Large yellow croakers used in this study were purchased from Ningde Fufa Fishing Co., Ltd, Ningde, Fujian Province, with the length measured as 18.0 ± 1.5 cm, weight as 60.0 ± 15.0 g. The fish were kept in the recirculated seawater system with 3,000-L tanks which maintained at 24-26°C and feeded with commercial diet. After temporary rearing for about two weeks, the healthy fish were used in the subsequent experiments as previously mentioned (Zou et al., 2019; Zou et al., 2020).

Human embryonic kidney 293T (HEK 293T) cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Invitrogen-Gibco) and 100 U/mL penicillin (P) and streptomycin (S) as described previously. The cells were then kept in an incubator at 37°C and 5% CO₂ as our previous reports (Zou et al., 2019; Zou et al., 2020). Large yellow croaker muscle (LYCMS) cells were maintained in L15 Medium (Boster, USA) containing 10% FBS and 100 U/mL PS at 27°C as our previous report (Chen et al., 2022). Epithelioma papulosum cyprinid (EPC) cells were cultured at 25°C in M199 medium (Procell, Wuhan, China) containing 10% FBS and 100 U/mL PS. Spring viraemia of carp virus (SVCV), which is infective in EPC cells, was propagated in EPC cells at 25°C. Followed the manufacturer’s instructions, Lipofectamine^®3000 (Invitrogen, Carlsbad, CA) was used to transfect the plasmids into the above-mentioned cells.

Based on the transcriptome information for the large yellow croaker on the National Center for Biotechnology Information website (NCBI, GenBank accession No. XM_027288056.1), specific primers were used to amplify the full-length ORF of large yellow croaker TRAF7. For eukaryotic expression and subcellular localization analysis, the full-length ORF of TRAF7 and its truncated forms were cloned and inserted into the pcDNA3.1/myc-His (-) A vector that contains a C-terminal c-Myc tag (Invitrogen, Carlsbad, CA, USA) and pTurboGFP-N vector that contains a C-terminal TurboGFP tag (Evrogen, Moscow, Russia), respectively. All plasmid constructs were confirmed by sequencing analysis in Sangon Biotech Co., Ltd. (Shanghai, China) and Western blotting analysis. Primers used in the present study are listed in Table 1 .

For ORF determination and protein sequence prediction of the obtained large yellow croaker TRAF7, the Translate tool at the ExPASy Bioinformatics Resource Portal (https://web.expasy:translate) was used (Duvaud et al., 2021), and the conserved domain structures were detected according to the Simple Modular Architecture Research Toll (SMART) (http://smart.embl.de) and the Conserved Domain Database (CDD) on the NCBI (http://www.ncbi.nlm.nih.gov/cdd) (Lu et al., 2020). The Basic Local Alignment Search Tool (BLAST) analysis tool on the NCBI (http://www.ncbi.nlm.nih.gov/blast) was utilized for the search of vertebrate TRAF7 orthologs. The Clustal X program was used to conduct amino acid alignments of vertebrate TRAF7, with the results edited using the GeneDoc software (Larkin et al., 2007). The phylogenetic tree of vertebrate TRAF7 was constructed using the MEGA7 software with the neighbor-joining method (Kumar et al., 2016). The vertebrate TRAF7 gene sequences were analyzed online using the Splign tool (https://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi) following the gene search on the NCBI genome database (https://www.ncbi.nlm.nih.gov/genome).

To understand the constitutive expression profiles of TRAF7 in large yellow croaker, various organs and tissues, such as the head kidney, trunk kidney, liver, gill, spleen, heart, muscle, blood, intestine, skin, and brain were dissected from randomly selected six healthy fish, then immediately frozen in liquid nitrogen for RNA extraction and qPCR analysis.

In order to determine the expression profiles of TRAF7 in large yellow croakers that subjected to a variety of immune stimuli, the fish were divided into five groups, each with 50 fish, and each fish of the group was separately taken an injection with different PAMPs (Zou et al., 2021a; Zou et al., 2021b). In brief, 100 μL of phosphate buffered saline (PBS) was injected intraperitoneally into each fish of the control group. Simultaneously, each fish in the challenge groups was taken an injection of the same amount of lipopolysaccharides (LPS) (L3024, Sigma, 0.5 mg/mL), polyinosinic-polycytidylic acid potassium salt (poly I:C) (P9582, Sigma, 1 mg/mL), peptidoglycan (PGN) (69554, Sigma, 1 mg/mL), or P. plecoglossicida suspension in PBS (5 × 10⁵ CFU/mL), respectively (Zou et al., 2021a; Zou et al., 2021b). After that, six fish from each group were randomly selected and anaesthetized in 0.01% eugenol at 6, 12, and 24 h post injection (hpi), the intestine, gill, blood, spleen, and head kidney were collected for the subsequent experiments as described above.

To further reveal the role of large yellow croaker TRAF7 in the host immune responses, especially on the expression profiles of antiviral and inflammatory-related genes including g-type lysozyme, IL-1β, TNF-α, ISG15, ISG56, IRF3, IRF7, and RSAD2, LYCMS cells seeded on the 6-well plates (5 × 10⁵ cells per well) overnight were transiently transfected with 5 μg of pcDNA3.1-TRAF7 or pcDNA3.1 empty vector (control), respectively. After that, the cells were collected at 48 h post-transfection (hpt), with the expression patterns of TRAF7 and the antiviral and inflammatory-related genes mentioned above detected by qRT-PCR analysis. All primers used are listed in Table 1 and could also refer to our previous reports (Chen et al., 2022; Luo et al., 2022).

Total RNA was isolated from the different organ/tissue and the LYCMS cell samples described above by using the Eastep™ Super Total RNA Extraction Kit (Promega, Beijing, China), then the first-stand cDNA synthesis kit (RevertAid First Stand cDNA Synthesis Kit, #K1622, Thermo Scientific™) was used to reverse transcribed the total RNA into cDNA according to the manufacturer’s protocol. The synthetic cDNA was then kept at -80°C and used as a template for full-length ORF cloning of target genes or qRT-PCR analysis.

qRT-PCR was performed on a Roche LightCycler^® 480 II quantitative real-time detection system (Roche, Switzerland) using Go Taq^® qPCR Master Mix (Promega, Madison, USA) with the following procedure: 5 min at 95°C, followed by 40 cycles of 20 s at 95°C, 15 s at 60°C, and 15 s at 72°C, with a final extension at 72°C for 10 min. Then, the melting curve was conducted and analyzed as the reaction temperature was raised from 72°C to 95°C at a rate of 0.5°C per second. All reactions were performed in a 384-well plate, with the mean value recorded. The comparative Ct method (2^-ΔΔCt) was used to analyze the relative expression of target gene, which was normalized to the expression of β-actin (Livak and Schmittgen, 2001; Zou et al., 2022).

To investigate the subcellular distribution of large yellow croaker TRAF7 and its truncated forms, HEK 293T cells seeded on cell slides in 6-well plates (2 × 10⁵ cells per well) overnight were separately transfected with 5 μg constructed plasmid of pTurbo-TRAF7-GFP, pTurbo-TRAF7-ΔRING-GFP, pTurbo-TRAF7-WD40-GFP, pTurbo-TRAF7-ΔWD40-GFP, pTurbo-TRAF7-RING-GFP, or pTurboGFP-N (control) using Lipofectamine 3000 in accordance with the manufacturer’s protocols. At 24 hpt, the cells on the coverslips were washed with 1 mL PBS, fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 10 min at room temperature, respectively. After that, the cell slides were washed with PBS, followed by overlaying with one drop of mounting media (VECTASHIELDR Hard Set^™ Mounting media with DAPI, Vector Laboratories, CA). Finally, a confocal microscope (Leica TCS SP8, Germany) was used for examining and photographing the cells.

To evaluate the roles of large yellow croaker TRAF7 on the induction of IRF3, IRF7, NF-κB, and IFN1 promoter activation, HEK 293T cells cultured in 24-well plates (1 × 10⁵ cells per well) for one night, and then the cells were transiently transfected with 100 ng of pGL4-IRF3-pro (Chinese invention patent number: ZL201710457836.8), pGL4-IRF7-pro (Chinese invention patent number: ZL201710457820.7), pNF-κB-luc (Clontech, Palo Alto, CA), or pGL4-IFN1-pro (Chinese invention patent application number: 201710456729.3), 10 ng of pRL-TK (Promega, Madison, WI) and together with pcDNA3.1-TRAF7 or pcDNA3.1 empty vector (control) at increasing concentrations (10, 50, 100, and 200 ng) using Lipofectamine 3000. At 24 hpt, the cells were lysed for subsequent luciferase activity assays using Dual-Luciferase Reporter System (Promega), with luciferase activity measured on a Promega GloMax^® 20/20 luminometer (Promega). The results were normalized to the Renilla luciferase activities and expressed as the fold in comparison to the control group.

To further determine the roles of various domains of large yellow croaker TRAF7 on IRF3, IRF7, NF-κB, and IFN1 promoter activation, HEK 293T cells were transiently transfected with 100 ng of pGL4-IRF3-pro, pGL4-IRF7-pro, pNF-κB-luc, or pGL4-IFN1-pro, 10 ng of pRL-TK together with 100 ng of pcDNA3.1-TRAF7, pcDNA3.1-TRAF7-ΔRING, pcDNA3.1-TRAF7-WD40, pcDNA3.1-TRAF7-ΔWD40, pcDNA3.1-TRAF7-RING, or pcDNA3.1 empty vector (control), respectively. At 24 hpt, the cells were collected for luciferase activity assays as described above.

For antiviral activity assays, EPC cells seeded in 6-well plates (2 × 10⁶ cells per well) overnight were transfected with 2.5 μg pcDNA3.1-TRAF7 or pcDNA3.1 empty vector (control). At 24 hpt, the transfected cells were washed and infected with SVCV at an MOI of 1, with the cells collected for total RNA extraction at 24 h post-infection, followed by subsequent mRNA expression pattern evaluation of SVCV Glycoprotein (SVCV-G), Matrix protein (SVCV-M), and Phosphoprotein (SVCV-P) by qRT-PCR analysis. The expression data was calculated using the comparative Ct method (2^−ΔΔCt) as described above, with the viral genes normalized to the β-actin of EPC. The primers used for qRT-PCR analysis are shown in Table 1 .

To confirm the expression of the constructed plasmids described above, Western blotting analysis was performed as per our previous reports (Zou et al., 2019; Zou et al., 2020). In brief, the cells transfected with the constructed plasmids were collected and lysed using the RIPA buffer (Beyotime, Shanghai, China), with the proteins separated by 10% SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membrane with pore size of 0.45 μm (Millipore Corporation). The transferred membrane was blocked with 5% non-fat dry milk in PBS for 1 h at room temperature. After one wash with PBS containing 0.1% Tween-20 (PBST) and two washes with PBS, each for 5 min, the membrane was incubated with rabbit polyclonal Anti-TurboGFP antibody (Evrogen, Moscow, Russia) or mouse monoclonal Anti-c-Myc antibody (Beyotime, Shanghai, China) that diluted with antibody diluent at 1:5000 at 4°C overnight, respectively. After that, the membrane was washed three times as described above, and then incubated with the secondary antibody (diluted with antibody diluent at 1:2000) for 1 h at room temperature, followed by washing for three times. Subsequently, the bands were visualized using WesternBright™ ECL HRP substrate (Advansta, San Jose, USA) and ECL Western blotting system (LAS-4000mini, Fujifilm, Tokyo, Japan) according to the manufacturers’ instructions.

All data acquired from the qRT-PCR as well as dual-luciferase reporter assays were recorded as mean of three repeated experiments, with the bars above the histogram representing the standard error (SE). One-way analysis of variance (ANOVA) was used for statistical analysis, followed by Duncan’s multiple range test on the SPSS version 25. The different superscripts above the bars indicate statistically different (P < 0.05), * P < 0.05, and ** P < 0.01 are considered statistically significant and remarkably significant, respectively.

The full-length ORF of TRAF7 gene was cloned and named as Lc-TRAF7 (GenBank Accession No. ON357640), which was constituted of 1,962 bp and encoded a 653 aa protein. It was revealed that Lc-TRAF7 contained a RING finger domain (101-134 aa), a coiled-coil domain (292-356 aa), and seven WD40 domains (368-407, 411-448, 451-487, 492-528, 531-568, 571-612, and 615-652 aa) ( Supplementary Figure 1 ).

The amino acid sequence of Lc-TRAF7 was compared with TRAF7 of other vertebrates, including grouper (Epinephelus coioides), medaka (Oryzias latipes), zebrafish (Danio rerio), fugu (Takifugu rubripes), gold fish (Carassius auratus), African clawed frog (Xenopus laevis), chicken (Gallus gallus), mouse (Mus musculus), and human (Homo sapiens) ( Supplementary Figure 1 ), which revealed a relatively conservative RING finger domain, coiled-coil domain, and WD40 domains. Additionally, the amino acid sequence of Lc-TRAF7 had an identity of 97% with the TRAF7 of grouper, 96% with fugu, 95% with medaka, 93% with zebrafish, 92% with gold fish, 87% with chicken, 86% with mouse, and 85% with human and African clawed frog ( Table 2 ).

To comprehend the phylogenic relationship of TRAF7 in vertebrates, a phylogenetic tree of TRAF7 was constructed using the amino acid sequences of Lc-TRAF7 and the TRAF7 orthologs from other vertebrates, such as teleosts, amphibians, birds, and mammals. It was shown that TRAF7 orthologs could be isolated into two gatherings, with various fishes bunching in one clade, while amphibians, birds, and mammals gathering into another clade ( Figure 1 ).

The TRAF7 genomic structures of the large yellow croaker, medaka, grouper, African clawed frog, chicken, mouse, and human were identified by comparing the vertebrate TRAF7 genomic sequences. It was demonstrated that the genomic sequences of TRAF7 ranged from 9,378 bp to 30,654 bp in length, with the longest sequence found in the African clawed frog and the shortest in the grouper ( Figure 2 ). The large yellow croaker TRAF7 gene was consisted of 20 exons and 19 introns, which was the same from that found in medaka, grouper, African clawed frog, chicken, mouse, and human ( Figure 2 ). In addition, the sizes of the exons of TRAF7 gene in large yellow croaker, grouper, and medaka were nearly the same, only the sixth exon showed some different. Meanwhile, the lengths of the exons of TRAF7 gene in African clawed frog, chicken, mouse, and human were also conserved, except for the first exon, the lengths of other exons were completely the same. However, the lengths of the introns were varied greatly from different species ( Figure 2 ).

The mRNA expression levels of TRAF7 in various organs/tissues were detected by qRT-PCR analysis. The findings demonstrated that Lc-TRAF7 was widely expressed in all the detected eleven organs/tissues of the healthy fish. In addition, the expression level of Lc-TRAF7 in the liver was significantly higher than that found in other organs/tissues, whereas the lowest expression level was observed in the brain ( Figure 3 ).

To further determine the role of Lc-TRAF7 in the host immune responses, qRT-PCR was used to detect the expression of Lc-TRAF7 in the gill, intestine, spleen, head kidney, and blood under different PAMPs stimulation or bacterial infection. The findings demonstrated that poly I:C stimulation significantly increased the mRNA expression level of Lc-TRAF7 in the gill, intestine, spleen, head kidney, and peripheral blood, with a 2.0-, 2.2-, and 3.0-fold increase at 6, 12, and 24 hpi in the gill ( Figure 4A ), a 5.0-fold increase at 12 hpi in the intestine ( Figure 4B ), a 2.4- and 2.7-fold increase at 12 and 24 hpi in the spleen ( Figure 4C ), a 4.1- and 2.4-fold increase at 6 and 12 hpi in the head kidney ( Figure 4D ), and a 2.8- and 3.7-fold increase at 6 and 12 hpi in the peripheral blood ( Figure 4E ), respectively. Upon LPS challenge, the expression levels of Lc-TRAF7 were also significantly increased, which was a 4.5-, 4.0-, and 2.8-fold increase at 6, 12, and 24 hpi in the gill ( Figure 4A ), a 2.9- and 3.6-fold increase at 6 and 12 hpi in the intestine ( Figure 4B ), a 4.5- and 3.6-fold increase at 6 hpi in the spleen and peripheral blood ( Figures 4C, E ), a 3.4- and 2.5-fold increase at 6 and 12 hpi in the head kidney ( Figure 4D ), respectively. In addition, Lc-TRAF7 was significantly up-regulated in response to PGN stimulation, with a 7.8-fold at 6 hpi in the gill ( Figure 4A ), a 2.1- and 3.1-fold increase at 6 and 12 hpi in the spleen ( Figure 4C ), a 2.1- and 1.9-fold increase at 12 and 24 hpi in the head kidney ( Figure 4D ), a 3.1- and 5.4-fold increase at 12 hpi in the intestine and peripheral blood ( Figures 4B, E ), respectively. Additionally, Lc-TRAF7 was also significantly induced upon P. plecoglossicida infection, with a 2.3- and 1.9-fold at 6 and 12 hpi in the gill ( Figure 4A ), a 2.9- and 2.1-fold at 6 and 12 hpi in the intestine ( Figure 4B ), a 1.9-fold at 6 hpi in the spleen ( Figure 4C ), a 2.0- and 1.9-fold at 12 hpi in the head kidney and peripheral blood ( Figures 4C, E ), respectively.

The results of dual-luciferase reporter assays revealed that Lc-TRAF7 overexpression could significantly induce the NF-κB, IRF3, IRF7, and IFN1 promoter activation, and such induction was presented as a plasmid dose-dependent manner ( Figures 5A–D ). To further ascertain the roles that various domains of Lc-TRAF7 play in the activation of NF-κB, IRF3, IRF7, or IFN1 promoter, four truncated forms of expression vectors were established, including TRAF7-ΔRING (with RING finger domain deletion), TRAF7-WD40 (only containing seven WD40 domains), TRAF7-ΔWD40 (deletion of seven WD40 domains), and TRAF7-RING (only containing RING finger domain) ( Figure 6A ). Along with the full-length TRAF7, it was found that the truncated forms of TRAF7-ΔRING and TRAF7-WD40 could significantly trigger the activation of NF-κB promoter, whereas TRAF7-ΔWD40 and TRAF7-RING could not induce the NF-κB promoter activation ( Figure 6B ). Moreover, the truncated form of TRAF7-ΔWD40 could effectively initiate IRF3, IRF7, and IFN1 promoter activation, whereas other truncated forms including TRAF7-ΔRING, TRAF7-WD40, and TRAF7-RING had no significant activity to induce such promoter activation ( Figures 6C–E ). Additionally, the expressions of target proteins including the full-length Lc-TRAF7 as well as its truncated forms were further detected by Western blotting analysis using Anti-c-Myc antibody, which confirmed the successful transfection and expression of the constructed plasmids ( Figure 6F ).

The full-length ORF of Lc-TRAF7 was subcloned into the pTurboGFP-N vector and then transfected into HEK 293T cells to determine the subcellular localization. The results showed that, except for the nucleus, the pTubo-TRAF7-GFP fusion protein was distributed throughout the cytoplasm, with a lot of bright green spots found around the nucleus ( Figure 7A ). To further ascertain the potential impact that various domains might have on the subcellular distribution of Lc-TRAF7, a panel of TRAF7 truncated forms including TRAF7-ΔRING, TRAF7-WD40, TRAF7-ΔWD40, and TRAF7-RING were also inserted into the pTurboGFP-N vector, followed by transiently transfected into HEK 293T cells. It was found that the mutants of TRAF7-ΔRING, TRAF7-ΔWD40, and TRAF7-RING had a similar subcellular localization as that found in the full-length Lc-TRAF7, which were all found in the cytoplasm with some aggregations around the nucleus. However, TRAF7-WD40 was not only distributed in the cytoplasm, but also located in the nucleus. Conversely, the control cells transfected with the pTurboGFP-N vector (control) had an entire GFP distribution throughout the whole cell ( Figure 7A ). In addition, the presence of the GFP fusion proteins, including the pTurboGFP (control), the full-length of Lc-TRAF7, TRAF7-ΔRING, TRAF7-WD40, TRAF7-ΔWD40, and TRAF7-RING was also confirmed by an Anti-TurboGFP polyclonal antibody using Western blotting analysis, which revealed the successful expression of target proteins ( Figure 7B ).

To determine the role of Lc-TRAF7 in the host antiviral response, EPC cells were transiently transfected with pcDNA3.1-TRAF7 or pcDNA3.1 empty vector (control) before being infected by SVCV. It was revealed that the EPC cells overexpression of Lc-TRAF7 could significantly decreased the mRNA expression levels of SVCV-G, SVCV-M, and SVCV-P compared to the control cells transfected with the empty pcDNA3.1 vector at 24 h post-infection, which was 0.16-, 0.28-, and 0.09-fold relative to the control cells, respectively ( Figure 8 ).

To further delineate the function of Lc-TRAF7 on the induction of downstream immune-related molecules, qRT-PCR analysis was performed to detect the expression profiles of antiviral and inflammatory-related genes including ISG15, ISG56, IRF3, IRF7, RSAD2, g-type lysozyme, IL-1β, and TNF-α under the overexpression of Lc-TRAF7. By transfecting the pcDNA3.1-TRAF7 plasmid into LYCMS cells, it was discovered that Lc-TRAF7 was successfully overexpressed ( Figure 9A ). Interestingly, the overexpression of Lc-TRAF7 significantly increased the mRNA expression level of the six detected immune-related molecules, such as IRF3, IRF7, ISG15, ISG56, RSAD2, and TNF-α, whereas the expression patterns of g-type lysozyme and IL-1β were not significantly affected ( Figure 9B ).