Functional Characterization of Potato UBC13-UEV1s Genes Required for Ubiquitin Lys63 Chain to Polyubiquitination

Ubiquitin-conjugating enzymes (E2s/UBC) are components of the ubiquitin proteasome system (UPS), and the ubiquitin-conjugating enzyme variant (UEV) is one of E2s (ubiquitin-conjugating enzymes, UBC) subfamily. The UEVs and UBC13 play an auxiliary role in mediating Lys63-linked polyUb chain assembly, which is correlated with target protein non-proteolytic functions, such as DNA repair or response to stress. However, the collaborative mechanism of StUBC13 (homologue of AtUBC13) and StUEVs (the UEVs in potato) involved in potato are not fully understood understood. Here, we identified two StUBC13 and seven StUEVs from potato genome. We analyzed protein motif and conserved domain, gene structure, phylogenetic features, cis-acting elements of StUBC13 and StUEVs. Subsequently, we screened StUBC13 partners protein and verified interaction between StUBC13 and StUEVs using yeast two-hybrid, split luciferase complementation (SLC) and bimolecular fluorescence complementation (BiFC) approach. The expression profile and qRT-PCR analysis suggested that StUBC13 and StUEVs gene exhibited a tissue-specific expression and were induced by different stress. Overall, this investigative study provides a comprehensive reference and view for further functional research on StUBC13 and StUEV1s in potato.


Introduction
The ubiquitin-proteasome system (UPS), a post-translational modification (PTM), is responsible for selectively removing most of the regulatory proteins of eukaryotes cells (80-90%) [1]. The UPS plays a critical role in virtually every aspect of the cytological and physiological processes of plant cells. Ubiquitin (Ub) is well established as a reusable recognition signal for protein degradation or reversible, non-proteolytic regulatory events. Ubiquitination proceeds through a sequential three-step enzymatic cascade reaction from activation to transfer (ubiquitin-activating enzyme E1→ubiquitin-conjugating enzymes E2→ubiquitin ligase enzymes E3), which results in Ub or Ub polymers covalently bonding to target proteins.
The -COOH group from the ubiquitin (Ub) C-terminus, activated by an ATP-dependent reaction, conjugates with the E1 Cys residue to form a stable intermediate E1~Ub (ATP~Ub→E1~Ub) via a high-energy thioester bond. Then, E1~Ub transfers Ub to the Cys residue of the E2 active site through a transesterification reaction to form the E2~Ub complex (E1~Ub→E2~Ub). E3 recruits the ubiquitin-loaded E2 enzyme into proximity with the target protein and facilitates the formation of isopeptide bonds between the ε-amino group of the target protein lysine (K) and the glycine from the Ub C-terminus. As a result, E3 ligase is responsible for recognizing the substrate, followed by either direct ubiquitin transfer (RING or U-box, CRLs) or thioester formation (HECT) with ubiquitin. In this process, the target protein is ubiquitinated by one or more Ub units (monoubiquitination or multi-monoubiquitination) or a Ub chain (defined as polyubiquitination).
Ubiquitin possesses several modification sites, Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 Lys63, and Met1 (N-terminal methionine) to form distinct homo-and heterotypic polyubiquitin chains [2]. Modified with Lys11, Lys29, and Lys48 polyUb chains are potent signals for target proteins degradation by the 26S proteasome [2,3]. Monoubiquitylation and Lys63-linked Ub polymers are implicated in nonproteolytic outcomes in plants, such as hormonal responses and development [4]. However, evidence from other studies suggests that Lys63 polyUb polyubiquitination may serve as a signal for target protein degradation by the 26S proteasome [5]. Lys63-linked polyUb chains are the second most abundant type of ubiquitin linkage with proteasome-independent functions in plants, and are involved in plant development, hormonal responses, nutritional responses, biotic interactions, DNA repair, and responses to environmental conditions [6].
E2 possesses a core UBC domain that contains a Cys active site mediating the ubiquitination of the target protein. UBC13, the only E2 enzyme identified that facilitate Lys63 polyUb chain, functions require a collaborator protein, a UBC E2 variation known as MMS2 or UEV1 [7]. Typically, UBC13 and UEV1/MMS2 form a heterodimer to catalyze the synthesis of K63 polyUb chains. In this process, the UBC13 active site Cys provides the binding site for the first Ub, whereas UEV1/MMS2 E2 non-covalently binds the second Ub to polymerize the Lys63-linked chain extension [8]. UBC13 and UEV1s have been identified and functionally characterized in several plants, indicating that they are conserved in terms of sequence and function [9]. Four Arabidopsis UEV1s (AtUEV1A-D) and rice UEV1s (OsUEV1A-D) were identified [10,11], which interacted with UBC13 (UBC13A, UBC13B). UBC13 diverges to undertake multiple functions in Arabidopsis, such as apical dominance, iron metabolism to promote development response, auxin signaling to affect root development, and response to low temperature and pathogens [9]. UEV1s fluctuate in different tissues and during abiotic stress at transcription level [12]. Recent studies have revealed that OsUEV1B is required to maintain Pi homeostasis in rice [13]. UBC13 interacts with UEV1s to form a stable heterodimer to assembly of Lys63-linked polyubiquitin chains, which play a critical role in error-free DNA-damage tolerance and repair [14]. In addition, UBC13 and UEV1s complexes coordinately assemble and transfer Lys63-linked polyubiquitin chains to target proteins through E3. AtUBC13A/AtUEV1B interacts with AtRGLG1 and AtRGLG2, which carries Lys63-linked Ub chains to AtPIN2 for regulating polar auxin transport and adaptive growth responses [6]. However, UBC13 and UEVs have not been characterized, and little is known about the roles of UBC13-UEV1s complexes in potato.
Here, we reported that seven StUEVs and two StUBC13 from potato, of which StUBC13 and StUEV1s were further described in molecular cloning and functionally characterized. We also exclusively screened StUBC13 partners and verified their interactions relationship. Moreover, RNA-seq data and qRT-PCR were used to analyzed expression patterns of StUBC13 and StUEVs. This study contributes to further explore functional diversity and molecular mechanisms of StUBC13 and StUEVs in potato.

Identification and Characterization of Potato UEVs
The integrity of the UBC domain (lack of Cys active site, UEV domain) was confirmed by InterProScan and SMART programs. There are seven StUEVs in potato which are named based on being matched to the genes of Arabidopsis UEVs: StUEV1B, StUEV1D-like, StUEV1D-like1, StCOP10, StVPS23/ELC, StELC-like, and StELC-like2. The potato UEVs were disproportionately distributed on chromosomes 1, 4, 9, and 10. The longest ORFs were StELC-like, encoding 407 amino acid residues. The StUEVs genes information is listed in Table 1.
The analysis of spatial structure of its homologue AtUEVs showed that different StUEVs are identical in the spatial configuration of UEV conserved domain but varied in other areas apart from UEV conserved domain in different subgroups (Figure 1b). To further investigate the functions of StUEVs, multiple sequence alignments were conducted on the conserved UBC domain (Figure 1c). The StVPS23 subfamily consists of N-terminal UEV and C-terminal VPS domain, among which the UEVs domain is comprised of α helices, 8 β sheets, and a few random coils. The StUEV1s subfamily contains a conserved UEV domain, while the C-terminus is variable. Most UEV1s have a TXY conserved motif at the C-terminus. However, the AtUEV1A/AtUEV1B/StUEV1B/OsUEV1A/BdUEV1A have feature C-terminal extensions with the CaaX motif ( Figure 1d). UEV domains are present in both the Arabidopsis and potato sequences of the StCOP10 subfamily, with a similarity of 61.54%.

The Cis-Regulatory Elements Analysis
Analysis of the cis-acting elements showed that a variety of the elements related to hormone pathways, such as, such as abscisic acid ABA (ABRE), gibberellin GA (P-box and GARE-motif), salicylic acid SA (TCA-element), auxin IAA (TGA-box/element), and methyl jasmonate MeJA (TGACG-motif and CGTCA-motif), of which six StUEVs contain cis-acting elements associated with MeJA responses (Figure 2a, Table S1). Five StUEVs contain ABA/GA/IAA responses associated with cis-acting elements, suggesting that most StUEVs are related to MeJA, ABA, GA, and IAA signaling pathways.

Conserved Motif and Gene Structure Analyses
The motifs in the same subfamily are similar in type and distribution ( Figure 2b). The types and distributions of motifs shown in different subgroups vary greatly, particularly between the VPS23 and UEV1s subgroups. The UEV1 subgroups have three distinct motifs, motif1, motif2, and motif3. The VPS23 subgroups contains three different specific motifs, including motif4, motif5, and motif6. In addition, motif1 was widely distributed in StUEVs, while only StELC-like2 is absent of motif1. Interestingly, StCOP1 contains only motif1. To better understand gene structural diversity, we examined the exon-intron distribution patterns of StUEVs genes. The StUEVs occupied positions in the same clade with similar intron-exon distribution patterns. UEV1 and COP1 subgroups were spliced with three introns and four exons, while StUEV1B contains four introns and five exons. Particularly, the VPS23 subgroups have no introns. These results suggest that StUEVs motifs and gene structure display diverse distribution patterns. (Figure 2c).

Identification and Characterization of Potato StUBC13
Based on a BLAST search, two StUBC13s were identified in potato, which were named corresponding to the Arabidopsis genes: StUBC35/StUBC13A, StUBC35/StUBC13A-like (Table S2). StUBC13A and StUBC13A-like are located on chromosome 10 and 7, respectively. There are segmental duplication events of the StUBC13 gene in potato, indicating that the StUBC13 gene participates in the evolution of large gene families. Phylogenetic tree analysis revealed that the StUBC13s of five plants descended from the same branch and were orthologs ( Figure 3a). Some species lost UBC13B throughout the evolution of UBC13s (such as tomato, potato, and rice), whereas others retained UBC13B. (such as Brachypodium). StUBC13A evolved from a common plant ancestor and is the closest relative to SlUBC13s of tomato and the most distant relative to BdUBC13B of Brachypodium.  Gene structure analysis showed that StUBC13s were consistent with the number and distribution of intron-exon ( Figure 3b). However, the length of the second exon (53 bp) of AtUBC13B was different from that of other species (28 bp). The lengths of other corresponding exons were consistent, and the nucleic acid sequences were highly conserved. The intron length and sequence identity at the same location varied greatly between species, while the intron lengths of StUBC13A and SlUBC13A, StUBC13A-like, and SlUBC13A-like at the same site were nearly identical. The sequence identity was the highest, 88.55%, indicating that UBC13s were highly conserved not only in the exon sequence but also in the intron sequence during potato and tomato evolution. The promoter region analysis showed that several cis-acting elements participate in in abiotic stress and hormones responsiveness (Table S1).
Compared with the amino acid sequence of the UBC13s of other organisms (Figure 3c), the active site Cys87 formed with the Ub thioester, the Met64 interacting with RING-type E3 ligase, and Glu55, Phe57 and Arg70 interacting with Mms2 (UEV1s) are conserved [9]. In comparison to other plants, the protein sequence of Arabidopsis thaliana AtUBC13B contains nine amino acid residues (GNFITSFDP) inserted between positions 21-29, and the S amino acid at position 30 differs from the A amino acid in other species.
BiFC and SLC assays were conducted to further verify StUBC13-StUEV1B and StUBC13-StRGLG1 interactions. The BiFC assay (bimolecular fluorescence complementation) showed that YFP fluorescence signals were observed in the nucleus and cell membrane in epidermal cells, which indicated that StUBC13 and StUEV1B interactions occurred in the nucleus, cytoplasm, and cell membrane (Figure 4b). We observed a specific reconstituted luciferase activity signal using SLC (split-luciferase complementation) assays when the combination of StUBC13 and StUEV1B/StRGLG1 was coexpressed in N. benthamiana leaves (Figure 4c). These data confirm that StUBC13 interacted with StUEV1B and StRGLG1 in vivo, respectively.
The GO analysis revealed that StUBC13 partners were significantly enriched to the biological process of response to stimulus (GO:0050896) ( Figure S1a). There was a significant statistical overlap between five biological processes of StUBC13 and its targets, including protein metabolic process (GO:0019538), cellular protein modification process (GO:0006464), response to chemicals (GO:0042221), and response to stress (GO:0006950). In addition, KEGG pathway annotation showed that UBC13 partners were significantly enriched in metabolism, genetic information processing, environmental information processing, cellular processing, and organismal system ( Figure S1b). BiFC and SLC assays were conducted to further verify StUBC13-StUEV1B a StUBC13-StRGLG1 interactions. The BiFC assay (bimolecular fluorescence complemen tion) showed that YFP fluorescence signals were observed in the nucleus and cell me brane in epidermal cells, which indicated that StUBC13 and StUEV1B interactions curred in the nucleus, cytoplasm, and cell membrane (Figure 4b). We observed a spec reconstituted luciferase activity signal using SLC (split-luciferase complementation) says when the combination of StUBC13 and StUEV1B/StRGLG1 was coexpressed in benthamiana leaves (Figure 4c). These data confirm that StUBC13 interacted with StUEV and StRGLG1 in vivo, respectively.
The GO analysis revealed that StUBC13 partners were significantly enriched to biological process of response to stimulus (GO:0050896) ( Figure S1a). There was a sign

Subcellular Localization of UBC13 and Its Partners
To further explore the role of StUBC13 in plant cells, StUBC13 CDS was fused with green fluorescence protein (eGFP) to produce fluorescence signals for subcellular localization. The sample was expressed in tobacco (N. benthamiana) leaves using the Agrobacterium-mediated infiltration method. Confocal imaging showed that 35S: StUBC13-EGFP was distributed in the nucleus and cell membrane, and continuous intensity fluorescent on the cell membrane ( Figure 5). Similarly, StUEV1B and RGLG1 also observed GFP fluorescence signals in the nucleus, cytoplasm, and cell membrane ( Figure 5). These results indicate that StUBC13, StUEV1B, and StRGLG1 were localized in the nucleus, cytoplasm, and cell membrane.

Expression Patterns of StUEVs and StUBC13
To further reveal the role of StUEVs and StUBC13, RNA-seq data was performed to analyze the tissue-specific expression level. There are different tissue-specific expression patterns in DM and RH. StUBC13A and StUBC13A-like in stamens, StUEV1D-like1 and StVPS23/ELC in petals, StELC-like2 in callus, StELC-like in mature fruits, and StCOP10 and StUEV1D-like1 in sepals displayed significant expression levels in DM, respectively (Figure 6a). StUEV1D-like1, StVPS23/ELC, StUBC13A-like in the petiole, StUBC13A in the shoot apex, StELC-like2 in root had the highest expression level in RH (Figure 6b). Other genes were not differentially expressed among tissues in DM and RH. The different expression profiles showed the various roles of StUEVs and StUBC13 in potato development and growth.
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 11 o In addition, KEGG pathway annotation showed that UBC13 partners were significan enriched in metabolism, genetic information processing, environmental information p cessing, cellular processing, and organismal system ( Figure S1b).

Subcellular Localization of UBC13 and Its Partners
To further explore the role of StUBC13 in plant cells, StUBC13 CDS was fused w green fluorescence protein (eGFP) to produce fluorescence signals for subcellular loc zation. The sample was expressed in tobacco (N. benthamiana) leaves using the Agrob terium-mediated infiltration method. Confocal imaging showed that 35S:StUBC13-EG was distributed in the nucleus and cell membrane, and continuous intensity fluoresc on the cell membrane ( Figure 5). Similarly, StUEV1B and RGLG1 also observed GFP orescence signals in the nucleus, cytoplasm, and cell membrane ( Figure 5). These resu indicate that StUBC13, StUEV1B, and StRGLG1 were localized in the nucleus, cytopla and cell membrane.

Expression Patterns of StUEVs and StUBC13
To further reveal the role of StUEVs and StUBC13, RNA-seq data was performed analyze the tissue-specific expression level. There are different tissue-specific express To further understand the function of StUEVs and StUBC13, the publicly available transcriptome data were performed an expression profile analysis of StUEVs and StUBC13 gene after drought, heat, mannitol, salt, ABA, IAA, GA3, BAP (Figure 6c). StELC-like2, StUEV1B, and StELC-like were obviously upregulated after heat stress. The expression levels of StUEV1D-like, StUEV1D-like1, and StUBC13A were higher under ABA stress. The transcript levels of StCOP10 and StVPS23/ELC were significantly higher under GA3 stress. StUEV1D-like1 and StVPS23/ELC were slightly upregulated following BAP stress. In addition, StUBC13A and StUBC13A-like were noticeably downregulated after BAP stress. All had no obvious expression changes under salt, ABA, mannitol stress. For drought stress, the expression levels of StUEV1D-like, StUEV1D-like1, StUEV1B, StELC-like were significantly induced in the early flowering Atlantic, while StUBC13A, StUBC13A-like, StCOP10 were less expressed in the early flowering Atlantic (Figure 6d). StVPS23/ELC and StELC-like were highly expressed in the falling flowering Qingshu, StUEV1D-like1 and StUBC13A-like were obviously expressed in the full flowering Qingshu.  To further understand the function of StUEVs and StUBC13, the publicly available transcriptome data were performed an expression profile analysis of StUEVs and StUBC13 gene after drought, heat, mannitol, salt, ABA, IAA, GA3, BAP (Figure 6c). StELC-like2, StUEV1B, and StELC-like were obviously upregulated after heat stress. The expression levels of StUEV1D-like, StUEV1D-like1, and StUBC13A were higher under ABA stress. The transcript levels of StCOP10 and StVPS23/ELC were significantly higher under GA3 stress. StUEV1D-like1 and StVPS23/ELC were slightly upregulated following BAP stress. In addition, StUBC13A and StUBC13A-like were noticeably downregulated after BAP stress. All had no obvious expression changes under salt, ABA, mannitol stress. For drought stress, the expression levels of StUEV1D-like, StUEV1D-like1, StUEV1B, StELC-like were significantly induced in the early flowering Atlantic, while StUBC13A, StUBC13A-like, StCOP10 were less expressed in the early flowering Atlantic (Figure 6d). StVPS23/ELC and StELC-

The qRT-PCR Analysis of StUEVs and StUBC13 in Potato
To further determine the expression patterns of StUEVs and StUBC13s, qRT-PCR was performed to analyze organ-specific expression levels in Atlantic (Figure 7). StUEV1D-like1, StUEV1B, StELC-like, and StELC-like2 in root had the highest transcript abundances. StUEV1B, StELC-like, and StUBC13A in tubers exhibited significant expression levels. StUEV1D-like1, StVPS23/ELC, StUBC13A, StUBC13A-like in stems had a significant expression level. Interestingly, StUEV1D-like was not specifically expressed between tubers, stems, roots and leaves. These results suggested that StUEVs and StUBC13 play an important role in potato development and growth.
To further explore the role of StUEVs and StUBC13 in potato, the expression patterns of StUEV1s and StUBC13 were determined by qRT-PCR under different stresses (Figure 8 like, and StELC-like were down-regulated under heat stress. StUEV1D-like1 was down-regulated under drought, salt, and mannitol stress, but was up-regulated by ABA, IAA, GA3, and heat stress. This suggests StUEV1s and StUBC13 were implicated in different stresses. Overall, StUEVs and StUBC13 participated in potato development and growth, and responses to different types of stress.

Discussion
Lys63-linked polyubiquitination is widely considered to be a non-canonical ubiquitation and is different from conventional Lys-48-linked polyubiquitination. The Lys63linked polyubiquitination process is a signal for a nonproteolytic rather than mediating Overall, StUEVs and StUBC13 participated in potato development and growth, and responses to different types of stress.

Discussion
Lys63-linked polyubiquitination is widely considered to be a non-canonical ubiquitation and is different from conventional Lys-48-linked polyubiquitination. The Lys63linked polyubiquitination process is a signal for a nonproteolytic rather than mediating the target protein degradation by 26S proteasome. Lys63-linked polyubiquitination is similar to phosphorylation and sumoylation, which influence target protein activities to play a critical role in several cellular processes. UBC13, the only known E2, can catalyze Lys-63-linked Ub chain assembly by forming stable heterodimers complex with UEV1s [15,16]. In this study, StUBC13A in potato was isolated and characterized in potato. The gene structure analysis showed that the distribution pattern of UBC13s introns/exons was highly conserved in different species (Figure 3b) [17,18]. The phylogenetic analysis and sequence alignment of amino acid was 98.69% identified between StUBC13A and StUBC13A-like (Figure 3a), which were separated from one gene duplication events [19]. Both tomato and potato have two StUBC13A, indicating that these two species have expanded StUBC13A during evolution, which is consistent with previous research on UBC13 gene duplication events (PGDD: http://chibba.agtec.uga.edu/duplication/index/locus, accessed on 22 September 2022). Therefore, StUBC13 genes were involved in genome duplication or gene fusion in potato during the evolution of the E2 gene family, which indicates that StUBC13 genes may be functionally redundant, consistent with previous studies [20]. Of great interest is the observation that the expression of StUBC13s is induced by different stress (Figure 8), which is similar to the BdUBC13A results in Brachypodium [20]. However, UBC13 was considered a housekeeping gene in both Arabidopsis and rice [17].
To date, UEVs have been identified in several plants, including eight UEVs in A. thaliana. However, only four and three UEV1s subfamilies have been identified in rice (O. sativa) and B. distachyon, respectively [11,21]. In this study, we characterized seven highly conserved StUEVs in potato (Table S1). The phylogenetic analysis showed that members of the StUEVs family were expanded/lost in potato during the evolution process, such as the loss of the UEV2 and the expansion of the VPS23 subgroups (Figure 1a). This suggests that, during the evolutionary process of adapting to the environment, StUEVs might respond to environmental changes and needs by selecting, eliminating, or extending their members. Sequence alignment analysis showed that StUEV1B included a special C-terminal extension containing the CaaX motif except for the typical UEV conserved domain (Figure 1c), which is similar to AtUEV1A, AtUEV1B, OsUEV1A, BdUEV1A sequence feature. The CaaX motif is considered to be the isoprene-specific site in plants, which plays a crucial role in the functional differentiation between AtUEV1A/B and AtUEV1C/D and affects the subcellular localization to confer other nuclear functions [12,21]. Similarly, we propose that the CaaX motif may be implicated in functional differentiation between StUEV1B and StUEV1D-like/StUEV1D-like1 and subcellular localization ( Figure 5). The distribution pattern and number of introns/exons and motifs is in accordance with in the same subfamily (Figure 2b,c), which implies little diversity between the same subgroups [10]. StUEV1s exhibited distinct expression patterns in different potato tissues (Figures 6 and 7). AtUEV1s are ubiquitously expressed in Arabidopsis, and AtUEV1D is highly expressed in shoots, which is consistent with the expression profiles of StUEV1B. Previous studies showed that AtUEV1s were derived from a segmental duplication gene, and the duplication events result in similarity expression profiles and conserved functions [10]. A cis-acting element in the StUEVs gene promoter region serves as a binding site for transcription factors and regulates gene expression. Distinct cis-acting components serve different purposes. qRT-PCR analysis showed that the StUEVs family specifically expressed in different tissue and induced expression under different stresses (Figure 8). This is associated with the cis-acting elements of hormonal and abiotic stress responses in the StUEVs promoter region (Figure 2a). Therefore, UEVs are involved in response to abiotic stress and development [10,13].
The formation of Lys63-linked polyubiquitination is required to the stabilize the heterodimer complex UBC13-UEV1s, which is involved in the regulation of DNA damage and response to stress. UBC13 interacted with Uev1s to form stable heterodimers, which confers different functions [21]. In this study, StUBC13 interacted with all three StUEV1s (Figure 4a), which suggested that StUEV1s and StUBC13 may be involved in DNA damage and response to stress [11]. Several partners of StUBC13 were screened by the yeast twohybrid, including StRGLG1/2, StNF-YA6, StCAM7, StS6PDH, StLOG2, StRD21, StNCL, and StPUB40 (Figure 4a), of which homologous partners play a crucial role in regulating plant growth and development [22][23][24][25][26][27][28][29]. The GO and KEGG analysis showed that the partners were involved in metabolism and response to stimulus ( Figure S1). For example, RGLG1/2 ubiquitin E3 ligase can interact with StUBC13 to form a E2-E3 complex in potato using Y2H, SLC, and BiFC technology assays, and the complex is involved in the iron deficiency response of Arabidopsis roots [30]. In this regard, future research will focus on how StUBC13-StUEV1s conjugates different E3s to form Lys-63-linked poly-Ub chains and influences target proteins activities to regulate multiple cellular processes, such as [8,[31][32][33][34][35].

Multiple Sequence Alignment, Gene Structure and Motif, and Cis-Acting Elements Analysis
The protein sequences of UBC13 and UEVs from several species were performed to multiple sequence alignment to analyse conserved domain features by ClustalW and visualized using GENEDOC [39]. The distribution of the exon-intron structure UEVs in potato were examined by Gene Structure Display Server (GSDS: http://gsds.cbi.pku.edu.cn, accessed on 15 September 2022). The UEVs conserved motifs analysis was performed by MEME online program (https://meme-suite.org/meme/doc/meme.html, accessed on 15 September 2022) [40]. The UEVs subfamily of the promoter regions (from 0 to −1000 bp of coding sequence) was performed to analyze cis-acting elements using the PlantCARE database (http: //bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 17 September 2022).

Yeast Two-Hybrid Assay
The yeast two-hybrid (Y2H) assay was performed as previously described [41]. The StUBC13 gene (StUBC13A) was recombined into the pGBKT7 vector as bait and used to screen the potential interaction proteins of StUBC13 according to instructions from Clontech. The StUEV1s gene were cloned into the pGADT7 vector as prey and were used to verify the interaction between StUBC13 and StUEV1s through co-transforming into yeast strain YH109 by one-to-one Y2H assay. The primer used in this study is listed in Table S4.

Gene Ontology (GO) and KEGG Analysis
To further correlate UBC13 with its partners, the Gene Ontology (GO) terms and KEGG were identified and enriched using OmicShare tools, a free online data analysis (https://www.omicshare.com/tools, accessed on 22 September 2022).

Subcellular Localization and BiFC, SLC Analysis
The StUBC13, StUEV1B and StRGLG1 gene were fused to the pCAMBIA1300-EGFP vector, respectively. Similarily, StUBC13, StUEV1B and StRGLG1 were separately recombined into the pSPYCE and pSPYNE vectors, and the pCAMBIA1300-CLuc and pCAMBIA1300-NLuc vectors. The different combinations were transformed into Nicotiana benthamiana leaves, respectively. The GFP and YFP fluorescence signal were observed by confocal microscope (Zeiss LSM780, Oberkochen, Germany). The LUC recombinant fluorescence signal was imaged by PlantView 100 (Guangzhou Biolight Biotechnology, Guangzhou, China). The primer used in this study is listed in Table S1.

Expression Profiles of StUBC13s and StUEV1s
The raw transcriptome data was downloaded from the publicly available transcriptome at NCBI (Project ID: PRJNA541096) and SpudDB (DM and RH potato) [36]. The RNA-seq data was analyzed and visualized using TBtools [42].

Plant Materials and Growth Condition
The potato cultivar Atlantic was cultured in MS medium supplemented with 0.75% agar and 3% sucrose under 22 • C, 16 h normal light/8 h dark. After 4 weeks of growth, the plants were transplanted into pots with soil mix (10 × 10 cm), and then placed in a growth chamber at 21 ± 2 • C under 16 h light/8 h dark and watered weekly. After another 4 weeks, the plants were classed into nine groups to be subjected to different treatments and controls groups. For the salt and mannitol stress treatment, the plants were irrigated with 200 mM NaCl and mannitol for 24 h, while the controls were irrigated with water. For hormone treatment, the plants leaves were sprayed with 50 µM ABA, 10 µM IAA, and 50 µM GA3 for 24 h, respectively, while the controls were sprayed with water. For the drought stress treatment, the plants were cultured without water to reach 25 ± 5% of soil field capacity, while the controls were watered weekly. For the heat stress treatment, the plants were stored at 35 • C for 24 h, while the controls were grown in normal condition. The differently treated samples were harvested and immediately frozen in liquid nitrogen, and stored at −80 • C.

RNA Extraction and qRT-PCR Assay
The total RNA of the samples was extracted by using the RNAprep Pure Plant Kit (Tiangen Biotechnology, Beijing, China) and then synthesized to first strand of cDNA by FastKing RT Kit (Tiangen Biotechnology, Beijing, China). The gene expression level was detected by qRT-PCR using SuperReal PreMix Plus (SYBR Green) (Tiangen Biotechnology, Beijing, China) with LightCycler ® 96 System. The reference gene Stef1α (GenBank accession: AB061263) was used for data normalization. The results were calculated using the 2 − CT method [43].

Conclusions
UBC13 and UEVs were required for the ubiquitin Lys63 chain to undergo polyubiquitination. In this study, we systematically identified and analyzed StUBC13 and StUEVs gene structure, conserved domains, cis-acting elements, interactions, and gene expression in potato. Our observations collectively suggest that StUBC13 interacted with StUEV1s to form complexes that may be involved in multiple cellular processes.

Conflicts of Interest:
The authors declare no conflict of interest.