# EIN3-binding F-box protein S1EBF3 modulates resistance against Botrytis cinerea and carotenoid biosynthesis by degradation of BBX20 in tomato

Zhuo Gao, Heng Deng, Xiaoqing He, Yanpeng Yin, Chengpeng Yang, Tianhao Mao, Jinyan Guo, Mondher Bouzayen, Mingchun Liu, Mengbo Wu

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Zhuo Gao, Heng Deng, Xiaoqing He, Yanpeng Yin, Chengpeng Yang, et al.. EIN3-binding F-box protein S1EBF3 modulates resistance against Botrytis cinerea and carotenoid biosynthesis by degradation of BBX20 in tomato. Horticulture research, 2025, 12 (11), pp.uhaf219. 10.1093/hr/uhaf219. hal-05507150

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Article

# EIN3-binding F-box protein SIEBF3 modulates resistance against Botrytis cinerea and carotenoid biosynthesis by degradation of BBX20 in tomato

Zhuo Gao<fnref n="1" />, Heng Deng<fnref n="2" />, Xiaoqing He<fnref n="1" />, Yanpeng Yin<fnref n="1" />, Chengpeng Yang ID <fnref n="1" />, Tianhao Mao<fnref n="1" />, Jinyan Guo<fnref n="1" />, Mondher Bouzayen<fnref n="3" />, Mingchun Liu ID <fnref n="1" /> and Mengbo Wu ID <fnref n="1" />,*,

## Abstract

EIN3 binding F-box (EBF) proteins have been reported to play important roles in ethylene signaling pathway by mediating the ubiquitin-dependent degradation of EIN3-Like (EIL) proteins, but little is known about their roles in postharvest disease resistance. Here, we showed that SIEBF3 confers resistance against Botrytis cinerea by ubiquitin-mediated degradation of SIBBX20. Overexpression of SIEBF3 enhanced resistance to B. cinerea and increased the expression levels of genes related to PR (pathogenesis-related) and JA (jasmonic acid) in tomato, while knockdown of SIEBF3 does not affect tomato resistance to B. cinerea. Further study demonstrated that SIEBF3 interacts with SIBBX20 the interaction between SIEBF3 and SIBBX20 promotes SIBBX20 degradation via the 26S proteasome, which confers enhanced resistance to B. cinerea through the JA signaling pathway mediated by the SIBBX20-SMYC2-SIMED25 module. Meanwhile, SIEBF3 extends fruit shelf life by remodeling cell wall composition and promoting cuticular accumulation. Additionally, SIEBF3 is involved in carotenoid metabolism regulation by interacting with SIBBX20, SIRIN, SIFUL1, and SITAGL1, which is independent of the degradation of EIL proteins. Overall, this study revealed the molecular mechanism by which SIEBF3 responds to JA signaling to regulate B. cinerea resistance, enriched the roles of SIEBF3 in the regulatory network of carotenoids metabolism, and provided new insights into the extension of fruit shelf life.

## Introduction

Botrytis cinerea is a necrotrophic phytopathogenic fungus that can infect hundreds of plants and cause significant economic and agricultural losses worldwide [1, 2]. As a leading commercial vegetable crop with global cultivation significance, tomato (Solanum lycopersicum) exhibits heightened susceptibility to B. cinerea infestation throughout its maturation phase. This pathogen causes gray mold disease, leading to significant postharvest losses and reduced yield [3, 4]. The use of chemical fungicides is currently an effective strategy for controlling B. cinerea, while overuse poses new threats to ecosystems and consumer health [5]. Consequently, understanding the molecular mechanisms of fruit resistance to B. cinerea is essential for the development of eco-friendly strategies for disease control, thereby preserving the postharvest quality and economic value of fruits.

The phytohormone jasmonic acid (JA) orchestrates diverse developmental pathways essential for plant morphogenesis and enhances defense responses against physical injury and microbial pathogenesis [6-9]. After decades of research, the molecular mechanism of JA signaling, consisting of multiple interacting proteins, has been elucidated. The bHLH transcription

factor MYC2 serves as a master regulator, integrating diverse JA-responsive pathways [10]. Without JA, JAZ proteins interact with the corepressor TOPLESS to form transcriptional repression complexes. These complexes inhibit MYC2-dependent gene activation by interfering with the ability of transcription factors to bind target DNA. When the bioactive JA ligand jasmonoyl-isoleucine (JA-Ile) is present, JAZ proteins bind with CORONATINE-INSENSITIVE1 (COI1), the F-box component of the SCFCO11 E3 ligase complex. This JA-Ile-dependent interaction initiates the polyubiquitination of JAZ proteins and their subsequent proteasomal degradation, thereby relieving the inhibition of MYC2. Subsequently, MYC2 interacts with the mediator subunit MED25 to form a functional transcriptional complex, which activates the expression of JA-regulated genes such as SJA2L (JA2-Like) and ERF.C3 [10-15]. BBX proteins are characterized by their zinc-finger domains and represent an important family of transcriptional regulators involved in diverse physiological processes. Recent studies demonstrate that BBX20 both regulates anthocyanin biosynthesis in fruit while suppressing JA-mediated defense responses through inhibiting the accumulation of MED25, consequently reducing resistance to B. cinerea [16].

<footnote n="1">¹Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, Sichuan 610065, China</footnote>

<footnote n="2">²School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, China</footnote>

<footnote n="3">³Laboratoire de Recherche en Sciences Végétales-Génomique et Biotechnologie des Fruits-UMR5546, Université de Toulouse, CNRS, UPS, Toulouse-INP, Toulouse, France</footnote>

<footnote>*Corresponding author. E-mail: mbwu@scu.edu.cn</footnote>
As a crucial phytohormone, ethylene (ET) serves dual biological functions not only by orchestrating the maturation of climacteric fruits but also by exerting significant influence on plant defense mechanisms and immune-related processes [17–21, 23]. The SIERF members has been extensively researched on resistance to B. cinerea. The susceptibility of fruit to B. cinerea increased with the knockout of SIERF.C1, whereas its overexpression (OE) improved resistance [21]. Silencing of SIERF.A1, SIERF.A3 (Pit4), SIERF.B4, and SIERF.C3 (Pit5) via virus-induced gene silencing (VIGA) increased susceptibility to B. cinerea in tomato leaves [24]. Ethylene Insensitive3 Like1(EIL1), an upstream of ethylene response factors (ERFs), targets and activates the JA metabolizing gene SICYP94C1, thereby negating JA-mediated resistance [25]. While SIERFs and SIEIL1 are well characterized in the defense against B. cinerea, the involvement of other ET signaling pathway members in the disease resistance is still uncertain.

Carotenoids are vital phytonutrients in tomato and their synthesis is mediated by various enzymes such as phytoene synthase (PSY) and phytoene desaturase among others. Tomato mutants with loss-of-function of SIPSY1 produce yellow fruits indicating that SIPSY1 plays a crucial role in regulating carotenoid synthesis [26, 27]. Transcription factors, including as SIBBX20, S1MYB72, S1MYB1, and HY5 have been shown to directly interact with the promoter of SIPSY1 to regulate carotenoid synthesis [28–31]. During tomato ripening, key transcription factors such as RIN, FUL1, TAGL1, and NOR play important functions in the regulation of carotenoid metabolism and have been widely studied [32–35]. Our recent finding indicate that SIEBF3 (EIN3 BINDING F-BOX 3) plays a role in climacteric fruit ripening by facilitating the degradation of EIL proteins [36]. However, the relationships between these EBF proteins and these key transcription factors regulating carotenoids synthesis are unknown.

This study reveals novel mechanisms of SIEBF3 in regulating tomato fruit resistance to B. cinerea, phytoene synthesis, and fruit ripening through interactions with BBX20, RIN, FUL1, and TAGL1. Additionally, SIEBF3 shows functional redundancy with other SIEBF genes in mediating resistance to B. cinerea, but exhibiting distinct regulatory effects on phytoene synthesis. Knockdown of SIEBF3 did not notably affect resistance to B. cinerea, but it significantly increased phytoene content compared to wild type (WT). OE of SIEBF3 markedly increased resistance to B. cinerea, accompanied by a notable reduction in phytoene content. In our study, we found that SIEBF3 responds to JA signaling through the degradation of SIBBX20 and thus regulates tomato resistance to B. cinerea. In addition, SIEBF3 interacts with RIN, FUL1, and TAGL1 to regulate phytoene synthesis and fruit ripening, while none of the above results were accomplished by degrading EIL. In conclusion, our study identified a novel function of SIEBF3 in regulating resistance against B. cinerea in tomato fruits and enriched the regulatory network of carotenoid synthesis and ripening in fruits, which provides a new strategy for maintaining postharvest fruit quality.

## Results

### SIEBF3 is induced by B. cinerea and enhances resistance to B. cinerea infection in tomato fruits

Previous research has shown that SIEBF3 participates in regulating fruit maturation and carotenoid biosynthesis by interacting with EILs and repressing it accumulation. However, its involvement in the response to B. cinerea remains unclear. To investigate whether SIEBFs responds to B. cinerea infestation, RT-qPCR assay was performed after infesting WT plants in tomato leaves with

B. cinerea. Similar with PR genes, SIEBFs were significantly upregulated (Fig. 1a), demonstrating that SIEBFs are typical B. cinerea response genes. Given that SIEBF3 exhibited the most pronounced changes among the three EBF genes, we speculate that EBF3 plays a pivotal role in responding to B. cinerea infection (Fig. 1a). To analyze the function of SIEBF3 in B. cinerea resistance, we generated SIEBF3 knockout (SIEBF3-KO) lines by CRISPR/Cas9 mediated genome editing technology and obtained two KO lines with 5-bp deletion and 16-bp insertion and resulting in truncated versions with 22 and 61 amino acids remaining, respectively (Fig. S1a). Two OE lines were used for further study (Fig. S1b). There was no effect on vegetable growth such as plant size (Fig. S2a and b). Apart from delayed fruit ripening in SIEBF3-OE lines (Fig. S2c and d), no significant differences in fruit morphology or pericarp thickness were observed among the WT, SIEBF3-KO, and SIEBF3-OE lines (Fig. S2e–g).

To assess the potential role of SIEBF3 in conferring resistance to B. cinerea during fruit development, we collected WT, SIEBF3-KO, and SIEBF3-OE fruits at MG (mature green), Br (breaker), and Br + 7 stages and inoculated them with B. cinerea. After 48 h post-inoculation (hpi), the area of the lesions was measured (Fig. 1b). As shown in Fig. 1, the mean lesion size in SIEBF3-OE fruits was significantly smaller than that in WT fruits at the MG, Br, and Br + 7 stages, with no differences observed between WT and SIEBF3-KO across these stages (Fig. 1c–e). We quantified the activities of two important antioxidant enzymes, superoxide dismutase (SOD), and peroxidase (POD). SIEBF3-OEs fruits demonstrated notably higher SOD and POD activities compared to WT fruits, whereas SIEBF3-KO fruits did not display any significant differences in these activities relative to WT fruits (Fig. 1f and g). To further assess the severity of the disease, we analyzed the spread of B. cinerea hyphae in infected fruits by WGA-PI staining. Intense green fluorescence was distributed along the cell wall and/or in the intercellular spaces in the tissues of WT and SIEBF3-KO fruits, whereas it was significantly reduced in the tissues of SIEBF3-OE fruits (Fig. 1h). These findings indicated that compared with WT and SIEBF3-KO, SIEBF3-OE fruits are less susceptible to B. cinerea infection. These findings indicate that SIEBF3 enhances resistance to B. cinerea infection.

### SIEBF3 enhances resistance to B. cinerea in tomato leaves

We evaluated the transgenic plant's resistance to B. cinerea in leaves. Four days after infestation, the lesion area on these leaves was measured. The mean lesion area of SIEBF3-OE leaves was significantly smaller compared to WT leaves, whereas no significant difference was observed between SIEBF3-KO and WT leaves (Fig. 2a and b). To visualize the difference in resistance between SIEBF3 transgenic plants and WT, detached leaves inoculated with B. cinerea for two days were stained with trypan blue. SIEBF3-OE leaves had smaller areas of dead cells compared to WT, while SIEBF3-KO leaves showed no significant difference from WT leaves (Fig. 2c and d). After 48 hpi with B. cinerea, SIEBF3-OEs leaves exhibited increased SOD and POD activities compared to WT (Fig. 2e and f).

Meanwhile, the expression levels of disease resistance-related genes SIPR1, SIPR1a, SIPR2b, SIPR5, and SIRBOHD were significantly increased in SIEBF3-OEs lines (Fig. 2g). These findings suggest that SIEBF3 positively influences tomato leaf resistance to B. cinerea, with its OE significantly enhances this resistance. The KO SIEBF3 did not impact tomato resistance to B. cinerea, suggesting potential functional redundancy among the three SIEBF3 genes in tomato. To verify this hypothesis, we examined the expression of SIEBF3
<figure><img src="image_2.png" /><figcaption>**Figure 1.** SIEBF3 is induced by B. cinerea and enhances resistance against B. cinerea infection in tomato. (a) Relative transcript levels of pathogenesis-related (PR) genes and SIEBFs in response to B. cinerea infection. CK, blank potato dextrose agar (PDA) media. BC, B. cinerea. Relative mRNA levels of each gene in WT before B. cinerea infection were normalized to 1. Data are shown as means ± SD (n = 3). Asterisks indicate statistical significance using Student's t-test, P < 0.05. B. cinerea infection were normalized to 1, and SIActin was used as an internal control. (b) Disease phenotype in WT, SIEBF3-KO lines, and SIEBF3-OE fruits after incubated with B. cinerea. Fruits were collected at MG, Br, and Br + 7 stages and then infected with B. cinerea. Photographs were taken at 48 h hpi/2 DPI. MG (mature green) stage, Br (Br stage), Br + 7, 7 d after the Br stage. Bar = 10 mm. (c–e) Lesion diameter in tomato fruits after infected with B. cinerea for 2 d at MG (c), Br (d), and Br + 7 (e) stages. Data are shown as means ± SD (n = 10). Asterisks indicate statistical significance using Student's t-test, P < 0.05. (f, g) Antioxidant enzymatic activities of SOD (f) and POD (g) in fruits at 2 DPI with B. cinerea. Data are shown as means ± SD (n = 10). Asterisks indicate statistical significance using Student's t-test, P < 0.05. (h) Fungal hyphae in planta were visible in the WGA-PI-stained pericarp from WT, SIEBF3-KO, and SIEBF3-OE fruits after infected with B. cinerea under the fluorescence microscopy at Br stages. Fungal hyphae show green fluorescence, cell walls of plant show magenta fluorescence (replacing red fluorescence with magenta). Bar = 100 µm.</figcaption></figure>

homologous family members SIEBF1 and SIEBF2 in SIEBF3-KO and SIEBF3-OE fruits after inoculation with B. cinerea. The results showed that SIEBF1 and SIEBF2 were significantly upregulated in SIEBF3-KO and downregulated in SIEBF3-OE after inoculation with B. cinerea (Fig. 2h), which demonstrated the functional redundancy of SIEBFs in regulating B. cinerea resistance.

## SIEBF3 affects the expression of disease resistance-related genes in tomato fruit

To better understand the potential molecular mechanism by which SIEBF3 contributes to B. cinerea resistance, we performed RNA-Seq on WT and SIEBF3-OE fruits that were harvested at the Br stage and infected with B. cinerea for 48 h, with three replicates per sample. In the comparison between SIEBF3-OE and WT fruits, a total of 6386 differentially expressed genes (DEGs) were identified, with 2570 genes were downregulated and 3816 genes upregulated, were found in SIEBF3-OE vs WT fruits (Fig. 3a; Dataset S2). KEGG

analysis revealed that SIEBF3-OE influences pathways associated with glyoxylate and dicarboxylate metabolism, glutathione metabolism, plant hormone signal transduction and plant-pathogen interaction were affected by SIEBF3-OE in B. cinerea-infected fruits (Fig. 3b; Dataset S3). In alignment with the disease progression, the expression levels of PR genes such as SIPR1, SIPR1a, SIPR2b, and SIPR5 and defense related (DR) genes such as Respiratory burst oxidase homolog D (SIRBOHD) were increased significantly in SIEBF3-OE fruits compared with WT following B. cinerea infection (Fig. 3c).

## SIEBF3 is induced by JA and positively regulates JA signaling

ET and JA can induce the expression of PR genes [37]. SIEBF3 plays a negative regulatory role in the ET signaling pathway; therefore, we hypothesize that the upregulation of PR genes in SIEBF3-OE fruits may be attributed to activation of the JA signaling pathway.
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 2.** SIEBF3 enhances resistance to *B. cinerea* in tomato leaves. (a) Disease symptom on detached leaves 4 days after infected with *B. cinerea* on WT, SIEBF3-KO, and SIEBF3-OE lines. Bar = 10 mm. (b) Lesion diameter in leaves at 4 DPI. At least 10 leaves from each line were infected with *B. cinerea* for lesion size measurement. Data are shown as means ± SD (n = 10). Asterisks indicate statistical significance using Student's t-test, P < 0.05. (c) Representative images of trypan blue staining for cell death in indicated tomato leaves at 2 DPI with *B. cinerea*. Bar = 10 mm. (d) Lesion diameter in leaves at 2 DPI. At least 5 leaves from each line were infected with *B. cinerea* for lesion size measurement. Data are shown as means ± SD (n = 5). Asterisks indicate statistical significance using Student's t-test, P < 0.05. (e, f) Antioxidant enzymatic activities of SOD (e) and POD (f) in leaves at 2 DPI with *B. cinerea*. Data are shown as means ± SD (n = 3). Asterisks indicate statistical significance using Student's t-test, P < 0.05. (g) Relative expression levels of disease resistance-related genes *SPIR1*, *SPIR1a*, *SPIR2b*, *SPIR5*, and *SIRBOHD* in WT, SIEBF3-KO, and SIEBF3-OE lines. (h) Relative expression levels of SIEBF1, SIEBF2 in WT, SIEBF3-KO, and SIEBF3-OE lines after infected with *B. cinerea*. The transcript levels in WT were normalized to 1, and *SlActin* were used as an internal standard. Data are shown as means ± SD (n = 3). Asterisks indicate statistical significance using Student's t-test, P < 0.05.</figcaption></figure>

Consequently, we investigated the expression profiles of JA sig-
naling and response-related genes in SIEBF3-OE fruits at 2 d post-
inoculation (DPI). Transcriptomic analysis revealed substantial
transcriptional upregulation of SIMYC2, SlMED25, SlJA2L, and SlTD
(Fig. 4a). We used RT-qPCR to examine the transcript levels of rel-
evant genes in fruits from WT, SIEBF3-KO, and SIEBF3-OE lines to
determine whether SIEBF3 influences tomato disease resistance
through JA signaling. The findings indicated that SIEBF3-OE lines
exhibited increased transcription of SlJA2L and SlTD compared to
WT lines, whereas no transcriptional differences were detected
between WT and SIEBF3-KO lines (Fig. 4b and c). Notably, the
transcript levels of these genes were consistently higher in SIEBF3-
OE lines than in WT and SIEBF3-KO lines (Fig. 4b and c). These
findings indicate that SIEBF3 promotes the expression of SlJA2L
and SlTD. The expression levels of SlJA2L and SlTD increased in
WT, SIEBF3-KO, and SIEBF3-OE lines after MeJA treatment (Fig. 4b
and c).

To examine whether SIEBF3 expression is responsive to JA,
we tested the transcript level of SIEBF3 in WT after treatment
with MeJA. These findings indicated a significant up-regulation

after JA treatment, which was consistent with the trend of
changes in the JA-responsive genes SIMYC2 and SlJA2L indicating
that SIEBF3 can be induced by JA (Fig. 4d). These results
suggest that SIEBF3 is induced by JA and positively regulates
JA signalling.

SIEBF3 interacts with SIBBX20 and attenuates its
suppression of SlMED25-SIMYC2 activation of
SlJA2L

To elucidate the molecular mechanisms by which SIEBF3
modulates JA signaling, we conducted a yeast two-hybrid (Y2H)
screening assay to identify potential interacting proteins. The
results indicated that SIEBF3 interacts with SIBBX20, a negative
regulator of JA signaling, but not with SIMYC2 or SlMED25
(Fig. 5a). Additionally, a bimolecular fluorescence complemen-
tation (BiFC) assay was conducted to validate the interaction
between SIEBF3 and SIBBX20. Tobacco leaves were coinjected with
Agrobacterium tumefaciens strains carrying plasmids for SIEBF3-
YNE and SIBBX20-YCE expression. Three days post-injection, flu-
orescence was observed in the experimental group but not in the
<figure><img src="image_8.png" /><figcaption>**Figure 3.** RNA-Seq analysis of WT and SIEBF3-OE fruits. (a) Volcano diagrams of DEGs in WT and SIEBF3-OE fruits at 2 DPI at Br stage. The block colored in red and green means the up-regulated genes and downregulated genes, respectively. (b) KEGG analysis of 6386 DEGs in WT and SIEBF3-OE fruits. (c) Expression levels of disease resistance-related genes SIPR1, SIPR1a, SIPR2b, SIPR5, and SIRBOHD in WT and SIEBF3-OE fruits. Data are shown as means ± SD (n = 3). Asterisks indicate statistical significance using Student's t-test, P < 0.05.</figcaption></figure>

control group (Fig. 5b). We conducted a co-immunoprecipitation (Co-IP) assay to confirm the interaction between the two proteins. The results revealed that SIBBX20-FLAG co-immunoprecipitated with SIEBF3-HA, but not GFP-FLAG (Fig. 5c). Taken together, our results demonstrate that SIEBF3 interacts with SIBBX20 in vitro and *in planta*.

It has reported that SIMYC2-SIMED25 can promote resistance to B. cinerea by activating the transcription of SJA2L, and SIBBX20 negatively regulates JA signaling through inhibition of SIMED25 protein accumulation. A dual luciferase assay was performed to evaluate the impact of SIEBF3 on the transcriptional activation of the SJA2L promoter by the SIBBX20-SIMED25-SIMYC2 complex. Full-length coding sequences of SIEBF3, SIMYC2, SIMED25, and SIBBX20 were amplified and inserted into the pGreen-62SK plasmid as the effector and the promoter of the JA-responsive gene SJA2L was fused to a Mini35S-LUC as a reporter (Fig. 5d). Tobacco leaves were co-infiltrated with A. tumefaciens strains containing the effector and reporter gene constructs and the LUC/REN ratio was measured after three days. The results showed that SIMED25-SIMYC2 could activate the SJA2L promoter and SIBBX20 could repress this transcriptional activation activity, which is consistent with the findings of previous studies [10, 16] (Fig. 5e). We found that SIEBF3 suppress the inhibitory effect of SIBBX20 on SIMED25-SIMYC2 activation of SJA2L and did not affect the activity of the SJA2L promoter alone (Fig. 5e). Collectively, these results demonstrate that SIEBF3 physically interacts with SIBBX20 to repress its regulatory function.

## SIEBF3 promotes SIBBX20 degradation through ubiquitin-mediated proteasomal degradation

To investigate whether the interaction between SIEBF3 and SIBBX20 affects SIBBX20 transcription, we analyzed the expression level of SIBBX20 in WT, SIEBF3-KO, and SIEBF3-OE lines. The results showed that SIEBF3 did not significantly affect SIBBX20 transcript levels (Fig. S3a). We subsequently examined the protein accumulation when SIBBX20 was expressed alone or co-expressed with SIEBF3. And found that SIBBX20 protein levels were significantly reduced when SIEBF3-HA and SIBBX20-FLAG were co-expressed in tobacco (Fig. 5f). SIEBF3 is a substrate-recruiting subunit of Skp1-cullin1-F-box (SCF)-type E3 ubiquitin ligases that are responsible for binding and ubiquitinating degradation substrates. Therefore, we speculate that the interaction between SIEBF3 and SIBBX20 results in the degradation of SIBBX20 by ubiquitinated. To validate this hypothesis, we detected the degree of ubiquitination with an anti-ubi antibody and found that there was a higher degree of ubiquitination in the presence of SIEBF3 (Fig. 5f). To validate these results, Ub-HA was co-expressed with SIBBX20-FLAG in tobacco, and the result of the Co-IP experiments showed that Ub could be bound by SIBBX20-FLAG (Fig. 5g).

To investigate the ubiquitination mechanism of SIBBX20, we analyzed the protein sequence and identified two lysine residues (K143 and K152) that were predicted as potential ubiquitination sites (https://sumo.biocuckoo.cn/; Fig. S3b). To map the specific residues of SIBBX20 responsible for its SIEBF3-dependent ubiquitination and proteasomal turnover, we conducted a site-directed mutagenesis analysis of 2 lysine residues in the SIBBX20
<figure><img src="image_9.png" /><figcaption>**Figure 4.** SIEBF3 is induced by JA and positively regulates JA signalling. (a) Relative transcript levels of SIMYC2, SIMED25, SJA2L, and SITD genes in WT and SIEBF3-OE fruits at 2 DPI at Br stage. (b, c) Relative expression of SJA2L (b) and SITD (c) in WT, SIEBF3-KO, and SIEBF3-OE lines after MeJA treatment. Expression levels were normalized to untreated WT controls (set as 1). Data represent mean ± SD (n = 3). Asterisks indicate statistical significance using Student's t-test, P < 0.05. (d) Relative expression of SIMYC2, SJA2L, and SIEBF3 in WT after MeJA treatment. Expression levels of each gene in WT were normalized to 1. Data represent mean ± SD (n = 3). Asterisks indicate statistical significance using Student's t-test, P < 0.05.</figcaption></figure>

protein (Fig. S3c). Co-expression of SIEBF3 with mutant SIBBX20 in tobacco leaves, the single mutations at K143 or K152 decreased the ubiquitination and increased the protein level of SIBBX20 (Fig. 5h). After mutating both K143 and K152, no ubiquitination of SIBBX20 was detected, and the protein level of SIBBX20 was twice as high as in the case of ubiquitination (Fig. 5h). To further demonstrate that the interaction between SIEBF3 and SIBBX20 promotes the degradation of SIBBX20, we performed protein degradation assays. Cycloheximide (CHX) was used in the co-expression system to assess the degradation rate of SIBBX20. SIBBX20 was rapidly degraded in the presence of SIEBF3-HA (Fig. 5i and j). In contrast, the degradation of SIBBX20 was greatly slowed when it was expressed alone (Fig. 5i and j). Taken together, these results demonstrate that SIEBF3 mediates the ubiquitin-dependent degradation of SIBBX20, thereby restoring the SlMED25-SIMYC2-mediated activation of SJA2L. Furthermore, our mutagenesis analysis identified lysine residues K143 and K152 as critical sites for SIEBF3-mediated ubiquitination and subsequent degradation of SIBBX20.

## SIEBF3 inhibits S1PSY1 activation by S1BBX20 and thus impacts phytoene synthesis

SIBBX20 was reported to regulate carotenoid metabolism by directly binding the promoter of S1PSY1 and activating its expression [28]. However, whether the interaction between SIEBF3 and S1BBX20 influences S1BBX20-mediated transcriptional regulation of S1PSY1 is unclear. We determined the carotenoid content by liquid chromatography-mass spectrometry (LC-MS) and detected a significant increase in total carotenoid content in SIEBF3-KO and a notable decrease in SIEBF3-OE compared to the WT (Fig. 6a). Notably, the expression of PSY1, a key carotenoid

biosynthetic gene, was significantly upregulated in the SIEBF3-KO lines but markedly downregulated in the SIEBF3-OE lines (Fig. 6b). A dual-luciferase reporter (DLR) assay was performed to investigate whether the physical interaction between SIEBF3 and S1BBX20 affects S1BBX20-mediated transcriptional regulation. The results of DLR indicated that S1BBX20 could activate S1PSY1, whereas SIEBF3 could inhibit the activation of S1PSY1 by S1BBX20 (Fig. 6c). Taken together, these data suggest that SIEBF3 affects the accumulation of carotenoids by inhibiting the transcriptional activity of S1BBX20 on S1PSY1 gene.

## SIEBF3 interacts with SlRIN, SlFUL1, and SlTAGL1 without undergoing ubiquitination and inhibits their upregulation of SlPSY1, SlACO1, and SlACS2

We have reported that SIEBF3 is involved in fruit ripening by regulating the accumulation of EILs [36]. However, it remains unclear whether SIEBF3 can regulate fruit ripening-related traits independently of its canonical role in EIL protein degradation. To explore whether there are other molecular mechanisms by which SIEBF3 regulates the fruit ripening process, we investigated the interactions between SIEBF3 and several ripening regulators. The yeast two-hybrid assay indicated that SIEBF3 interacts with SlRIN, SlFUL1, and SlTAGL1, but not with SlNOR (Fig. 6d). We performed a Co-IP assay after transiently co-infiltrating of SIEBF3-HA with SlRIN/SINOR/SIFUL1/SITAGL1-FLAG constructs in N. benthamiana leaves to validate these interactions in vivo. The findings indicated that SIEBF3-HA co-precipitated with SlRIN-FLAG, SlFUL1-FLAG, and SlTAGL1-FLAG, but not with SlNOR-FLAG, which aligns with the yeast two-hybrid results (Fig. 6e). Furthermore, direct physical interactions between SIEBF3 and SlRIN/SIFUL1/SITAGL1 were confirmed through BiFC assay (Fig. 6f). These findings demonstrate
<figure><img src="image_10.png" /><figcaption>**Figure 5.** SIEBF3 interacts with SIBBX20 and attenuates its suppression of SIMED25-SIMYC2 activation of SIJA2L. (a) Y2H assay of interaction between SIEBF3 and SIBBX20, SIMYC2, and SIMED25. SIBBX20, SIMYC2, and SIMED25 were used as bait, SIEBF3 was used as prey. Interactions between P53 and T7 and between SIEBF3-BD and empty AD were used as positive and negative controls, respectively. (b) BiFC assay of interaction between SIEBF3 and SIBBX20. Bar = 50 µm. (c) In vivo Co-IP assays of SIEBF3 with SIBBX20. SIBBX20-FLAG was co-infiltrated with SIEBF3-HA in N. benthamiana leaves by Agrobacterium-mediated infiltration. Total proteins were extracted and immunoprecipitated with anti-FLAG antibody. GFP-FLAG was used as negative control. Immunoblots were probed with anti-FLAG antibody to detect SIBBX20 and anti-HA antibody to detect SIEBF3. (d) A schematic illustration of the LUC reporter and effector constructs used in transient expression assays. (e) Influence of SIEBF3-62sk on the expression of SIJA2L in transient expression assays. The LUC/REN ratio represents the relative activity of the SIJA2L promoter. Data are shown as means ± SD (n = 3). Values marked with different letters indicate statistically significant differences as analyzed by a one-way ANOVA test from three independent biological replicate experiments, P < 0.05. (f) Effect of SIEBF3 on the protein stability of SIBBX20 and in vivo ubiquitination of SIBBX20 by SIEBF3. The SIBBX20-Flag was expressed in the presence (+) or absence (−) of SIEBF3-HA in N. benthamiana leaves. After 3 days, protein was extracted from tobacco leaves. The SIEBF3-HA and SIBBX20-FLAG proteins were detected by immunoblotting. Proteins were analyzed by immunoblotting with anti-FLAG, anti-HA, and anti-actin antibodies. Total proteins extracted from transformed leaves were immunoprecipitated with anti-flag beads, the ubiquitination was detected by anti-ubiquitin antibody. The arrow indicates a band containing the SIBBX20 protein. The numbers under the bands indicate the ratio of SIBBX20 protein to actin. HA is pBTEX-HA empty vector used as a negative control. (g) In vivo detection of ubiquitination of SIBBX20-FLAG by Co-IP. (h) Identification for SIBBX20 site ubiquitinated by SIEBF3 via in vivo ubiquitination assay. The two predicted lysine (K) sites were mutated individually and together to arginine (R). The SIBBX20-Flag, SIBBX20K152R-Flag, SIBBX20K143R-Flag, and SIBBX20K143R/K152R-Flag were expressed in the presence of SIEBF3-HA in N. benthamiana leaves and subjected to ubiquitination analysis as described in (d). (i, j) Degradation rate analysis of SIBBX20 in the presence (+) or absence (−) of SIEBF3. Co-expression of SIBBX20-Flag with or without SIEBF3-HA was performed in N. benthamiana leaves, followed by treatment with translation inhibitor CHX. The total proteins were extracted for immunoblotting with anti-FLAG and anti-HA antibody at an indicated time point after treatment. The intensity of protein bands was quantified using ImageJ. Data are shown as means ± SD (n = 3).</figcaption></figure>

that SIEBF3 interacts with SIRIN, SIFUL1, and SITAGL1 both in vitro and in vivo.

SIPSY1, SIACO1, and SIACS2, which were significantly downregulated in SIEBF3-OE lines (Fig. S4), are direct targets of SIRIN,

SIFUL1, and SITAGL1. To investigate whether the interaction between SIEBF3 and SIRIN, SIFUL1 and SITAGL1 affects the activation of their target genes, a DLR assay was conducted. It showed that SIBBX20 activate SIPSY1, while SIEBF3 inhibit the activation
<figure><img src="image_11.png" /><figcaption>**Figure 6.** SIEBF3 involved in fruit ripening and carotenoid metabolism. (a) Carotenoid contents in fruits of the WT, SIEBF3-KO lines, and SIEBF3-OE at Br + 7 stage. Each value represents the mean of three biological replicates. Data are shown as means ± SD. Asterisks indicate statistical significance using Student's t-test, *P* < 0.05. (b) Relative expression levels of SIPSY1 in WT, SIEBF3-KO, and SIEBF3-OE lines. Expression levels were normalized to untreated WT controls (set as 1) and SlActin was used as an internal control. Data are shown as means ± SD (n = 3). Asterisks indicate statistical significance using Student's t-test, *P* < 0.05. (c) SIEBF3 represses the activation of SIPSY1 by SIBBX20. The LUC/REN ratio represents the relative activity of the SIPSY1 promoter. Data are shown as means ± SD (n = 3). Values marked with different letters indicate statistically significant differences as analyzed by a one-way ANOVA test from three independent biological replicate experiments, *P* < 0.05. (d) Screening for ripening regulators that interact with SIEBF3 by yeast two-hybrid. SIRIN, SINOR, SIFUL1, and SITAGL1 were used as bait, SIEBF3 was used as prey. Interactions between P53 and T7 and between SIEBF3-BD and empty AD were used as positive and negative controls, respectively. (e) *In vivo* Co-IP assays of SIEBF3 with SIRIN, SINOR, SIFUL1, and SITAGL1. SIRIN-FLAG, SINOR-FLAG, SFUL1-FLAG, and SITAGL1-FLAG were co-infiltrated with SIEBF3-HA in *N. benthamiana* leaves by Agrobacterium-mediated infiltration. Total proteins were extracted and immunoprecipitated with anti-FLAG antibody. GFP-FLAG was used as negative control. Immunoblots were probed with anti-FLAG antibody to detect SIRIN, SINOR, SFUL1, SITAGL1, and anti-HA antibody to detect SIEBF3. (f) BiFC assay of interaction between SIEBF3 and SIRIN, SIFUL1, and SITAGL1. Bar = 50 µm. (g) Influence of SIEBF3-62sk, SIRIN-62sk, SIFUL1-62sk, and SITAGL1-62sk on the expression of SIPSY1, SlACO1, and SlACS2 in transient expression assays. The LUC/REN ratio represents the relative activity of the SIPSY1, SlACO1, and SlACS2 promoter. Data are shown as means ± SD (n = 3). Values marked with different letters indicate statistically significant differences as analyzed by a one-way ANOVA test from three independent biological replicate experiments, *P* < 0.05. (h) Effect of SIEBF3 on the protein stability of SIRIN, SIFUL1, and SITAGL1. The SIRIN-FLAG, SIFUL1-FLAG, and SITAGL1-FLAG were expressed in the presence (+) or absence (−) of SIEBF3-HA in *N. benthamiana* leaves. Proteins were detected by immunoblotting and analyzed by immunoblotting with anti-FLAG, anti-HA, and anti-actin antibodies.</figcaption></figure>

of SIPSY1, SlACO1, and SlACS2 by SIRIN, SIFUL1, and SITAGL1 (Fig. 6g). Taken together, these data suggest that SIEBF3 modulates carotenoid metabolism and fruit ripening by interacting with SIRIN, SIFUL1, and SITAGL1 to suppress their transcriptional activation of the SIPSY1, SlACO1, and SlACS2 promoters.

Next, we wonder whether the interaction between SIEBF3 and SIRIN/SIFUL1/SITAGL1 affects their protein abundance. After SIEBF3-HA was transiently co-expressing with SIRIN-FLAG, SIFUL1-FLAG, and SITAGL1-FLAG in *N. benthamiana* leaves, we tested the accumulation of proteins, the results showed
that SIEBF3 specifically reduced SITAGL1 protein accumulation without affecting SIRIN and SIFUL1 protein levels (Fig. 6h). To verify whether ubiquitination occurs during the interaction of SIEBF3 with SIRIN/SIFUL1/SITAGL1, we examined the ubiquitin binding of SIRIN-FLAG, SIFUL1-FLAG, and SITAGL1-FLAG in vivo by Co-IP. Ub-HAs were co-expressed with SIRIN-FLAG, SIFUL1-FLAG, and SITAGL1-FLAG in tobacco, and the results of the Co-IP experiments showed that SIRIN-FLAG, SIFUL1-FLAG, and SITAGL1-FLAG could not be bound by Ub (Fig. S5a). Subsequently, we examined the protein stability of SIRIN/SIFUL1/SITAGL1 under the influence of the proteasome inhibitor MG132. The data showed that MG132 treatment did not significantly affect the protein degradation rates of SIRIN, SIFUL1, and SITAGL1 (Fig. S5b), suggesting that SIRIN, SIFUL1, and SITAGL1 may not be degraded through the 26S ubiquitin-proteasome system. The above results indicate that SIRIN, SIFUL1, and SITAGL1 cannot be ubiquitinated in vivo. To further examine the functional interplay between EBF3 and transcription factors, we performed electrophoretic mobility shift assays (EMSA). And found that TAGL1 can bind to the PSY1 promoter region alone; however, the binding of TAGL1 to the PSY1 promoter was abolished upon the addition of EBF3 (Fig. S5c). These results indicate that the interaction between EBF3 and TAGL1 inhibits TAGL1's ability to bind the promoter of its downstream target gene. Thus, we propose that EBF3 represses transcriptional activation of downstream target genes by inhibiting the transcription factor's binding to their promoters.

## SIEBF3-OE fruits extend shelf life by increasing cuticle thickness and the content of cellulose in pericarps.

To assess the role of SIEBF3 in fruit shelf life, we initially measured the fruit firmness in WT, KO, and OE lines. No statistically significant variations in firmness were observed at the BR stage. However, the firmness of fruits from SIEBF3-OE lines was higher than that from WT and SIEBF3-KO lines at Br + 7 stage (Fig. S6). The cellulose content of the fruit is one of the most significant factors affecting fruit firmness [23]. To investigate whether SIEBF3 affects cellulose content, we specifically stained cellulose in the cell wall with Congo red. The results showed that SIEBF3-OE fruits showed markedly higher cellulose fluorescence in cell walls than WT and SIEBF3-KO fruits (Fig. 7a). The contents of key cell wall components such as hemicellulose, total pectin, protopectin, and soluble pectin were determined (Fig. 7b, Fig. S7). Cellulose content was elevated in SIEBF3-OE fruits than in WT and SIEBF3-KO fruits, aligning with the observed cellulose fluorescence levels (Fig. 7b). The results showed that the contents of hemicellulose and total pectin in SIEBF3-KO and SIEBF3-OE did not change significantly compared with WT (Fig. S7). In SIEBF3-KO, the contents of protopectin and soluble pectin did not change significantly compared to WT, whereas in SIEBF3-OE, the contents of protopectin were higher and soluble pectin lower in the fruits of SIEBF3-OE compared to those of WT (Fig. 7b). Protopectin is water-insoluble and is known to increase fruit firmness, while soluble pectin is water-soluble, associated with reduced fruit firmness. These results indicate that SIEBF3 enhances fruit firmness by modulating cell wall composition.

Disease resistance and storage tolerance are both critical indicators of postharvest quality of tomato fruits, both of which significantly influence the shelf life of fruits. To investigate whether SIEBF3 affects tomato fruit storage tolerance, we examined the role of SIEBF3 in postharvest fruit storage. Fruits of WT, SIEBF3-KO, and SIEBF3-OE lines were collected at Br + 7 stage to assess water loss rate changes over 40 days postharvest at room

temperature. The results showed that SIEBF3-OE fruits exhibited reduced pericarp shrinkage and withering, along with a lower water loss rate over 40 days compared to WT and SIEBF3-KO fruits (Fig. 7e and f). To further elucidate the potential mechanism by which SIEBF regulates fruit water loss, we examined cuticular deposition in fruits at Br + 7 stage by Sudan IV staining. SIEBF3-OE lines exhibited significantly increased cuticle thickness compared with WT, whereas SIEBF3-KO showed no difference (Fig. 7g). RT-qPCR was conducted to confirm the transcript levels of several key genes involved in cuticle synthesis such as ECERIFERUM6 (CER6), LONG CHAIN ACYL-COA SYNTHETASE 1 (LACS1), and GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE 6 (GPAT6). The expression level of SIGPAT6, SILACS1, and SICER6 were notably elevated in SIEBF3-OE fruits (Fig. 7f). These findings suggest that SIEBF3 promote the synthesis of fruit cuticles by increasing SIGPAT6, SILACS1, and SICER6 expression, thereby reducing fruit water loss during storage.

## Discussion

EIN3-binding F-box proteins (EBFs) are crucial components in ET signaling. In Arabidopsis, EBF1 and EBF2 can target EIN3 and EIN3-like 1 (EIL1) for degradation. The functional roles of EBF1 and EBF2 are temporally distinct: EBF1 mediates early ET responses, whereas EBF2 contributes to subsequent signaling events [38–40]. In tomato, four SIEBF genes have been identified that have been demonstrated to play both overlapping and distinct roles in regulating ET signaling [20, 41]. While the function of SIEBF in the regulation of maturation has been largely elucidated, its importance in other biological processes remains uncertain. Here, we demonstrate that SIEBF3 responds to JA signaling and enhances postharvest resistance to B. cinerea in tomato fruit by interacting with and suppressing SIBBX20, a known negative regulator of JA signaling and resistance to B. cinerea (Fig. 8). The interaction of SIEBF3 and SIBBX20 inhibited the accumulation of SIBBX20 and restored its inhibitory effect on the activation of SJA2L by SIMED25-SIMYC2 to improve postharvest resistance in tomato fruit. Furthermore, our results demonstrate that SIEBF3 regulates carotenoid synthesis by inhibiting the activation of SIPSY1 through interactions with SIBBX20, SIRIN, SIFUL1, and SITAGL1. Additionally, SIEBF3 modulates fruit ripening by interacting with SIRIN, SIFUL1, and SITAGL1 to suppress their activation of SIACO1 and SACS2 (Fig. 8). In conclusion, our findings identify a new role for SIEBF3 in modulating JA signaling, enhancing B. cinerea resistance in tomato fruits and expanding the regulatory network for carotenoid metabolism and fruit maturation (Fig. 8).

SIEBF3 ubiquitinates and degrades SIBBX20, restoring its inhibitory effect on SIMED25-SIMYC2 activation of the JA-responsive gene SJA2L, which then enhances the expression of SJA2L and improves tomato resistance to B. cinerea. SIEBF3 also inhibits activation of SIPSY1 by SIBBX20, which in turn inhibits phytoene synthesis. In addition, SIEBF3 can interact with SIRIN, SIFUL1, and SITAGL1 without ubiquitination, but it can inhibit the activation of SIPSY1, SIACO1, and SACS2 by SIRIN, SIFUL1, and SITAGL1 to regulate the synthesis and ripening process of phytoene in tomato fruit. SIEBF3 can also positively regulate cuticle thickness and cellulose content, thereby maintaining the postharvest quality of tomato fruits. Solid lines indicate SIEBF3-mediated enhancement, dashed lines indicate SIEBF3-mediated inhibition.

The relationship between EBFs exhibit considerable complexity in their regulatory networks [39, 42]. SIEBFs exhibit both overlapping and distinct functions in controlling tomato fruit ripening [41]. In this study, our data suggest that no statistically meaningful
<figure><img src="image_12.png" /><figcaption>**Figure 7.** OE of SIEBF3 prolongs fruit shelf life. (a) Pericarp sections stained with Congo red in WT, SIEBF3-KO, and SIEBF3-OE fruits. Bar = 50 µm. (b) Cellulose, protopectin, and water-soluble pectin contents in pericarps of WT, SIEBF3-KO1, SIEBF3-KO5, SIEBF3-OE11, and SIEBF3-OE12 fruits. Data are shown as means ± SD (n = 3). (c, d) Water loss in WT, SIEBF3-KO1, SIEBF3-KO5, SIEBF3-OE11, and SIEBF3-OE12 fruits. WT, SIEBF3-KO1, SIEBF3-KO5, SIEBF3-OE11, and SIEBF3-OE12 fruits harvested at BR + 10 stage and stored at room temperature. Photographs were taken at 40 d of storage. Bar = 10 mm. Data are shown as means ± SD (n = 15). (e) Cuticle deposition analysis in WT, SIEBF3-KO, and SIEBF3-OE11 fruits by Sudan IV staining. (f) Relative expression levels of cuticle synthesis-related genes GPAT6, CER6, and LACS1 in WT, SIEBF3-KO, and SIEBF3-OE lines. The mRNA levels of each gene in WT were standardized to 1 and normalized with actin genes. Data are shown as means ± SD (n = 3). Asterisks indicate statistical significance using Student's t-test, *P* < 0.05.</figcaption></figure>

differences were detected in the transcriptional profiles of disease resistance, ripening, or cuticle-related genes between WT and KO plants (Figs 2g and 4b, c, and f). These findings suggest that SIEBFs exhibit complete functional redundancy in regulating disease resistance, ripening processes, and cuticle development. Notably, SIPSY1 expression was significantly upregulated, accompanied by an increase in carotenoid content, in SIEBF3-KO fruit compared with WT fruit. In contrast, SIPSY1 expression was downregulated and carotenoid content was decreased in SIEBF3-OE fruit (Fig. 5a and b). These results suggest that SIEBFs are not fully functionally redundant in the regulation of carotenoid metabolism. The redun-

dant and specific functions of SIEBFs in different phenotypes demonstrate the flexibility and breadth of SIEBFs in the regulation of fruit ripening, metabolism, and postharvest quality, but the linkage and division of labour among SIEBF family members and the specific regulatory networks need to be further explored and validated. In conclusion, our results provide further evidence for the intricate regulatory relationships among SIEBF family members, revealing both significant functional redundancy and distinct functional specificity.

JA plays crucial roles in plant defense mechanisms [10, 43–45]. SIMYC2 is a core transcription factor of the JA signaling response
<figure><img src="image_13.png" /><figcaption>**Figure 8.** Working model of SIEBF3-mediated regulation of *B. cinerea* resistance, carotenoid biosynthesis, and fruit ripening in tomato.</figcaption></figure>

that can interact with the multitalented Mediator subunit MED25 to form a transcriptional activation complex that activates the transcription of JA-responsive genes such as SJA2L and SIERF.C3, which all positively regulate tomato *B. cinerea* resistance [10, 14, 15]. This study shows that SIEBF3 positively regulates the JA response to *B. cinerea* infection. The expression of SIEBF3 was significantly upregulated after MeJA treatment (Fig. 3a), indicating that SIEBF3 can be induced by JA. The SIEBF3-OE strain showed a significant induction in the expression of JA-responsive genes SJA2L and SITD (Fig. 3b and c). Transcriptomic analysis revealed an up-regulation of JA signaling and response genes in SIEBF3-OE following *B. cinerea* inoculation (Fig. 2c). Collectively, these findings indicate that SIEBF3 enhances the JA signaling pathway to regulate gray mold resistance.

F-box proteins are crucial elements of SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase complexes, and act as specificity determinants for substrate recognition. These proteins catalyze the polyubiquitination of specific substrates, leading to their eventual destruction via the 26S proteasome pathway. Through this regulatory mechanism, F-box proteins significantly influence diverse physiological processes, particularly in plant hormone signaling pathways and immune response regulation [46]. During ET signaling, EIN2 and EIN3 are regulated by their respective F-box proteins, ETP1/2 and EBF1/2, whereas EIL1-4 can be degraded by EBF3 in tomato [36, 38–40]. In auxin signal transduction, Aux/IAA proteins are targeted by TIR1/AFB F-box proteins, leading to their ubiquitination and subsequent degradation via the SCF-26S proteasome system [47–50]. In the JA signaling pathway, COI1 can mediate ubiquitination degradation of the JAZ repressor, thereby derepressing MYC2 [12–14]. SIBBX20 can be modified by ubiquitination and can negatively regulate the JA signaling pathway and *B. cinerea* resistance. In addition, SIBBX20 can interact with eight SIJAZ proteins and SIJAZ proteins can also interact with SIMTBs (MYC2 TARGETED BHLH) [16, 29]. In this study, SIEBF3 can affect JA signaling and thus regulate *B. cinerea* resistance by binding and degrading SIBBX20. Whether SIEBF3 can affect other components of the transcriptional repressor complex consisting of SIBBX20, SIMTB, and SIJAZ proteins in the same way needs to be further investigated and demonstrated. In addition, SIEBF3 was able to interact with SIRIN, SIFUL1, and SITAGL1 without undergoing

ubiquitination (Fig. 6), which is a novel pathway for SIEBF3 to regulate maturation without degrading SIEILs proteins. Further research and verification are needed to elucidate the molecular mechanisms by which SIEBF3 affects the activation of target genes by SIRIN, SIFUL1, and SITAGL1.

Both postharvest resistance and storage tolerance of tomato fruit are important factors in maintaining postharvest fruit quality. Excessive postharvest softening of fruit results in reduced resistance to pathogens and increased water loss, which in turn leads to fruit deterioration [51]. We found that SIEBF3-OE fruits could reduce water loss and extend shelf life by altering cell wall composition (Fig. 7), and that postharvest changes in fruit texture and disease resistance were closely related, with specific linkages that need to be further explored in future studies. Overall, our study revealed the molecular associations and potential mechanisms by which SIEBF3 affects JA signaling during postharvest resistance to *B. cinerea* in tomato fruits, and also enriched the regulatory network of SIEBF3 affecting the ripening process and carotenoid metabolism in tomato fruits. In addition, SIEBF3 could be a potential candidate gene for the genetic improvement of fruit postharvest disease resistance and storage tolerance to extend fruit shelf life.

## Materials and methods

### Plant materials

Tomato plants 'MicroTom' (MT) was used as WT. Three SIEBF3-OE lines were obtained from Deng [36]. Independent guide RNA of SIEBF3 were designed using the application tool CRISPR-P and inserted into the plasmid of pFASTCas9/ccdB to construct the CRISPR/Cas9 genome editing plasmid vector of SIEBF3. The recombinant plasmid pFASTCas9/ccdB-SIEBF3 was verified through Sanger sequencing before being introduced into 'MicroTom' tomato plants using Agrobacterium-mediated transformation [21].

### B. cinerea infection assay

Ten tomato fruits of each group at MG, Br, and Br + 7 stages were punctured with a sterile needle, and then the mycelial plugs were placed over the wound with mycelial plugs from the edge of fungal
cultures. For leaf incubation, mycelial plugs were applied to the front side of 35- to 45-day-old leaves that has been pierced with an anatomical needle. Inoculated fruits and leaves were kept at 20°C in darkness. Diameter of each lesion on fruits or leaves were measured 48 or 96 h postincubation (hpi).

## Measuring fruit firmness

For WT and each transgenic line, a minimum of 15 fruits were randomly selected at both the Br stage and Br + 7 stage. Fruit firmness measurements were conducted with the TA.XTC-18 (BOSIN, Shanghai).

## Histological analysis of fruit pericarp

For cellular morphology analysis, equatorial sections (3 mm in thickness) of breaker-stage (Br) fruits were prepared. After brief staining with 0.5% toluidine blue (30 s), samples were photographed using a stereomicroscope (M205FA, Leica, Germany). Fruits at Br and Br + 7 stages were collected for cell wall characterization. Sequential staining with safranin O and fast green for 4–6 s each preceded neutral balsam mounting and brightfield microscopy (Olympus VS200, Japan). Cellulose detection was achieved through Congo Red (Servicebio, China) staining of paraffin sections, with fluorescence visualization using a Leica DM4 B microscope (Germany). Quantitative analysis of cellular parameters (including cell number, size, and pericarp thickness) was conducted utilizing ImageJ.

## Measuring fruit cellulose, hemicellulose, total pectin, protopectin, and soluble pectin contents

At least 10 fruits per line were harvested at the Br + 7 stage. Samples were snap frozen in liquid nitrogen and ground into powder. Three portions of cell wall material were weighed, and cellulose, hemicellulose, total pectin, protopectin, and soluble pectin contents were measured using a Cell Wall Assay kit (Solarbio, Beijing) according to Pei et al.

## Quantification of postharvest water loss

A shelf-life analysis was conducted on tomato fruits from three genetic backgrounds: WT, SIEBF3-KO, and SIEBF3-OE. Fifteen fruits of each line were stored in 22°C. Fruits weight was recorded initially and then every 10 days over a 40-day period. Water loss was quantified as the percentage decrease in fruit mass relative to the initial weight [52].

## Measuring fruit color

Fruits from WT, SIEBF3-KO, and SIEBF3-OE fruits were collected at Br + 7 stage. Fruit color was quantitatively measured with a Konica Minolta chromameter (CR-400; Konica Minolta, Tokyo, Japan) following established protocols [53].

## Measuring carotenoid contents

Carotenoid content in WT, SIEBF3-KO, and SIEBF3-OE fruits at the Br + 7 stage were measured following the method outlined by You [54]. Freeze-dried fruit powder (100 mg) from different samples was extracted with a mixture of n-hexane, acetone, and ethanol (2:1:1, v/v/v). Each sample was vortexed briefly, sonicated at 25°C for 20 min, centrifuged (13000 g), and the supernatant collected; this process was repeated one time. The supernatants were concentrated in a Concentrator Plus (Eppendorf) and reconstituted in methanol and 1% (v/v) methylene chloride. Carotenoid content was determined by LC-MS. Carotenoid composition was determined by HPLC analysis using authentic standards.

## RNA extraction and RT-qPCR

Following the manufacturer's instructions, total RNA was isolated from fruit pericarp and leaves with a TRIzol Reagent Kit (Takara, Otsu, Japan). A HiScript II 1st Strand cDNA Syn-thesis Kit (Vazyme, Nanjing, China) was used to reverse transcribe RNA into cDNA. The primer sequences used in this assay are listed in Dataset S1.

## Yeast two-hybrid assay

The coding sequences for SIEBF3 was cloned into pGBK7 and the coding sequences for SIBBX20, SIRIN, SINOR, SIFUL1, SITAGL1, SIMYC2, and SIMED25 were cloned into pGADT7. Then transformed the AD and BD plasmids into AH109 yeast competent cells and screened on SD medium without Leu and Trp (SD-LW) for 3d. Interaction was detected in SD-4 (SD-LWAH) medium.

## Co-IP assay

The complete coding sequences of SIBBX20, SIRIN, SINOR, SIFUL1, and SITAGL1 were cloned into the pBTEX-FLAG vector, while SIEBF3 was cloned into the pBTEX-HA vector, resulting in the creation of transient expression vectors: SIBBX20-FLAG, SIRIN-FLAG, SINOR-FLAG, SIFUL1-FLAG, SITAGL1-FLAG, and SIEBF3-HA, respectively. The constructs were transiently co-expressed in N. benthamiana leaves, which were harvested 48–72 h post-infiltration. Immunoprecipitation was performed using FLAG M2 magnetic beads (Sigma, M8823) at 4°C for 16 h. Proteins were examined followed by 4-h incubation with antibodies (anti-HA #2367 or anti-FLAG #14793; Cell Signaling Technology).

## Electrophoretic mobility shift assay

Electrophoretic mobility shift assays (EMSAs) were performed as previously described [21–23]. The full-length coding sequence of SITAGL1 was cloned into pet28a and the full-length coding sequence of SIEBF3 was cloned into pGEX-4 T-1. SITAGL1-His and SIEBF3-GST recombinant proteins were produced in E. coli BL21 (DE3) cells. Proteins were purified using His-tag Protein Purification Kit (Beyotime, #P2247) and GST-tag Protein Purification Kit (Beyotime, #P2262). Probes containing probes from the SIPSY1 promoter were labeled with biotin using the 3'- Biotin Labeling Kit (Thermo Fisher; 89818). EMSA experiments were subsequently performed using the EMSA/Gel-Shift kit (Beyotime) and the Light-Shift Chemiluminescent EMSA kit (Thermo).

## In vivo ubiquitination assay

The assay of ubiquitination in vivo was performed as described by Wang [55]. The two predicted lysine (K) sites were mutated individually and together to arginine (R). SIBBX20K143R-Flag, SIBBX20K152R-Flag, and SIBBX20K143R/K152R-Flag were cloned into pBTEX-FLAG. Tobacco leaves were used for co-expression assays. Anti-FLAG (CST, #14793), anti-HA (CST, #2367), and anti-ubiquitin (Abmart, PL026378S) were used to test the protein signal.

## Protein accumulation assays

The assay of protein stability and degradation were performed according to the experimental protocol of Wang *et al.* with some improvements [55]. For protein stability analysis, leaf tissues were harvested 36 h post-infiltration, treated with 50 µM MG132 to protein extraction. To assess degradation kinetics, leaves were exposed to 250 µM CHX at 36 hpi and sampled after 2-h incubation. Immunoblotting was performed using anti-FLAG (#14793; CST) and anti-HA (#2367; CST) antibodies. Protein band densitometry was performed using ImageJ analysis software,
with final values expressed as mean ± SD derived from triplicate
experimental replicates.

## Luciferase reporter assay

The full-length CDS of SIEBF3, SIMYC2, SIMED25, SIBBX20, SIRIN, SIFUL1, and SITAGL1 were cloned into the pGreenII 62sk vector as effectors. The promoter sequences of SIJA2L, SIPSY1, SIACO1, and SLACS2 were cloned into pGreen-Mini35S-LUC vectors as reporters. The experimental procedures were conducted as previously described [56]. Vectors were introduced into N. benthamiana using GV3101. Transient expression was then conducted in tobacco leaves. The Dual-Luciferase Reporter Assay System (Promega, E1910) was employed to assess LUC and REN activities.

## Bimolecular fluorescence complementation

The BiFC method for studying protein-protein interactions utilized the pSPYCE and pSPYNE vectors as described in prior research [57]. The coding sequences of the SIEBF3 and SIBBX20, SIRIN, SIFUL1, and SITAGL1 were inserted into the pSPYNE and pSPYCE plasmid. The resulting cultures were used for co-expression in N. benthamiana leaves. YFP fluorescence signal was observed and imaged using the fluorescence microscope (DM4 B, Leica, Germany).

## MeJA treatment

Four-weeks-old tomato leaves from WT and transgenic lines were treated with 100 µM MeJA. The leaves were collected after 1 h. The transcription levels of SIEBF3, SIMYC2 and SIJA2L was quantified using RT-qPCR. (Table S1).

## Wheat germ agglutinin and propidium iodide staining

As described by Fernandez-Alvarez, we visualized fungal hyphae in planta by wheat germ agglutinin and propidium iodide (WGA-PI) staining to evaluate the severity of fruits infection by B. cinerea [58]. Following 48-h B. cinerea infection, fruit pericarp tissues were preserved in Carnot's fixative. Tissue sections were subsequently stained with a WGA-PI dual staining solution, then imaged using an Olympus VS200 digital slide scanning system (Olympus Corporation, Japan).

## Antioxidant enzyme activity

The enzymatic activities of antioxidant enzymes were assessed with detection kits (Solarbio, Beijing). Absorbance was read at 560 nm (SOD) and 470 nm (POD) wavelengths using a Thermo Multiskan GO.

## RNA-Seq analysis

RNA was extracted from WT and SIEBF3-OE fruits at the BR stage after B. cinerea inoculation, using three biological replicates for each genotype. Purified RNA samples were subsequently processed for cDNA library preparation and sequenced using MGI-DNB Seq platform (Shenzhen, China). DEGs were identified using a threshold of P < 0.05 and |FC| ≥ 1.5 [59].

## Statistical analysis

All data were analyzed using GraphPad Prism version 9. Results are expressed as mean values ± standard deviation (SD) from a minimum of three separate experiments. Intergroup comparisons were analyzed by Student's t-test, with statistical significance set at P < 0.05 (denoted by asterisks).

## Acknowledgements

This work was supported in part by the National Natural Science Foundation of China (nos 32302622, 32372780, 32172643, and 32172271), the Technology Innovation and Application Development Program of Chongqing (no. cstc2021jscx-cylhX0001), the Natural Science Foundation of Sichuan Province, China (nos 2023NSFSC1991 and 2024NSFSC1302), and the China Postdoctoral Science Foundation (2023 M732486).

## Author contributions

M.W., M.L., and M.B. planned and designed the research. Z.G., H.D., X.H., Y.Y., C.Y., G.J., and T.M. performed experiments. Z.G. analyzed data. Z.G. and M.W. wrote the manuscript.

## Data availability

The authors confirm that all the experimental data are available and accessible via the main text and/or the supplemental data.

## Conflict of interest statement

The authors declare no conflict of interest.

## Supplementary data

Supplementary data is available at *Horticulture Research* online.

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