVE3 to inhibit viral infection. These results demonstrate the plant defensive strategies and\nviral counterdefensive strategies. The natural variation is the results of arms race between plants and pathogens, providing\ngenetic resources in biotech breeding. This is a nice work. I recommend this paper for Nature Communications which\nneeded major revision.\nMajor comments\n1. Line 147 “The pathogenicity was assessed based on symptom severity”, However, they only showed the results of\naccumulation level of CP for evaluation of ToCV resistance in Figure 1c-e and Figure S1c-e. At least, it is not suitable for\ntobacco breeding utilization. The standard estimations of ToCV resistance should be showed in the revised manuscript.\nThey should also revised other similar issues.\n2. They said that “p59 expression enhanced ToCV pathogenicity.” Does the authors test the function of p59 as an RNA\nsilencing inhibitor? I think they should add the corresponding experiments.\n3. Line 183, the authors should show the Y2H results, in the other word, they should show other potential p59-interacted\nproteins and why they chose AVE3?\n4. Line 258, “SlAVE2 enhanced SlAVE3 activity more potently than generic E2.” “Unlike SlAVE3 (Fig. S9d), transcript levels\nof SlAVE2 remained unchanged during ToCV infection” Thus, they had the conclusion “SlAVE2 functions primarily through\nSlAVE3 in mediating ToCV resistance”. However, the WB results in Figure 2m demonstrated that silencing AVE3 in ave3 #1\npromotes the viral accumulation compared to silencing EV in ave3 #1? It is the reason that I suggest the authors to add the\nstandard estimation and the repeat of WB analysis.\n5. Line 266-275, does AVE2 interacted with WRKY6? AVE3 can not directly ubiquitinate WRKY6? It seems that AVE2 is\nnecessary for the ubiquitination process.\n6. Line 344: “p59 acts as an effector to induce SlAVE3-dependent degradation of SlCAT1, thereby promoting ToCV\ninfection.” It is a important results. However, have the authors tested the competitive interaction of p59 and CAT1 to AVE3?\nMaybe p59 could interfere the interaction between AVE3 and CAT1? They should confirm this issue.\n7. Similarly, WRKY might also interfere the interaction of p59-AVE3 and AVE3-CAT1.\nMinor comments\n1. Figure S7c, the authors should add the control group of AVE3-nYFP and p59-cYFP.\n2. Figure 2d, in the cell-free degradation assays, they should repeat the experiment at least three times and add statistical\nanalysis. They should also revised similar issues.\n3. Line 259, “We silenced SlAVE2 in both WT and slave3 backgrounds (Fig. S12d)”. They only showed the results of\nsilencing SlAVE2 in WT.\n4. The authors demonstrated that “The p59 hijacks and degrades SlCAT1 via the SlAVE2-SlAVE3 UPS to facilitate ToCV\npathogenicity”. They have constructed ToCV-Δp6, ToCV-ΔHsp70h, and ToCV-Δp59. Does ToCV-Δp59 or other ToCV\nmutants changed the CAT1 activity and plant H2O2 content? They could staine the leaves using DAB staining after\ninoculated with ToCV-ΔCP, ToCV-ΔCPm, ToCV-Δp6, ToCV-ΔHsp70h, and ToCV-Δp59.\n5. Similarly, they should also show the H2O2 content using DAB staining in the AVE3 knockout plants and TRV-AVE2\nplants.\n6. Line 776 “All experiments should be conducted with at least two independent biological replicates with similar results”. Is\nthis a typing error? At least three???", "reviewer_3": "Key results\nOverall, the authors demonstrate strong proficiency in molecular biology and present well-founded hypotheses regarding the\nmolecular mechanisms underlying ToCV pathogenicity and plant antiviral resistance. The study is original and holds\nsignificant potential impact, as ToCV is a major viral disease that severely reduces tomato yield. Through a comprehensive\nsuite of molecular and biochemical experiments, including transformation, CRISPR-Cas9 mutagenesis, yeast two-hybrid,\nco-immunoprecipitation, bimolecular fluorescence complementation, luciferase complementation imaging, and cell-to-cell\nmovement assays, the authors systematically elucidate the pathogen (ToCV) and host (tomato) interaction network. The\nfindings include:\n1. p59 Function: p59 is identified as a central pathogenic determinant essential for virion assembly, plasmodesmatal\ntargeting, and intercellular movement. It localizes to the plasma membrane and plasmodesmata, modulates callose\ndeposition to widen intercellular pores, and functions as a viral movement protein, thereby enhancing viral spread and\npathogenicity.\n2. SlAVE3 Role: The tomato E3 ubiquitin ligase SlAVE3 specifically recognizes and ubiquitinates the viral effector p59,\ndirecting it to proteasomal degradation and restricting ToCV infection. The C-terminal RING domain of SlAVE3 and the\nLeu255 residue of p59 are critical for this interaction, establishing SlAVE3 as a key antiviral regulator that limits viral\nmovement and enhances resistance.\n3. Regulatory Network: SlWRKY6 functions as a transcriptional repressor of SlAVE3 by binding to W-box motifs in its\npromoter. The SlAVE2–SlAVE3 ubiquitin–proteasome complex degrades SlWRKY6, relieving repression and forming a\npositive feedback loop that amplifies SlAVE3 expression and fortifies antiviral defense.\n4. Redox Manipulation: p59 further promotes infection by hijacking and degrading the host antioxidant enzyme SlCAT1 via\nthe SlAVE2–SlAVE3 complex, disrupting redox homeostasis and creating a cellular environment favorable for viral\nreplication.\n5. Evolutionary Insight: SlAVE3 was subjected to selection during tomato domestication, producing three haplotypes from\ntwo non-synonymous mutations. Among them, SpAVE3, derived from S. pimpinellifolium, exhibits stronger binding to p59\nbut weaker interaction with SlCAT1, designating it as an antiviral haplotype associated with enhanced ToCV resistance.\nTogether, these findings establish a comprehensive interaction model (Figure 6) in which p59 drives ToCV pathogenicity by\nfacilitating virion assembly, intercellular movement, and host redox disruption, while the tomato AVE2–AVE3–WRKY6\nregulatory module orchestrates an antiviral feedback mechanism that recognizes and degrades p59, thereby restoring\ncellular homeostasis and conferring viral resistance.\nValidity\nOverall, the data interpretation is carefully executed and, in general, well supported by the presented evidence. The\nexperimental design convincingly demonstrates the proposed molecular mechanism of the interaction between ToCV and\ntomato and the conclusions drawn from these data are appropriate. The integration of molecular, biochemical, and\nphenotypic analyses supports and strengthens to the study’s main claims.\nOne area of concern lies in the interpretation of selection and domestication analyses. The inference of a selection sweep\nbased on phylogenetic reconstruction is not methodologically robust for detecting signals of domestication. Phylogenetic\ntrees describe evolutionary relationships but do not quantitatively measure selective pressures or genetic diversity within\npopulations. To strengthen this aspect, the authors should focus on population genetic statistics, such as nucleotide diversity\n(π), Tajima’s D, or the ratio of non-synonymous to synonymous substitutions (dN/dS), which would provide a more rigorous\nand quantitative framework for evaluating domestication-related selection.\nSignificance\nThis study uncovers a previously uncharacterized antiviral mechanism in tomato that counters ToCV, a major threat to global\ntomato production. The authors identify the viral effector p59 as a central determinant of virion assembly and cell-to-cell\nmovement, and demonstrate that the tomato E3 ubiquitin ligase SlAVE3 targets p59 for proteasomal degradation, thereby\nrestricting viral spread. The elucidation of the SlAVE2–SlAVE3–SlWRKY6 regulatory module reveals a self-reinforcing\nantiviral feedback loop in which degradation of SlWRKY6 enhances SlAVE3 expression and fortifies host defense. The\nstudy further shows that p59 manipulates host redox homeostasis through SlCAT1 degradation, linking viral infection to\noxidative stress responses. Importantly, evolutionary analysis identifies natural SlAVE3 haplotypes under selection during\ntomato domestication, including an antiviral allele (SpAVE3) that confers enhanced ToCV resistance. Collectively, this work\nprovides a comprehensive molecular framework for ToCV–tomato interactions, highlighting the AVE2–AVE3–WRKY6 axis\nas a critical regulatory node in plant antiviral immunity and offering valuable genetic insights for breeding virus-resistant\ncultivars.\nData and methodology\nWhile the experimental design is comprehensive, three methodological aspects would benefit from additional detail and\nclarification to improve reproducibility and rigor:\n1. Quantification Using ImageJ\nThe manuscript should specify the exact procedures and parameters used in ImageJ for image quantification. This includes\noutlining which plugins or measurement tools were applied (e.g., area, intensity, or co-localization analyses), how regions of\ninterest were defined and whether background correction or normalization steps were performed. Providing this information\nwould enhance transparency and allow accurate comparison or replication by other researchers.\n2. Plant Material of Nicotiana benthamiana\nThe genetic background of the N. benthamiana used in this study should be included.\n3. cDNA Libraries Used for Yeast Two-Hybrid Screening\nThe manuscript should provide details about the cDNA libraries employed in the yeast two-hybrid assay. Specifically, the\norigin of the library (species, tissue type, developmental stage, or stress condition), the vector system used, and the\ncoverage or complexity of the library should be reported. These details are important to assess the comprehensiveness of\nthe interaction screen and the biological relevance of the identified protein–protein interactions.\nAnalytical approach\nThe statistical analyses are appropriately conducted and effectively support the study’s conclusions by addressing the\nresearch questions with sufficient rigor.\nSuggested improvements\n1. Considering that pathogen–host interactions function through complex molecular networks, it would be highly valuable to\ninvestigate the associated gene regulatory network integrating RNA-seq analysis. Using transgenic materials that share a\nmore uniform genetic background with the wild type can minimize background variation between genotypes and provide\nmore reliable insight into network-level changes. In addition, it would be beneficial to propose or experimentally validate\ncandidate proteins that may participate in this network, such as other E3 ligases, transcription factors, or redox-regulating\nenzymes interacting with the SlAVE2–SlAVE3 UPS, to strengthen the mechanistic model and broaden understanding of the\nantiviral defense network.\n2. The study should also address the functional performance of heterozygous SpAVE haplotypes. In modern tomato\ncultivars, which are typically F₁ hybrids, understanding whether SpAVE alleles act dominantly or recessively is essential for\npractical breeding applications. If SpAVE exhibits dominance, the presence of one functional allele may be sufficient for the\nphenotype, whereas a recessive effect would limit its utility in hybrid cultivars. Experimental or genetic data addressing\nthis question would strengthen the manuscript and clarify the translational potential of these findings.\nClarity and context\nOverall, the manuscript is clearly written and logically structured, presenting the research in a coherent and convincing\nmanner. However, the suggestions below would further improve the quality and readability of the paper.\n1. Clarification of the SlAVE2–SlAVE3 Complex formation\nThe authors propose that the SlAVE2–SlAVE3 UPS complex mediates the degradation of SlWRKY6. However, the\nmolecular mechanism underlying the formation and functional dynamics of the SlAVE2–SlAVE3 complex remains\ninsufficiently explained. The manuscript would benefit from a clearer characterization of this complex, specifically, whether\nSlAVE2 and SlAVE3 form a stable heteromeric complex or interact transiently during ubiquitin-mediated degradation.\n2. Importance of Effector Compartmentation for Pathogenicity\nThe compartmentation of effectors within host cells is a critical determinant of pathogenicity. The authors have conducted\nseveral localization assays to determine the subcellular positions of both effector and host proteins, which is commendable.\nHowever, the discussion should better integrate how these localization patterns contribute mechanistically to virulence.\nSpecifically, clarify whether the spatial distribution of SlAVE2 and SlAVE3 affects their capacity to interact with SlWRKY6 or\nto recruit the ubiquitin–proteasome machinery. Emphasizing this compartmentalization will help link the molecular data to\nthe overall pathogenic strategy of the effector.\nReferences\nThe manuscript appropriately references previous literature. The cited works are relevant, up to date, and provide sufficient\ncontext for the study’s rationale and experimental design. The authors effectively integrate prior findings to support their\nhypotheses and discuss their results in relation to existing knowledge, demonstrating a solid understanding of the field."}, "author_response": "Response to Editor and Reviewers\nDear Editor and Reviewers,\nWe are very pleased to receive your positive feedback and consider it an honor to have\nour work viewed so favorably. We have carefully reviewed all of the comments you provided\nand believe they are highly valuable for enhancing the quality of our manuscript.\nAccordingly, in this revised version, we have fully addressed each of your suggestions\nand made corresponding improvements point by point. We sincerely appreciate your\nappreciate your\nthorough and insightful guidance, which has greatly helped us refine this work.\nIn addition, we have also revised some grammatical, spelling errors, and inappropriate\ndescriptions in this manuscript.\nThank you once again for your support and constructive input.\nSincerely,\nBiao Gong\nPoint by point revision\nReviewer #1 (Remarks to the Author):\nUnderstanding how plants detect viruses, and how viruses evade this detection, is key to breeding\nresistant crops. Here, the authors characterize a new ToCV effector, p59, and demonstrates that it\nis recognized by the host receptor SlAVE3. In turn, p59 exploits this interaction to disable the host\nCAT1. Natural variation in AVE3, fixed during tomato domestication, inadvertently facilitates this\nviral strategy, offering a clear example of co-evolution and pointing to the use of wild SpAVE3 for\nresistance. Overall, this is a solid and systematic study that significantly advances our\nunderstanding of plant–virus dynamics. Only a few minor points need clarification:\nI truly appreciate your comprehensive and positive evaluation.\nLine 119-128: The paragraph explaining the general role of ROS in plant–virus interactions is more\nsuitable for the Discussion section; please consider moving it there to keep the Introduction\nfocused on the specific knowledge gap addressed in this study.\nAs recommended, the description of ROS has been moved from the Introduction to the\nDiscussion. The revised structure improves the manuscript by enhancing the focus on the key\nscientific issues of this research.\nLine 152: Please change the citation \"Fig. 1c–e\" to \"Fig. S1c–e\". In addition, Fig. S2c–d lacks\nphotographs of N. benthamiana plants infected with ToCV and ToCV-Δp59; please supply these\nimages or revise the results accordingly.\nIn response to the comment regarding changing \"Fig. 1c–e\" to \"Fig. S1c–e\", we have made the\nrequested revision.\nAs shown in Fig. S2c-d (now Fig. S1,km), we have included the treatment groups infected with\nToCV and ToCV-Δp59 (red boxes and arrows).\nLine 153: Please add the citation “Fig. 1c–e” immediately after “PVX-p59” to correctly link the text to\nthe Figures.\nThanks, the issue has been addressed in the revised manuscript.\nLine 154-155: Please rearrange the lane order in Fig. 1c, so that PVX-EV/ToCV and\nPVX-p59/ToCV are adjacent; this will allow direct visual comparison and align with the description\nsequence in the text.\nThank you for your suggestion. In the revised version, we have reorganized this picture for\neasier comparison and observation.\nLine 259: Please quantify SlAVE2 transcript levels individually for both the slave3 mutant and WT\nbackgrounds in Fig. S12 to allow direct comparison.\nThanks. We determined the transcriptional levels of SlAVE2 in both the WT, slave3, and\nSlAVE3-OE lines, and found that theSlAVE3 did not affect SlAVE2 transcription. These data have\nbeen included in Fig. S10f.\nLine 294-298: Drop-in CAT activity appears to exacerbate ToMV viral symptoms. Please briefly\naddress whether the CAT-activity decrease is a broad plant-virus response or specific to\nToCV/ToMV pathogenicity in the Discussion.\nThank you for your valuable feedback. We have addressed your points in our Discussion.\nLine 370: In Fig. 5h, the SlCAT1 blot does not show an obvious reduction; please quantify the band\nintensities, plot a degradation curve to compare the activities of SlAVE3 vs SpAVE3.\nThank you for your suggestion. All WB quantification analyses in this study were performed\nwith three replicates. Since initial manuscript submission did not require raw data, we initially\npresented only representative results. Following initial review, we have now consolidated and\npresented the statistically aggregated data from all three replicates. This revision has been\nuniformly implemented throughout the manuscript.\nLine 374: Please verify whether “restore ToCV susceptibility” is correct; if the plants actually\nshowed reduced symptoms, change it to “ToCV resistance.”\nThanks, the issue has been addressed in the revised manuscript.\nFig. 6: Please merged the two panels into a single model that depicts AVE3-mediated\nco-degradation of SlCAT1 and P19 and emphasizes the central role of AVE3 in the tomato–ToCV\ninteraction. Additionally, show SlCAT1 being translated and hijacked in the cytosol (not exported\nfrom peroxisomes). For WRKY, replace the arrowhead with a blunt-end line (⊣) to indicate that\nWRKY negatively regulates ToMV progression.\nWe have, in accordance with your requirements, revised the mechanism model in a\nstreamlined manner.\nReviewer #2 (Remarks to the Author):\nThe authors revealed a p59-AVE3-CAT1 module affected the H2O2 homeostasis in tomato and a\npositive feedback loop of SlAVE2-SlAVE3-WRKY6-SlAVE3 to inhibit viral infection. These results\ndemonstrate the plant defensive strategies and viral counterdefensive strategies. The natural\nvariation is the results of arms race between plants and pathogens, providing genetic resources in\nbiotech breeding. This is a nice work. I recommend this paper for Nature Communications which\nneeded major revision.\nI truly appreciate your comprehensive and positive evaluation.\nMajor comments\n1. Line 147 “The pathogenicity was assessed based on symptom severity”, However, they only\nshowed the results of accumulation level of CP for evaluation of ToCV resistance in Figure 1c-e and\nFigure S1c-e. At least, it is not suitable for tobacco breeding utilization. The standard estimations of\nToCV resistance should be showed in the revised manuscript. They should also revised other\nsimilar issues.\nThank you for your suggestion. We have comprehensively adopted disease symptoms,\nincidence, index, CP gene expression, and CP protein levels to evaluate ToCV resistance in tomato\nplants. This approach can comprehensively reflect the resistance level of the host and the\npathogenicity of the pathogen.\n2. They said that “p59 expression enhanced ToCV pathogenicity.” Does the authors test the\nfunction of p59 as an RNA silencing inhibitor? I think they should add the corresponding\nexperiments.\nThanks for your suggestion. We evaluated the potential of p59 to function as an RNA silencing\nsuppressor. The results indicate that p59 does not possess this activity; please refer to Fig S2.\n3. Line 183, the authors should show the Y2H results, in the other word, they should show other\npotential p59-interacted proteins and why they chose AVE3?\nThe Y2H screening results for p59 have been included as Supplementary Dataset. Our revised\ntext now also explains that SlAVE3 was chosen as the target gene based on the well-established\nfunction of E3 ubiquitin ligases in directly recognizing and mediating the degradation of their\nsubstrate proteins.\n4. Line 258, “SlAVE2 enhanced SlAVE3 activity more potently than generic E2.” “Unlike SlAVE3\n(Fig. S9d), transcript levels of SlAVE2 remained unchanged during ToCV infection” Thus, they had\nthe conclusion “SlAVE2 functions primarily through SlAVE3 in mediating ToCV resistance”.\nHowever, the WB results in Figure 2m demonstrated that silencing AVE2 in ave3 #1 promotes the\nviral accumulation compared to silencing EV in ave3 #1? It is the reason that I suggest the authors\nto add the standard estimation and the repeat of WB analysis.\nThank you for your suggestion. All WB quantification analyses in this study were performed\nwith three replicates. Since initial manuscript submission did not require raw data, we initially\npresented only representative results. Following initial review, we have now consolidated and\npresented the statistically aggregated data from all three replicates. This revision has been\nuniformly implemented throughout the manuscript.\n5. Line 266-275, does AVE2 interacted with WRKY6? AVE3 can not directly ubiquitinate WRKY6? It\nseems that AVE2 is necessary for the ubiquitination process.\nWe performed Y2H and LCI assays, which showed no interaction between SlAVE2 and\nSlWRKY6 (See below figure). In contrast, SlAVE3 directly bound to SlWRKY6 independently of E1\nand E2. Despite this E1/E2-independent binding, successful ubiquitination of SlWRKY6 by SlAVE3\nrequires the full E1-E2-E3 enzymatic cascade. Thus, omitting either E1 or E2 abolishes\nubiquitination, as evidenced in Fig. 3b (lane 2 and lane 3).\n6. Line 344: “p59 acts as an effector to induce SlAVE3-dependent degradation of SlCAT1, thereby\npromoting ToCV infection.” It is a important results. However, have the authors tested the\ncompetitive interaction of p59 and CAT1 to AVE3? Maybe p59 could interfere the interaction\nbetween AVE3 and CAT1? They should confirm this issue.\nThanks, we have addressed this point by demonstrating that p59 promotes the interaction\nbetween SlAVE3 and SlCAT1 (Fig. 4d,e; Fig. S16d-g).\n7. Similarly, WRKY might also interfere the interaction of p59-AVE3 and AVE3-CAT1.\nIn response to your comment, we have added these data (Fig. S16i-l). As shown, both LCI and\nCo-IP assays confirm that SlWRKY6 has no effect on the interaction of SlAVE3-p59 or\nSlAVE3-SlCAT1. We had in fact thoroughly evaluated these interactions during our initial\nexperiments but had omitted the results from the manuscript as they were negative.\nMinor comments\n1. Figure S7c, the authors should add the control group of AVE3-nYFP and p59-cYFP.\nThank you. We have already corrected this oversight.\n2. Figure 2d, in the cell-free degradation assays, they should repeat the experiment at least three\ntimes and add statistical analysis. They should also revised similar issues.\nThank you for your suggestion. All WB quantification analyses in this study were performed\nwith three replicates. Since initial manuscript submission did not require raw data, we initially\npresented only representative results. Following initial review, we have now consolidated and\npresented the statistically aggregated data from all three replicates. This revision has been\nuniformly implemented throughout the manuscript.\n3. Line 259, “We silenced SlAVE2 in both WT and slave3 backgrounds (Fig. S12d)”. They only\nshowed the results of silencing SlAVE2 in WT.\nGiven that the silencing efficiency of VIGS is highly dependent on primer design, the target\nsequences of all designed primers were validated for their silencing efficiency prior to the formal\nexperiments. Therefore, the VIGS primers and corresponding vectors used in this study have been\nconfirmed to achieve satisfactory silencing efficiency.\n4. The authors demonstrated that “The p59 hijacks and degrades SlCAT1 via the SlAVE2-SlAVE3\nUPS to facilitate ToCV pathogenicity”. They have constructed ToCV-Δp6, ToCV-ΔHsp70h, and\nToCV-Δp59. Does ToCV-Δp59 or other ToCV mutants changed the CAT1 activity and plant H2O2\ncontent? They could staine the leaves using DAB staining after inoculated with ToCV-ΔCP,\nToCV-ΔCPm, ToCV-Δp6, ToCV-ΔHsp70h, and ToCV-Δp59.\nWhile several studies have established that viral pathogens can employ specific proteins to\nhijack plant CAT, our study is specifically designed to investigate the role of p59, rather than other\nviral proteins such as CP, CPm, p6, or Hsp70h. Introducing assays on these additional effectors\nwithout elucidating their underlying mechanisms would inevitably complicate our findings, dilute the\npaper's focus, and compromise the rigor of our conclusions. Consequently, this could lead to\nsubstantial confusion for readers. We therefore hope you can appreciate our rationale for\nconcentrating this work on the defined interaction between p59 and the SlAVE3 signaling pathway.\nWe believe that Reviewer #1's initial comment, which suggested removing the broader ROS\nintroduction, was similarly intended to help sharpen the focus of our manuscript.\n5. Similarly, they should also show the H2O2 content using DAB staining in the AVE3 knockout\nplants and TRV-AVE2 plants.\nIn response to your request, we have evaluated the role of the SlAVE2-SlAVE3 UPS on CAT\nactivity and H O content. The corresponding data have been added as suggested (Fig. S17i,j).\n2 2\n6. Line 776 “All experiments should be conducted with at least two independent biological\nreplicates with similar results”. Is this a typing error? At least three???\nThe number of biological replicates was determined by the nature of experiments. For\nqualitative assays like the Y2H, it is standard practice to perform independent biological replicates\nfor both interacting partners, which is why we used the term \"at least.\" In contrast, the vast majority\nof our quantitative experiments were conducted with a minimum of three independent biological\nreplicates to ensure statistical rigor.\nReviewer #3 (Remarks to the Author):\nKey results\nOverall, the authors demonstrate strong proficiency in molecular biology and present well-founded\nhypotheses regarding the molecular mechanisms underlying ToCV pathogenicity and plant antiviral\nresistance. The study is original and holds significant potential impact, as ToCV is a major viral\ndisease that severely reduces tomato yield. Through a comprehensive suite of molecular and\nbiochemical experiments, including transformation, CRISPR-Cas9 mutagenesis, yeast two-hybrid,\nco-immunoprecipitation, bimolecular fluorescence complementation, luciferase complementation\nimaging, and cell-to-cell movement assays, the authors systematically elucidate the pathogen\n(ToCV) and host (tomato) interaction network. The findings include:\n1. p59 Function: p59 is identified as a central pathogenic determinant essential for virion assembly,\nplasmodesmatal targeting, and intercellular movement. It localizes to the plasma membrane and\nplasmodesmata, modulates callose deposition to widen intercellular pores, and functions as a viral\nmovement protein, thereby enhancing viral spread and pathogenicity.\n2. SlAVE3 Role: The tomato E3 ubiquitin ligase SlAVE3 specifically recognizes and ubiquitinates\nthe viral effector p59, directing it to proteasomal degradation and restricting ToCV infection. The\nC-terminal RING domain of SlAVE3 and the Leu255 residue of p59 are critical for this interaction,\nestablishing SlAVE3 as a key antiviral regulator that limits viral movement and enhances\nresistance.\n3. Regulatory Network: SlWRKY6 functions as a transcriptional repressor of SlAVE3 by binding to\nW-box motifs in its promoter. The SlAVE2–SlAVE3 ubiquitin–proteasome complex degrades\nSlWRKY6, relieving repression and forming a positive feedback loop that amplifies SlAVE3\nexpression and fortifies antiviral defense.\n4. Redox Manipulation: p59 further promotes infection by hijacking and degrading the host\nantioxidant enzyme SlCAT1 via the SlAVE2–SlAVE3 complex, disrupting redox homeostasis and\ncreating a cellular environment favorable for viral replication.\n5. Evolutionary Insight: SlAVE3 was subjected to selection during tomato domestication, producing\nthree haplotypes from two non-synonymous mutations. Among them, SpAVE3, derived from S.\npimpinellifolium, exhibits stronger binding to p59 but weaker interaction with SlCAT1, designating it\nas an antiviral haplotype associated with enhanced ToCV resistance.\nTogether, these findings establish a comprehensive interaction model (Figure 6) in which p59 drives\nToCV pathogenicity by facilitating virion assembly, intercellular movement, and host redox\ndisruption, while the tomato AVE2–AVE3–WRKY6 regulatory module orchestrates an antiviral\nfeedback mechanism that recognizes and degrades p59, thereby restoring cellular homeostasis\nand conferring viral resistance.\nI truly appreciate your comprehensive and positive evaluation.\nValidity\nOverall, the data interpretation is carefully executed and, in general, well supported by the\npresented evidence. The experimental design convincingly demonstrates the proposed molecular\nmechanism of the interaction between ToCV and tomato and the conclusions drawn from these\ndata are appropriate. The integration of molecular, biochemical, and phenotypic analyses supports\nand strengthens to the study’s main claims.\nI truly appreciate your comprehensive and positive evaluation.\nOne area of concern lies in the interpretation of selection and domestication analyses. The\ninference of a selection sweep based on phylogenetic reconstruction is not methodologically robust\nfor detecting signals of domestication. Phylogenetic trees describe evolutionary relationships but do\nnot quantitatively measure selective pressures or genetic diversity within populations. To\nstrengthen this aspect, the authors should focus on population genetic statistics, such as\nnucleotide diversity (π), Tajima’s D, or the ratio of non-synonymous to synonymous substitutions\n(dN/dS), which would provide a more rigorous and quantitative framework for evaluating\ndomestication-related selection.\nThank you for your suggestion. We have provided the nucleotide diversity and Tajima's D\nanalysis as per your request. Please refer to Fig. S18a,b for details.\nSignificance\nThis study uncovers a previously uncharacterized antiviral mechanism in tomato that counters\nToCV, a major threat to global tomato production. The authors identify the viral effector p59 as a\ncentral determinant of virion assembly and cell-to-cell movement, and demonstrate that the tomato\nE3 ubiquitin ligase SlAVE3 targets p59 for proteasomal degradation, thereby restricting viral spread.\nThe elucidation of the SlAVE2–SlAVE3–SlWRKY6 regulatory module reveals a self-reinforcing\nantiviral feedback loop in which degradation of SlWRKY6 enhances SlAVE3 expression and\nfortifies host defense. The study further shows that p59 manipulates host redox homeostasis\nthrough SlCAT1 degradation, linking viral infection to oxidative stress responses. Importantly,\nevolutionary analysis identifies natural SlAVE3 haplotypes under selection during tomato\ndomestication, including an antiviral allele (SpAVE3) that confers enhanced ToCV resistance.\nCollectively, this work provides a comprehensive molecular framework for ToCV–tomato\ninteractions, highlighting the AVE2–AVE3–WRKY6 axis as a critical regulatory node in plant\nantiviral immunity and offering valuable genetic insights for breeding virus-resistant cultivars.\nI truly appreciate your comprehensive and positive evaluation.\nData and methodology\nWhile the experimental design is comprehensive, three methodological aspects would benefit from\nadditional detail and clarification to improve reproducibility and rigor:\n1. Quantification Using ImageJ\nThe manuscript should specify the exact procedures and parameters used in ImageJ for image\nquantification. This includes outlining which plugins or measurement tools were applied (e.g., area,\nintensity, or co-localization analyses), how regions of interest were defined and whether\nbackground correction or normalization steps were performed. Providing this information would\nenhance transparency and allow accurate comparison or replication by other researchers.\nAs suggested, the description of the quantification using ImageJ has now been incorporated\ninto the \"Total protein extraction and immunoblotting\" section within the Methods.\n2. Plant Material of Nicotiana benthamiana\nThe genetic background of the N. benthamiana used in this study should be included.\nWe have revised this. The Nicotiana benthamiana LAB cultivar is commonly used in botanical\nexperiments.\n3. cDNA Libraries Used for Yeast Two-Hybrid Screening\nThe manuscript should provide details about the cDNA libraries employed in the yeast two-hybrid\nassay. Specifically, the origin of the library (species, tissue type, developmental stage, or stress\ncondition), the vector system used, and the coverage or complexity of the library should be reported.\nThese details are important to assess the comprehensiveness of the interaction screen and the\nbiological relevance of the identified protein–protein interactions.\nAs suggested, the description of the library construction has now been incorporated into the\n\"Yeast two-hybrid (Y2H) screening and verification\" section within the Methods.\nAnalytical approach\nThe statistical analyses are appropriately conducted and effectively support the study’s conclusions\nby addressing the research questions with sufficient rigor.\nI truly appreciate your comprehensive and positive evaluation.\nSuggested improvements\n1. Considering that pathogen–host interactions function through complex molecular networks, it\nwould be highly valuable to investigate the associated gene regulatory network integrating\nRNA-seq analysis. Using transgenic materials that share a more uniform genetic background with\nthe wild type can minimize background variation between genotypes and provide more reliable\ninsight into network-level changes. In addition, it would be beneficial to propose or experimentally\nvalidate candidate proteins that may participate in this network, such as other E3 ligases,\ntranscription factors, or redox-regulating enzymes interacting with the SlAVE2–SlAVE3 UPS, to\nstrengthen the mechanistic model and broaden understanding of the antiviral defense network.\nThank you for this excellent suggestion! As SlWRKY6 is the core transcription factor in this\nstudy, it is essential to investigate its regulatory network and potential antiviral response\nmechanisms. Considering your advice and the focus of our manuscript, we have decided to\nsupplement transcriptomic analyses under three treatment conditions: TRV-EV, TRV-EV+ToCV,\nand TRV-SlWRKY6+ToCV. The rationale for this experimental design is as follows:\n(1) Among the key genes studied here, only SlWRKY6 is a transcription factor. RNA-seq can\nreveal the critical transcriptional networks it regulates, which aligns with established methodologies\nin plant science research. In contrast, functional studies of enzymes such as SlAVE3 and SlCAT1\nare more suited to strategies like protein-protein interaction analysis.\n(2) By comparing TRV-EV and TRV-EV+ToCV, we can analyze the natural response patterns\nof tomato to ToCV infection. The comparison between TRV-EV+ToCV and TRV-SlWRKY6+ToCV,\non the other hand, reflects the SlWRKY6-mediated immune processes under infection conditions.\nThe overlapping genes identified from these two comparisons represent key target genes regulated\nby SlWRKY6 during the natural viral response. Elucidating these target genes will help us\nunderstand the antiviral significance of SlAVE3-mediated degradation of SlWRKY6 and the\npotential molecular pathways involved in the context of ToCV infection.\nIn summary, we have supplemented the relevant RNA-seq work and included it as Fig. S12.\n2. The study should also address the functional performance of heterozygous SpAVE haplotypes.\nIn modern tomato cultivars, which are typically F⊣ hybrids, understanding whether SpAVE alleles\nact dominantly or recessively is essential for practical breeding applications. If SpAVE exhibits\ndominance, the presence of one functional allele may be sufficient for the phenotype, whereas a\nrecessive effect would limit its utility in hybrid cultivars. Experimental or genetic data addressing\nthis question would strengthen the manuscript and clarify the translational potential of these\nfindings.\nThank you for your valuable feedback. Regarding your question, our data indicate that\ncompared to SlAVE3, SpAVE3 exhibits a stronger ability to ubiquitinate and degrade p59 but a\nweaker capacity to degrade SlCAT1. As a result, homozygous SpAVE3 lines demonstrate\nenhanced resistance to ToCV compared to homozygous SlAVE3 lines—a finding we have\nexperimentally validated.\nAs for the heterozygous SpAVE3/SlAVE3 plants, we would like to highlight that AVE3 functions\nas an enzyme, and enzymatic reactions are typically highly efficient. For instance, in the process of\ngenerating slave3 mutants, we also obtained several heterozygous mutants, which exhibited a viral\nresistance phenotype consistent with that of the WT. This further supports the high enzymatic\nactivity of SlAVE3.\nIn the case of SpAVE3/SlAVE3 heterozygous plants, we speculate that the presence of\nSpAVE3 would efficiently degrade p59. Since we have previously demonstrated that p59 acts as an\neffector that hijacks SlCAT1 and leads to its degradation by AVE3, it is reasonable to infer that the\nSpAVE3/SlAVE3 heterozygous lines would also exhibit a ToCV-resistant phenotype due to the\nefficient degradation of SlCAT1.\nHowever, within the current timeframe of this study, it has not been feasible to generate and\nvalidate the F hybrid plants. We fully acknowledge that this represents an interesting direction for\n1\nfuture breeding applications, though the absence of such data does not undermine the conclusions\nor novelty of our present work. We sincerely appreciate your understanding regarding this limitation\nand thank you once again for your insightful comments.\nClarity and context\nOverall, the manuscript is clearly written and logically structured, presenting the research in a\ncoherent and convincing manner. However, the suggestions below would further improve the\nquality and readability of the paper.\n1. Clarification of the SlAVE2–SlAVE3 Complex formation\nThe authors propose that the SlAVE2–SlAVE3 UPS complex mediates the degradation of\nSlWRKY6. However, the molecular mechanism underlying the formation and functional dynamics\nof the SlAVE2–SlAVE3 complex remains insufficiently explained. The manuscript would benefit\nfrom a clearer characterization of this complex, specifically, whether SlAVE2 and SlAVE3 form a\nstable heteromeric complex or interact transiently during ubiquitin-mediated degradation.\nThank you for your question. It pertains to a fundamental concept in this field. The role of the\nUPS as an unstable complex is well-established. Accordingly, please find a detailed explanation\nbelow.\n(1) Foundational Concept. Hershko and Ciechanover (1998; The ubiquitin system. Annual\nReview of Biochemistry, 67, 425-479.) establish the overarching principle of dynamic interactions\nwithin the ubiquitin cascade. This review outlines the entire ubiquitination cascade. While\nfoundational, it implicitly describes a system of unstable complexes: the ubiquitin thioester bond is\na high-energy intermediate that is passed from E1 to E2 and then to the substrate, with complexes\ndissociating after each transfer step. It details the ATP-dependent formation of the E1~Ub thioester\nbond, establishing that the entire process is driven by energy consumption, which is a hallmark of\ntransient, catalytic interactions rather than stable complex formation.\n(2) Mechanisms Underlying Ubiquitination. Berndsen and Wolberger (2014; New insights into\nubiquitin E3 ligase mechanism. Nature Structural & Molecular Biology, 21, 301-307) delves into the\nmolecular mechanics of ubiquitin transfer, explicitly highlighting the dynamic nature of E2-E3 and\nE3-substrate interactions. It explains how E3 ligases, particularly RING types (SlAVE3 in this study\nis a RING type E3), often act as molecular scaffolds that catalyze ubiquitin transfer by stabilizing a\nreactive conformation of the E2~Ub complex only momentarily, following which the complex\ndissociates. This \"proximity-driven catalysis\" is a defining feature of a transient complex.\n(3) Structural Biology Evidence. Plechanovová et al., (2012; Structure of a RING E3 ligase and\nubiquitin-loaded E2 primed for catalysis. Nature, 489, 115-120.) provided a high-resolution\nstructure of a RING E3 (RNF4) in complex with an E2~Ub (UbcH5A~Ub) conjugate, trapped in a\n\"pre-catalytic state.\" The structure revealed a limited protein-protein interface between the E3 and\nthe E2~Ub. Biochemical analyses confirmed that this complex is highly dynamic and unstable in\nsolution, directly providing structural proof of its transient nature.\n(4) Structural Insights into SCF-Mediated Ubiquitin Transfer. Duda et al., (2013; Structural\ninsights into the role of the SCF ubiquitin ligase in catalyzing ubiquitin transfer. eLife, 2, e00884.)\nfocuses on the structural mechanism of a large, multi-subunit cullin-RING E3 ligase complex (SCF).\nThe structures show how the E2~Ub is recruited to a flexible, distal site of the cullin scaffold. This\narchitecture functions as a dynamic platform that brings the E2~Ub into close proximity with a\nsubstrate for a brief period, facilitating ubiquitin transfer before the complex rearranges for the next\ncatalytic cycle.\n(5) Biophysical & Kinetic Evidence. Metzger et al., (2014; A conserved allosteric element\ncontrols the activation and polyubiquitination of Ube2S. Nature Structural & Molecular Biology, 21,\n876-883.) uses detailed biochemical and kinetic analysis to dissect the mechanism of the E2\nenzyme Ube2S. By measuring binding affinities (K ) and catalytic rates (K ), the study\nd cat\ndemonstrates that E3 binding accelerates ubiquitin transfer by lowering the activation energy rather\nthan forming a stable E2-E3-substrate complex. The rapid cycle of binding, catalysis, and\ndissociation is direct kinetic evidence for a transient complex.\n(6) Single-Molecule Observation of Ubiquitin Chain Assembly. According to Branigan et al.,\n(2020; Single-molecule studies of ubiquitin chain assembly by the anaphase-promoting\ncomplex/cyclosome (APC/C). Nature Communications, 11, 1-14.), single-molecule techniques are\nideal for observing transient events directly. This study visualizes ubiquitin chain formation by the\nmassive APC/C E3 ligase in real time. The authors directly observe and time the brief binding and\nrapid dissociation events of E2 enzymes (UBE2C and UBE2S) with the APC/C. These events occur\non a timescale of seconds or less, providing a visual and quantitative demonstration of the inherent\ninstability of the E2-E3 complex.\n2. Importance of Effector Compartmentation for Pathogenicity\nThe compartmentation of effectors within host cells is a critical determinant of pathogenicity. The\nauthors have conducted several localization assays to determine the subcellular positions of both\neffector and host proteins, which is commendable. However, the discussion should better integrate\nhow these localization patterns contribute mechanistically to virulence. Specifically, clarify whether\nthe spatial distribution of SlAVE2 and SlAVE3 affects their capacity to interact with SlWRKY6 or\nto recruit the ubiquitin–proteasome machinery. Emphasizing this compartmentalization will help link\nthe molecular data to the overall pathogenic strategy of the effector.\nThank you. This is indeed an interesting topic. We have included it in the Discussion. Or see\nbelow:\nThe spatiotemporal dynamics of protein subcellular localization are fundamental to the\nplant-virus interaction battlefield. Our findings that SlAVE3 localizes to the plasma membrane,\ncytoplasm, nucleus, plasmodesmata, and peroxisomes highlight a compelling strategy in plant\nimmunity: a decentralized defense network. By positioning this key immune component at multiple\nstrategic locations, including the primary boundary (plasma membrane), communication channels\n(plasmodesmata), genetic center (nucleus), and metabolic organelles (peroxisomes), the plant is\npoised to intercept viral pathogens at various critical entry and propagation points. This model\nprovides a spatial context for the interactions of SlAVE3 with partners like p59, SlCAT1, and\nSlWRKY6. The efficiency of the UPS in the cytoplasm is thus leveraged precisely because the\nubiquitination machinery is strategically pre-positioned. Furthermore, the viral effector p59's ability\nto hijack SlCAT1 from peroxisomes to the cytoplasm is not merely a manipulation for its own benefit;\nit inadvertently brings the substrate into the very compartment where the UPS machinery, primed\nby SlAVE3, is most active. This spatial hijacking is counteracted by the plant's spatially distributed\ndefense, explaining the enhanced degradation of SlCAT1 during infection compared to its relative\nstability in the non-infected state. Consequently, understanding these intricate localization patterns\nis not just about mapping proteins—it is about deciphering the tactical positioning of defenses and\nthe counter-tactics of pathogens within the cellular landscape.\nReferences\nThe manuscript appropriately references previous literature. The cited works are relevant, up to\ndate, and provide sufficient context for the study’s rationale and experimental design. The authors\neffectively integrate prior findings to support their hypotheses and discuss their results in relation to\nexisting knowledge, demonstrating a solid understanding of the field.\nI truly appreciate your comprehensive and positive evaluation."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have extensively revised the manuscript and most issues have been resolved. However, my original concern regarding the subcellular trafficking of CAT1 (Fig. 6) remains unaddressed. CAT1 is cytosolically translated and imported into peroxisomes; the data do not distinguish whether p19 blocks this import or retrieves CAT1 via retrograde export. The model should either provide evidence for export or be revised to depict cytosolic retention.", "reviewer_2": "The authors have adequately addressed most of the comments raised in the previous review.", "reviewer_3": "I've checked the entire revision and the point-by-point responses. The authors have satisfactorily addressed my previous questions and have substantially improved the manuscript. I believe the revisions have strengthened the work, and I now recommend the manuscript for acceptance."}, "author_response": "REVIEWERS' COMMENTS ROUND 2\nReviewer #1 (Remarks to the Author): The authors have extensively revised the\nmanuscript, and most issues have been resolved. However, my original concern\nregarding the subcellular trafficking of CAT1 (Fig. 6) remains unaddressed. CAT1 is\ncytosolically translated and imported into peroxisomes; the data do not distinguish\nwhether p19 blocks this import or retrieves CAT1 via retrograde export. The model\nshould either provide evidence for export or be revised to depict cytosolic retention.\nResponse: In accordance with your and editor’s requirements, we have revised\nthe mechanism model (Figure 6) and changed the corresponding description in\nthe main text and legends of Figure 6.\nReviewer #2 (Remarks to the Author): The authors have adequately addressed most\nof the comments raised in the previous review.\nResponse : We are very pleased to receive your positive feedback.\nReviewer #3 (Remarks to the Author): I've checked the entire revision and the\npoint-by-point responses. The authors have satisfactorily addressed my previous\nquestions and have substantially improved the manuscript. I believe the revisions have\nstrengthened the work, and I now recommend the manuscript for acceptance.\nResponse: We are very pleased to receive your positive feedback."}]

Decision letter