- Research article
- Open Access
Genome-wide analysis of genes encoding core components of the ubiquitin system in soybean (Glycine max) reveals a potential role for ubiquitination in host immunity against soybean cyst nematode
- Chunyu Zhang†1, 2,
- Li Song†3,
- Mani Kant Choudhary1, 2,
- Bangjun Zhou1, 2,
- Guangchao Sun2, 4,
- Kyle Broderick1,
- Loren Giesler1 and
- Lirong Zeng1, 2Email authorView ORCID ID profile
© The Author(s). 2018
- Received: 1 December 2017
- Accepted: 9 July 2018
- Published: 18 July 2018
Ubiquitination is a major post-translational protein modification that regulates essentially all cellular and physiological pathways in eukaryotes. The ubiquitination process typically involves three distinct classes of enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3). To date, a comprehensive identification and analysis of core components comprising of the whole soybean (Glycine max) ubiquitin system (UBS) has not been reported.
We performed a systematic, genome-wide analysis of genes that encode core members of the soybean UBS in this study. A total of 1431 genes were identified with high confidence to encode putative soybean UBS components, including 4 genes encoding E1s, 71 genes that encode the E2s, and 1356 genes encoding the E3-related components. Among the E3-encoding genes, 760 encode RING-type E3s, 124 encode U-box domain-containing E3s, and 472 encode F-box proteins. To find out whether the identified soybean UBS genes encode active enzymes, a set of genes were randomly selected and the enzymatic activities of their recombinant proteins were tested. Thioester assays indicated proteins encoded by the soybean E1 gene GmUBA1 and the majority of selected E2 genes are active E1 or E2 enzymes, respectively. Meanwhile, most of the purified RING and U-box domain-containing proteins displayed E3 activity in the in vitro ubiquitination assay. In addition, 1034 of the identified soybean UBS genes were found to express in at least one of 14 soybean tissues examined and the transcript level of 338 soybean USB genes were significantly changed after abiotic or biotic (Fusarium oxysporum and Rhizobium strains) stress treatment. Finally, the expression level of a large number of the identified soybean UBS-related genes was found significantly altered after soybean cyst nematode (SCN) treatment, suggesting the soybean UBS potentially plays an important role in soybean immunity against SCN.
Our findings indicate the presence of a large and diverse number of core UBS proteins in the soybean genome, which suggests that target-specific modification by ubiquitin is a complex and important part of cellular and physiological regulation in soybean. We also revealed certain members of the soybean UBS may be involved in immunity against soybean cyst nematode (SCN). This study sets up an essential foundation for further functional characterization of the soybean UBS in various physiological processes, such as host immunity against SCN.
- Ubiquitin system (UBS)
- Ubiquitin-activating enzyme (E1)
- Ubiquitin-conjugating enzyme (E2)
- RING domain
- U-box domain
- F-box domain
- Soybean cyst nematode
Ubiquitination is a major post-translational protein modification that plays an important role in many cellular and physiological processes in eukaryotes . It involves covalently attaching ubiquitin, a highly conserved small protein, to substrate through sequential reactions that are catalyzed by three classes of enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) . In the enzymatic cascade, the E1 enzyme first activates free ubiquitin in presence of ATP hydrolysis, leading to the formation of a thioester-linkage in which the C-terminal glycine of the ubiquitin molecule is linked with the cysteine residue at the active center of E1. The activated ubiquitin is then transferred to a conserved cysteine residue of the E2 enzyme. In the final step, the ubiquitin molecule is transferred from the E2-ubiquitin intermediate to the substrate protein with the assistance of an E3 ligase. The ubiquitin molecule is usually attached to the ε-amino group of lysine residues of a substrate . The enzymatic cascade can be repeated after the first ubiquitin is attached to the substrate protein, resulting in a polymeric ubiquitin chain being linked to the substrate protein where the linkage between ubiquitin moieties determines the substrate’s fate in the cell .
As the enzyme catalyzing the first step of the ubiquitin conjugation cascade, E1s regulate the rate of ubiquitination thus govern the overall ubiquitin function inside the cell . So far, E1 genes and their proteins have been isolated and characterized from rabbit , yeast , wheat , mice , human , Arabidopsis thaliana  and tobacco . Multiple E1 genes have been identified in each of the plant and animal genomes analyzed, whereas the yeast genome contains only a single E1 gene. The E1 proteins from all kingdoms possess a similar size ranging from 110 to 125 kDa and share regions of high homology that generally contain four different characteristic structural units: the adenylation domain composed of two ThiF-homology motifs ; the catalytic cysteine domain composed of the FCCH and SCCH half-domain (for “first” and “second” catalytic cysteine half-domain, respectively) ; a four-helix bundle (4HB) that immediately follows the FCCH; and the C-terminal ubiquitin-fold domain (UFD) [11, 13]. The specificity of an E1 towards E2s depends in part on the UFD, which is responsible for recruiting cognate E2s .
The E2 enzymes were originally defined as proteins capable of accepting ubiquitin from an E1 through thioester linkage with a cysteinyl sulfhydryl group . All E2s possess a highly conserved domain of about 140–150 amino acids called the ubiquitin-conjugating (UBC) domain where the cysteinyl residue of the active site resides . Currently, 11, 50 and 40 ubiquitin E2 proteins are known to exist in the yeast (Saccharomyces cerevisiae), human (Homo sapiens) and tomato (Solanum lycopersicum), respectively [17–19]. In addition to 37 ubiquitin E2 proteins [17, 20], a UBC domain is also identified in two RUB-conjugating enzymes (RCE1, At4g36800 and RCE2, At2g18600) and a SUMO-conjugating enzyme (SCE1, At3g57870) in Arabidopsis thaliana . Additionally, there are eight other Arabidopsis UBC proteins that lack the active site cysteinyl residue required for thioester formation . Previously, the E2s were often considered as ‘ubiquitin carriers’ with auxiliary roles. However, recent studies have suggested that E2s control the switch from chain initiation to elongation and govern the topology of ubiquitin chains formed, thereby determine the fate of the substrate proteins being modified .
The E3 ubiquitin ligases are the largest and most diverse group among the three classes of enzyme that catalyze the ubiquitination cascade. They recruit the target proteins for ubiquitination and are the main factor that determines the specificity of ubiquitination . In the Arabidopsis and human genome, more than 1300 and 600 genes are predicted to encode E3-related components, respectively [24, 25]. The E3 ligases can be either single-polypeptide proteins or multi-subunits complexes. Based on the mechanism of action and the presence of different protein domains responsible for E3 ligase activity, the single-polypeptide ubiquitin ligases can be divided into three defined types, the homology to the E6-associated protein C-terminus (HECT)-, really interesting new gene (RING)-, or U-box-domain containing E3s. The HECT-type E3s are single-subunit proteins characterized by having a C-terminal, approximately 350-amino-acid HECT domain that is involved in both accepting ubiquitin from an E2 protein and transferring it to the substrate protein . A unique feature of the HECT-type E3 ligases is a conserved and catalytic cysteine residue that serves as the site for forming a thioester-linked ubiquitin-E3 intermediate. In these E3 ligases, E2 charges the cysteine residue with ubiquitin prior to it being transferred to the substrate. To date, plant HECT-type E3s have been analyzed in Arabidopsis thaliana only, which contains seven HECT genes named UPL1 - UPL7 . Evolution analysis indicated the number of HECT genes has been kept quite constant in different plant genomes . Unlike the HECT-type E3s, RING and U-box proteins noncovalently interact with E2 carrying thioester-linked ubiquitin via the conserved RING or U-box domain to facilitate the transfer of ubiquitin to the substrate . RING and U-box ligases are structurally related and use zinc-chelating domain and hydrogen bonds /salt bridges, respectively to transfer ubiquitin [20, 29]. The RING-type E3s are the most abundant among single-subunit ubiquitin ligases [30, 31]. The U-box domain is a modified RING domain that lacks conserved Zn-coordinating residues . The U-box-type E3 ubiquitin ligases are characterized by the conserved ~ 70 amino acid U-box domain originally identified in the yeast UFD2 protein . In addition to being typically single-polypeptide E3s, the RING domain-containing proteins can also be a subunit of complex, multi-subunits E3s, including the Skp1-Cullin-F-box (SCF), the anaphase-promoting complex/cyclosome (APC/C) and the Cullin-Elongin-BC-VHL (CBC VHL)-type E3 ligases . In the well-studied SCF-type multi-subunits ligase, the RING domain-containing protein RBX/ROC/HRT is responsible for binding to E2, whereas the F-box protein confers the substrate recognition . A F-box protein contains at least one F-box domain that spans about 40 amino acids at their N-terminus, which binds the SKP1 to create the SCF complex . F-box proteins have been identified in both prokaryotes  and eukaryotes. In plants, the F-box gene family is also one of the largest gene families, suggesting they may regulate many important biological processes [38, 39].
Ubiquitination was originally identified as a principal signal for selective protein degradation in the cell. However, the functions of ubiquitination have extended far beyond that since its discovery over three decades ago. The importance of ubiquitination in the regulation of myriad cellular and physiological processes in animal, human and plant has been increasingly appreciated in the past three decades [31, 40]. Soybean (Glycine max) is a major crop and the dominant oil-seed in world. Diseases have been a major constraint on soybean yield . Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) has consistently been the most economically important pathogen of soybean worldwide, and causes approximately $1 billion in annual yield loss in the United States . Although a few subfamilies of E3 ligases have been studied individually in soybean [43–46], a comprehensive knowledge on core components of the whole ubiquitin system (UBS) has not been reported yet. In the present study, a genome-wide analysis of core components of the soybean UBS was performed. Through an array of bioinformatics analyses, 4 ubiquitin E1 genes, 71 ubiquitin E2 genes, 1356 genes encoding ubiquitin E3s including 760 RING domain-, 124 U-box domain- and 472 F-box domain-containing E3s were identified with high confidence from the soybean genome . Dozens of components of soybean UBS were cloned, and their enzymatic activities were examined. Moreover, analyses of RNA-seq data and real time quantitative PCR (real time qPCR) results indicated the expression patterns of many components in the soybean UBS were significantly changed under the soybean cyst nematode (SCN) treatment, which supports the notion that soybean UBS may play a role in host immunity against SCN. These results provide a valuable foundation for further functional characterizations of key components of soybean UBS in various physiological processes including their roles in soybean immunity against SCN.
The soybean genome possesses four ubiquitin E1 genes
All eukaryotic E1s contain an adenylation domain composed of two ThiF-homology motifs that are derived from the bacterial ThiF proteins . The ThiF motif is considered to be a minimal module for ubiquitin- and ubiquitin-like protein (UBL)-E1 recognition and adenylation activities . Thus, the consensus sequence of the ThiF motif (PF00899) from NCBI conserved domain database (CDD) was employed as query to perform BLAST search against the Phytozome v.12.1 database of the soybean genome (Glycine max Wm82.a2.v1). A total of 37 transcripts from 20 genes encoding ThiF motif-containing proteins were identified, apparently due to some of the genes have multiple annotated transcripts (Additional file 1: Table S1). Among them, seven transcripts from four genes encode proteins with more than 1000 amino acids and a molecular weight (Mw) around 120 kD (Additional file 1: Table S1), similar to the ubiquitin E1 proteins that have been characterized in other plant species [4, 7].
Seventy-one ubiquitin E2s encoded by the soybean genome are classified into eleven groups
Summary of the number of soybean UBS components identified after HMMER analysis, BLAST search against the Pfam and NCBI databases, and manual validation
Besides ubiquitin E2, other proteins such as ubiquitin-conjugating enzyme variant proteins (UEV), Related to Ubiquitin (RUB)-conjugating enzymes (RCE), Small Ubiquitin-like Modifier (SUMO)-conjugating enzyme (SCE), ELCH (ELC homolog) and Ubiquitin-fold modifier 1-conjugating enzyme (UFC1) also contain the UBC domain . To distinguish ubiquitin E2 from those proteins, we generated the phylogeny of soybean and Arabidopsis UBC domain-containing proteins (Additional file 5: Figure S2). The phylogenetic analysis indicated that, of the 91 genes, 71 encode ubiquitin E2 proteins, 11 encode UEV proteins (including homolog of the AtCOP10), two encode RUB E2 proteins (RCE), four encode putative SUMO E2, one encodes ELC and two encode UFC1 E2 proteins (Fig. 3b).
The Arabidopsis ubiquitin E2 proteins were largely subdivided into 12 groups . In addition, the AtUBC37 was assigned to group XIII due to its homology to tomato UBC37 . Based on the phylogenetic analysis of UBC domain-containing proteins in Arabidopsis and soybean, the soybean does not have close homologs to the group V E2s and AtUBC37 in Arabidopsis (Additional file 5: Figure S2). Therefore, the 71 soybean ubiquitin E2 proteins were classified into 11 groups (Additional file 6: Figure S3). Domain organization analysis using the Pfam and the NCBI database indicated that, except for Glyma.04G199200 and Glyma.06G166300, the 71 ubiquitin E2 proteins contain a UBC domain only (Fig. 3c). Both of Glyma.04G199200 and Glyma.06G166300 also contain an additional domain called ubiquitin-associated (UBA) domain at their C-terminuses (Fig. 3c). The UBA domain has been found to mediate protein-protein interactions through binding of ubiquitin molecules .
Identification of genes encoding RING-, U-box- and F-box-domain containing E3s in soybean genome
To identify genes that encode RING-, U-box- and F-box-type E3 ligases in soybean, the HMM profiles of these domains (Additional file 2: Table S2) from Pfam were used as the query files. A total of 1234, 158 and 579 homologs of RING, U-box and F-box proteins, respectively were identified in soybean genome by HMMER analysis (Table 1). To verify these identified proteins, all sequences in FASTA format were uploaded and searched against the Pfam and NCBI databases for detection of the RING, U-box, and F-box domain, respectively. Combined the BLAST results against the Pfam and NCBI databases, 1034, 145, and 547 genes that encode putative RING domain-, U-box domain- and F-box domain-containing proteins were obtained after removing redundant sequences (Table 1).
A typical RING has the consensus, 40–60 amino acids linear sequence of C-X2-C-X[9–39]-C-X[1–3]-H-X[2–3]-C-X2-C-X[4–48]-C-X2-C where the highly conserved Cysteine (C) and Histidine (H) residues form two cross-brace structure to bind two zinc ions and X can be any of the twenty amino acids . Two canonical RING-types (C3H2C3 and C3HC4) that differ in the presence of either a Cys or His at the fifth Cys residue were well characterized . We extracted the sequence of the RING domain from all soybean RING domain-containing proteins that were manually validated. We then performed the alignment of the sequences and generated graphical sequence consensus logos using the Weblogo3 algorithm online (Fig. 4a) . The conserved Cys and His residues that have been known to be responsible for stabilizing two loop regions through coordinating the two zinc ions, as well as a central conserved α-helix that connects the first and second loops are presented in the sequence consensus logos [30, 59] (Fig. 4a). A Trp or other hydrophobic residue that is often found at the α-helix region and has been implicated in interaction with E2s is also presented  (Fig. 4a). Unlike the RING domain, the U-box domain lacks the zinc-binding sites. The hydrogen-bonding networks that contain hydrophobic and polar amino acids are proposed to maintain the U-box scaffold . The consensus sequence generated by the Weblogo3 algorithm using sequences of the identified soybean U-box domains displays two α-helices and three β-strands in its secondary structure, which is consistent to the consensus structure of known plant U-box domains, as manifested by the Arabidopsis U-box protein AtPUB14  (Fig. 4b). In the U-box domain, three hydrophobic E2 binding sites and two hydrophobic cores have been shown to be essential for the function of U-box domain . These amino acid residues are identified in the soybean U-box domain consensus sequence generated by Weblogo3 as well (Fig. 4b).
The F-box domain is the signature structure of F-box proteins that act as a subunit of the SCF catalytic core through interacting with Skp1 . Several conserved residues that are known for contributing to protein-protein interaction and structure stability were used for verifying the soybean F-box proteins. In human Skp2 protein, Pro113, a hallmark amino acid residue of F-box domain, assists to launch α-helix while Leu124 and Try139 contribute to the packing of the F-box helices . These amino acid residues are highly conserved in soybean F-box proteins (Fig. 4c). In addition, the Skp1 binding residues of the Skp2 in human were conserved in F-box domain-containing proteins from soybean and other plant species [63–65] (Fig. 4c).
The soybean ubiquitin E1 protein and majority of the ubiquitin E2s examined are enzymatically active
Randomly selected soybean RING and U-box genes encode proteins that possess E3 ubiquitin ligase activity
Expression profile of soybean UBS genes during plant development and after stress treatments
To elucidate the potential roles of soybean UBS genes under biotic or abiotic stress, we analyzed the gene expression using previous RNA-seq datasets that were generated from experiments in which soybean plants were treated by various stresses [72–74]. Genes were considered differentially expressed if the fold changes are ≥2 or ≤ 0.5 between treated and control plants at a P-value of less than 0.05 (or false discovery rate ≤ 0.001 in the dataset that rhizobium strains were inoculated). The identified up-regulated and down-regulated genes are shown in Additional file 19: Figure S14 and Additional file 20: Table S6. The transcript level of 196, 45 and 112 soybean USB genes were significantly altered after abiotic stress (i.e. drought and salt) treatment, Fusarium oxysporum and rhizobium strains inoculation, respectively. Among them, the expression of 41 genes was significantly affected under both drought and salt, and 12 were in response to both F. oxysporum and rhizobium strains. Further analysis of the overlap between the biotic and abiotic stresses revealed that the transcript level of 3 RING-type E3 ligases (Glyma.03G215500, Glyma.06G150400 and Glyma.12G112000) were significantly changed under all stresses tested.
The expression level of many soybean UBS genes change significantly upon treatment with SCN
A genome-wide identification of genes that encode core components of the soybean UBS would be an essential step towards further functional characterizations of these genes in soybean. Previous studies have reported identification of a few individual ubiquitin E3 ligase gene families in soybean, including the HECT , the RBR (a subset of RING) , the U-box  and the F-box  genes. However, a systematic identification and analysis of genes that encode the core components of the entire soybean (Glycine max) ubiquitin system (UBS) in soybean have hitherto not been performed. To address this knowledge gap, we performed a comprehensive identification and analysis of soybean UBS genes in this study. Through an array of bioinformatics protocols for gene identification and analyses of their corresponding proteins, we pinpointed with high confidence 4 ubiquitin E1-encoding genes, 71 ubiquitin E2-encoding genes and 1356 genes encoding components of three families of ubiquitin E3 ligases (including 760 RING genes, 124 U-box genes and 472 F-box genes) from the soybean genome using the latest soybean genome database Wm82.a2.v1.
Generally, the ubiquitin E1 enzymes are monomeric proteins with a molecular weight of 110–125 kDa and contain two ThiF motifs that is involved in adenylation . In the present study, we identified 20 genes that encode ThiF motif-containing proteins from the soybean genome. However, only four of these genes encode proteins with a predicted molecular weight of more than 110 kDa and the presence of E1-specific catalytic Cys domain (UBA_e1_thiolCys) and ubiquitin-fold domain (UFD). The UBA_e1_thiolCys domain that is also called SCCH  contains a cysteine residue responsible for ubiquitin thioester linkage, while UFD confers specificity of E1 in recruiting ubiquitin E2s . Consistent with the domain organization information, phylogenetic analysis indicated the ThiF motif-CCD-UFD domain-containing proteins encoded by the four soybean genes also fall into the same clade of known ubiquitin E1s in Arabidopsis and human. We also identified 71 ubiquitin E2 genes out of 91 UBC domain-containing genes in soybean. The 71 ubiquitin E2s were classified into 11 groups, I-XII except V according to the grouping of Arabidopsis counterparts . We did not identify the homolog of AtUBC37 in the soybean genome. The absence of UBC37 and group V E2s in the list of soybean E2s identified in present study can either due to the genome is not completely sequenced thus the sequence for those genes are not presented in the soybean genome database or there are indeed no such E2s exist in the genome. The proteins encoded by a soybean ubiquitin E1 gene (GmUBA1) and four ubiquitin E2 genes (GmUBC2, 8, 19 and 21) were used to perform in vitro thioester assay. The results provide proofs that GmUBA1 functions as an active E1 enzyme to activate free ubiquitin to form thioester-linked ubiquitin. Thioester-linked ubiquitin is then transferred to the active E2 enzymes (GmUBC2, 8 and 19) to generate a thioester-linked E2-ubiquitin adduct.
The ubiquitin E3 ligases are the most diverse group in the ubiquitin system and are responsible for the substrate specificity of ubiquitination. Based on the mechanism of action and their structural features, E3 ligases can be grouped into single-subunit including HECT, RING and U-box-types  and multi-subunit including SCF (Skp1-Cullin-F-box), Cullin-Elongin-BC-VHL (CBC VHL) and the APC (Anaphase Promoting Complex) types . We did not include in this study the identification of HECT-type of soybean E3s as previous evolutionary analysis indicated the number of HECT genes has been kept quite constant in different plant genomes and 19 were predicted in the soybean genome [28, 45]. Instead, we focused on the three types of E3 (RING, U-box and F-box) that constitute the largest groups of E3 in plant genomes. In present study, 760 RING genes were identified in the soybean genome, which is almost 2 times of the numbers found in other eukaryotes such as Arabidopsis, human and mouse genomes that encode for 469, 385 and 305 RING proteins, respectively [80, 81]. Recently, 24 genes encoding RBR (RING1-IBR-RING2; a subset of RING proteins) domain-containing proteins were identified from the soybean genome . These RBR genes were among the 1234 RING genes identified by our HMMER3.1 analysis (Additional file 3: Table S3). However, only five of these RBR genes were among the list generated by BLAST against the NCBI & Pfam databases and none of them fall into the list after manual validation due to their highly atypical RING domain. The RING proteins that serve as a subunit of the Cullin-RING-like (CRL)-type of multiple-subunit ubiquitin E3s and the RBR proteins were also excluded from our final list of soybean RING E3s after manual validation. The Arabidopsis and rice (Oryzae sativa) genomes contain 64 and 77 predicted U-box proteins, respectively [29, 82]. Recently, 125 U-box genes were identified in the soybean genome . Our analysis identified 124 U-box genes, of which 119 (96%) are among the list that was reported in that study , five extra U-box genes were revealed by our study but were missed in the former study, and three other U-box genes reported by the that study were eliminated from our list after manual validation (Additional file 3: Table S3) . A close look of the three eliminated U-box genes revealed that the highly conserved amino acid residues at the N-terminus of the U-box domain were missed in the proteins encoded by those genes (data not shown). In plants, 694, 687, 337 and 156 F-box genes have been identified in Arabidopsis, rice, popular (Populus trichocarpa) and grape (Vitis vinifera), respectively [39, 83]. In present study, 472 F-box genes were identified, of which 440 (93%) were also identified by another group in a recent study . However, 32 F-box genes revealed in present study were missed in that study whereas 64 F-box genes that were reported in that study were eliminated from our final list after manual validation in this study even though they are actually on the list after HMMER 3.1 analysis (Additional file 3: Table S3). The overlap of the vast majority of the E3 genes we identified in present study with the previous reports indicates the effectiveness of the algorithms we used for our genome-wide gene identification. On the other hand, we combined in present study the HMMER 3.1 analysis, protein domain detection tools in the NCBI and, Pfam databases, and manual validation for the identification of genes of interest, which is more stringent than previous studies that involved HMMER analysis and/or BLAST only and may explain why some of the genes identified in those studies are not on our final list.
So far, a few soybean RING and U-box proteins have been shown to possess E3 ubiquitin ligase activity [44, 84, 85]. However, commercially available, non-soybean E1 and E2s were used for the in vitro ubiquitination assays in those studies to examine the E3 activity. In this study, a ubiquitin E1 gene GmUBA1 and three E2 genes GmUBC2, 8 and 19 are proved to encode active ubiquitin E1 and E2 enzymes by thioester assay but GmUBC21 failed to form adducts with ubiquitin in the assay. Similarly, the Arabidopsis homologs of GmUBC2, 8 and 19, AtUBC2, 8 and 19 have also been shown to carry thioester-linked ubiquitin  but AtUBC21 did not show E2 activity in thioester assay . Using components of the soybean ubiquitin system, four RING proteins and three U-box proteins were tested to be true E3 ubiquitin ligases when GmUBC8 was employed as the cognate ubiquitin E2 enzyme. Similar to the Arabidopsis AtPUB10 that is capable of performing autoubiquitination using AtUBC2 as the cognate E2 enzyme , the soybean GmPUB10 was also found to display E3 activity in the presence of soybean E2 GmUBC2. Demonstration of these randomly selected proteins of the soybean UBS as enzymatically active validates the algorithms we used for the identification at genome scale of components that constitute the soybean UBS.
Gene expression analyses can provide key information about the potential functions of soybean UBS genes. Accordingly, we analyzed the expression profile of UBS genes during plant development and under abiotic and biotic stresses using publicly-available RNA-seq datasets. The transcript of 1034 UBS genes could be detected in at least one of 14 soybean tissues examined, further suggesting the effectiveness of the algorithms we used for our genome-wide gene identification. Meanwhile, the expression level of 338 soybean USB genes were significantly changed after either abiotic (drought and salt) or biotic (F. oxysporum and rhizobium strains) stress treatment, implying they may play a role in these processes. Among biotic stresses, SCN (Heterodera glycines Ichinohe) has consistently been a major pest on soybean worldwide, which cause soybean yield loss of 15–30% yearly. Breeding and planting SCN-resistant cultivars is the most effective strategy to control SCN . There has hitherto been very limited study on the ubiquitin system (UBS) in soybean immunity against SCN and other pathogens. To expand our understanding of the functions of ubiquitination-related genes in soybean immunity, we examined their expression profiles after SCN treatment by employing publicly-available RNA-seq datasets . Based on the analysis of the RNA-seq datasets, 180 soybean UBS genes including 22 E2 genes and 158 E3 genes were found to have significantly altered their abundance in transcripts after incubation with SCN. Among these genes identified by RNA-seq analysis, six out of ten randomly selected ones were validated by real time qPCR using the SCN-susceptible soybean cv. Williams 82 after incubation with SCN. These results support the notion that UBS likely plays an important role in soybean immunity against SCN. Until now most soybean cultivars being resistant to SCN are derived from limited resistance sources and SCN race has begun evolving to overcome the resistance . Therefore, engineering novel SCN resistance may serve as an intriguing strategy for the management of SCN infection. To this end, pinpointing and characterizing members of the soybean UBS identified by present study that play key roles in soybean immunity should be the next experiments. Considering the omnipresence of ubiquitination in the regulation of plant growth, development, and biotic and abiotic stress responses, further functional characterization of the soybean UBS components identified in present study would also facilitate in-depth understanding of many other plant physiological processes.
In this study, genes encoding core components of the soybean ubiquitin system (UBS) were systematically identified by an array of bioinformatics protocols. A total of 4 ubiquitin E1 genes, 71 ubiquitin E2 genes and 1356 E3 ligase genes were identified from the soybean genome. The presence of such a large and diverse number of UBS proteins suggests that target-specific modification by ubiquitin is a complex and important part of cellular and physiological regulation in soybean. More than a dozen of proteins encoded by the identified soybean E1, E2 and E3 genes were randomly selected for biochemical tests and the enzymatic activity was validated for the majority of them. Combined the analysis of RNA-seq data and real time qPCR results indicate that the expression level of a large number of soybean UBS genes changed significantly after the SCN treatment, which suggests the involvement of UBS components in the soybean-SCN interactions. The present study has built a foundation and presented an essential framework for further functional characterization of soybean UBS genes in various physiological processes, including their role and the underlying molecular mechanism in the regulation of soybean immunity against SCN.
Identification of soybean UBS genes
The search for ubiquitin E1 enzyme-coding genes in soybean was performed using a consensus sequence of ThiF motif as query and the BLASTP algorithm against the latest soybean proteome database (Phytozome 12.1, https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Gmax). The consensus sequence for the ThiF motif (PF00899) was downloaded from the NCBI CDD database (http://www.ncbi.nlm.nih.gov/cdd/). To confirm the obtained proteins, the Pfam database (http://pfam.xfam.org/)  was used to further examine the presence of ThiF motif in the candidate proteins.
To identify potential members of ubiquitin E2 enzymes and E3 ligases in soybean, the HMM profiles (Additional file 2: Table S2) of corresponding domains were downloaded from the Pfam database. The HMMER3.1  program was then employed to search against the soybean proteome database (Wm82.a2.v1) at the Soybase (http://www.soybase.org/) [47, 71] using these HMM profiles as queries. The complete protein sequences were extracted from Soybase based on the HMMER search results, and then submitted to the Pfam and NCBI CDD databases to validate the presence of domains of interest. To finally determine these predicted proteins, we processed manual validation based on alignment of the sequence of domain of interest in candidate proteins and their corresponding consensus sequences that are downloaded from CDD database. Those proteins that lack the highly conserved key amino acids or secondary structures were excluded from the final dataset.
On the basis of the results of BLASTP searches in the soybean genome database of Phytozome, we obtained information on the chromosomal locations, cDNA sequences, CDS sequences, protein sequences, and alternative splicing events. The molecular weight was calculated using ProtParam (http://web.expasy.org/protparam/). The expressed sequence tag (EST) was identified by NCBI blast. If more than one transcript existed for a gene in the Soybase, the primary transcript was used for all subsequent analyses.
Phylogenetic, sequence conservation and gene duplication analysis
The phylogenetic trees were constructed using MUSCLE aligned full-length amino acids sequences and the Neighbor-joining (NJ) method in the MEGA6 program with parameters of p-distance, gaps treated by partial deletion, and 1000 bootstrap replicates .
To analyze the sequence features of the domain of interest, the sequences of the corresponding domain in the predicted proteins were extracted based on NCBI blast results, and the consensus sequences of the UBC, RING, U-box, and F-box domain were downloaded from CDD database. The multiple sequence alignments were performed by CLUSTAL2.1 , and visualized using the ESPript3 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi)  and BoxShade (http://www.ch.embnet.org/software/BOX_form.html) . The secondary structures were also generated by the ESPript3 according to the reference sequences. The sequence logos were produced from the multiple sequence alignment using the online program WebLogo3 (http://weblogo.threeplusone.com/create.cgi)  with the default parameters.
To inspect domain organization of the identified proteins, the amino acid sequences of the proteins with FASTA format were searched against the Pfam and CDD database. The information of conserved domains was extracted for analyzing the domain organization.
To analyze RING and F-box collinear paralogues, MCScanX was employed as previous described . The highest scoring path was identified by dynamic programming with standard settings. Gene loci were classified as whole genome duplications (WGD) /segmental, tandem, proximal or dispersed duplications based on the number of matching hits and positions in chromosomes and scaffolds.
Proteins expression and purification
The full-length coding sequences of the selected genes were cloned into the pDEST15 vector using the Gateway cloning system (Invitrogen), and transformed into the E. coli strain BL21 (DE3). The primers used for this assay are listed in Additional file 18: Table S5. GST-tagged fusion proteins were expressed in BL21 and purified using Glutathione Sepharose 4 Fast Flow beads (GE Healthcare) by following the protocol provided by the manufacturer. Briefly, the E. coli cells were harvested by centrifugation, suspended with lysis buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mg/mL lysozyme and cocktail), and disrupted using sonicator. For purification, 200 μL Glutathione Sepharose 4 Fast Flow beads was added to cleared supernatant and incubated on a rotator overnight at 4 °C. Beads were washed 3 times with 10 mL washing buffer (1 × PBS, 1 mM EDTA and 0.5% Triton X-100), and then eluted with 4 mL of elution buffer (10 mM reduced glutathione in 50 mM Tris-HCl pH 8.8). The purified proteins were further desalted and concentrated in the protein storage buffer (50 mM Tris-HCl, pH 8, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, and 0.5 mM PMSF) using the Amicon Centrifugal Filter (Millipore). Glycerol was added to the recombinant protein to a final concentration of 40% for storage at − 80 °C until being used. The concentration of purified protein was measured using protein assay agent (Bio-Rad).
The E1 ubiquitin-activating activity and E2 ubiquitin-conjugating activity were detected by in vitro thioester assays as previously described . The assays were conducted in a total reaction volume of 20 μL, consisting of 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 1 mM ATP. 40 ng of soybean E1 (GST-GmUBA1) was preincubated with 2 μg of FLAG-ubiquitin in the 20 μL reaction at 28 °C for 10 min. An approximate 100 ng of GST-fused E2 protein was added into the reaction and continued for 15 min. The reactions were split into two half-volume after incubation and terminated by the addition of SDS sample buffer with 100 mM dithiothreitol (DTT) or 4 M urea sample buffer without DTT (−). The reactions were probed with mouse monoclonal anti-FLAG M2-peroxidase-conjugated antibody (Sigma-Aldrich) before being detected using an ECL kit (Pierce, now Thermo Fisher).
In vitro ubiquitination assay
The in vitro ubiquitination assay was performed as described previously . In briefly, in a total of 30 μL, 40 ng of soybean E1 (GST-GmUBA1), an approximate 100 ng of GST-fused E2, 2 μg of GST-E3 ligase and 2 μg of ubiquitin were combined in ubiquitination buffer (50 mM Tris-HCl (pH 7.5), 5 mM ATP, 5 mM MgCl2, 2 mM DTT, 3 mM creatine phosphate, and 5 μg/mL creatine phosphokinase). After 1.5 h at 30 °C, the reactions were terminated by adding SDS sample loading buffer with 100 mM DTT, and boiled at 100 °C for 5 min. Products of the reactions were separated by 10% SDS-PAGE gel and detected by immunoblot using mouse monoclonal anti-ubiquitin M2-peroxidase-conjugated (horseradish peroxidase) antibody (Sigma-Aldrich).
Plant materials and gene expression analysis after SCN inoculation
Roots from three-week-old soybean Williams 82 plants were independently inoculated with two soybean cyst nematode (SCN, Heterodera glycines Ichinohe) populations, race 155 (HG Type 2.5.7) and race 117 (HG Type 126.96.36.199.6.7). One week after SCN inoculation, roots from three soybean plants were collected and immediately frozen in liquid nitrogen and ground to a fine powder for RNA extraction. Roots of three uninoculated Williams 82 plants were collected for the negative control.
Total RNA was extracted using the RNeasy Plant Mini Kit with DNase treatment (Qiagen) following the manufacturer’s procedure. Two micrograms of total RNA was then used as template for the first-strand cDNA synthesis in the presence of SuperScript III reverse transcriptase and oligo (dT) primer (Life Technologies). The cDNA population were diluted 10 times with sterilized ddH2O before being used for real time quantitative PCR (qPCR). The real time-qPCR was conducted on the LightCycler 480 Instrument II (Roche) with SYBR Green (Life Technologies) and gene-specific primers. The soybean EF1a gene, GmEF1a (Glyma.19G052400) was used as an internal control (Additional file 23: Table S8).
This work was supported by fund from the Nebraska Soybean Board (grant 1719) to LZ.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplemental data files.
LS, BZ and CZ performed bioinformatics analysis for the identification of soybean UBS genes. MC cloned and purified the recombinant proteins for part of the selected soybean E2 and E3 genes. GS performed the genome-wide gene duplication analyses for the RING and F-box gene families. KB and LG grew soybean plants and inoculated soybean roots with SCN. CZ performed the majority of experiments, analyzed data, and wrote the article. LZ designed experiments, analyzed the data, wrote and edited the article. All authors read and approved the final manuscript.
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Consent for publication
The authors declare that they have no competing interests.
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- Ye Y, Rape M. Building ubiquitin chains: E2 enzymes at work. Nat Rev Mol Cell Biol. 2009;10(11):755–64.View ArticlePubMedPubMed CentralGoogle Scholar
- Callis J. The ubiquitination machinery of the ubiquitin system. Arabidopsis Book. 2014;12:e0174.View ArticlePubMedPubMed CentralGoogle Scholar
- Komander D, Rape M. The ubiquitin code. Annu Rev Biochem. 2012;81:203–29.View ArticlePubMedGoogle Scholar
- Hatfield PM, Gosink MM, Carpenter TB, Vierstra RD. The ubiquitin-activating enzyme (E1) gene family in Arabidopsis thaliana. Plant J. 1997;11(2):213–26.View ArticlePubMedGoogle Scholar
- Ciechanover A, Elias S, Heller H, Hershko A. “Covalent affinity” purification of ubiquitin-activating enzyme. J Biol Chem. 1982;257(5):2537–42.PubMedGoogle Scholar
- McGrath JP, Jentsch S, Varshavsky A. UBA 1: an essential yeast gene encoding ubiquitin-activating enzyme. EMBO J. 1991;10(1):227–36.PubMedPubMed CentralView ArticleGoogle Scholar
- Hatfield PM, Vierstra RD. Multiple forms of ubiquitin-activating enzyme E1 from wheat. J Biol Chem. 1992;267(21):14799–803.PubMedGoogle Scholar
- Mitchell MJ, Woods DR, Tucker PK, Opp JS, Bishop CE. Homology of a candidate spermatogenic gene from the mouse Y chromosome to the ubiquitin-activating enzyme E1. Nature. 1991;354(6353):483–6.View ArticlePubMedGoogle Scholar
- Handley PM, Mueckler M, Siegel NR, Ciechanover A, Schwartz AL. Molecular cloning, sequence, and tissue distribution of the human ubiquitin-activating enzyme E1. Proc Natl Acad Sci U S A. 1991;88(1):258–62.View ArticlePubMedPubMed CentralGoogle Scholar
- Takizawa M, Goto A, Watanabe Y. The tobacco ubiquitin-activating enzymes NtE1A and NtE1B are induced by tobacco mosaic virus, wounding and stress hormones. Mol Cells. 2005;19(2):228–31.PubMedGoogle Scholar
- Walden H, Podgorski MS, Schulman BA. Insights into the ubiquitin transfer cascade from the structure of the activating enzyme for NEDD8. Nature. 2003;422(6929):330–4.View ArticlePubMedGoogle Scholar
- Szczepanowski RH, Filipek R, Bochtler M. Crystal structure of a fragment of mouse ubiquitin-activating enzyme. J Biol Chem. 2005;280(23):22006–11.View ArticlePubMedGoogle Scholar
- Huang DT, Hunt HW, Zhuang M, Ohi MD, Holton JM, Schulman BA. Basis for a ubiquitin-like protein thioester switch toggling E1-E2 affinity. Nature. 2007;445(7126):394–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Jin J, Li X, Gygi SP, Harper JW. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging. Nature. 2007;447(7148):1135–8.View ArticlePubMedGoogle Scholar
- Glickman MH, Ciechanover A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev. 2002;82(2):373–428.View ArticlePubMedGoogle Scholar
- Vierstra RD. The expanding universe of ubiquitin and ubiquitin-like modifiers. Plant Physiol. 2012;160(1):2–14.View ArticlePubMedPubMed CentralGoogle Scholar
- Bachmair A, Novatchkova M, Potuschak T, Eisenhaber F. Ubiquitylation in plants: a post-genomic look at a post-translational modification. Trends Plant Sci. 2001;6(10):463–70.View ArticlePubMedGoogle Scholar
- Jiang YH, Beaudet AL. Human disorders of ubiquitination and proteasomal degradation. Curr Opin Pediatr. 2004;16(4):419–26.View ArticlePubMedGoogle Scholar
- Zhou B, Mural RV, Chen X, Oates ME, Connor RA, Martin GB, Gough J, Zeng L. A subset of ubiquitin-conjugating enzymes is essential for plant immunity. Plant Physiol. 2017;173(2):1371–90.View ArticlePubMedGoogle Scholar
- Kraft E, Stone SL, Ma L, Su N, Gao Y, Lau OS, Deng XW, Callis J. Genome analysis and functional characterization of the E2 and RING-type E3 ligase ubiquitination enzymes of Arabidopsis. Plant Physiol. 2005;139(4):1597–611.View ArticlePubMedPubMed CentralGoogle Scholar
- Kurepa J, Walker JM, Smalle J, Gosink MM, Davis SJ, Durham TL, Sung DY, Vierstra RD. The small ubiquitin-like modifier (SUMO) protein modification system in Arabidopsis. Accumulation of SUMO1 and −2 conjugates is increased by stress. J Biol Chem. 2003;278(9):6862–72.View ArticlePubMedGoogle Scholar
- Windheim M, Peggie M, Cohen P. Two different classes of E2 ubiquitin-conjugating enzymes are required for the mono-ubiquitination of proteins and elongation by polyubiquitin chains with a specific topology. Biochem J. 2008;409(3):723–9.View ArticlePubMedGoogle Scholar
- Sadanandom A, Bailey M, Ewan R, Lee J, Nelis S. The ubiquitin-proteasome system: central modifier of plant signalling. New Phytol. 2012;196(1):13–28.View ArticlePubMedGoogle Scholar
- Smalle J, Vierstra RD. The ubiquitin 26S proteasome proteolytic pathway. Annu Rev Plant Biol. 2004;55:555–90.View ArticlePubMedGoogle Scholar
- Schwartz AL, Ciechanover A. Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology. Annu Rev Pharmacol Toxicol. 2009;49:73–96.View ArticlePubMedGoogle Scholar
- Rotin D, Kumar S. Physiological functions of the HECT family of ubiquitin ligases. Nat Rev Mol Cell Biol. 2009;10(6):398–409.View ArticlePubMedGoogle Scholar
- Downes BP, Stupar RM, Gingerich DJ, Vierstra RD. The HECT ubiquitin-protein ligase (UPL) family in Arabidopsis: UPL3 has a specific role in trichome development. Plant J. 2003;35(6):729–42.View ArticlePubMedGoogle Scholar
- Marin I. Evolution of plant HECT ubiquitin ligases. PLoS One. 2013;8(7):e68536.View ArticlePubMedPubMed CentralGoogle Scholar
- Yee D, Goring DR. The diversity of plant U-box E3 ubiquitin ligases: from upstream activators to downstream target substrates. J Exp Bot. 2009;60(4):1109–21.View ArticlePubMedGoogle Scholar
- Deshaies RJ, Joazeiro CA. RING domain E3 ubiquitin ligases. Annu Rev Biochem. 2009;78:399–434.View ArticlePubMedGoogle Scholar
- Vierstra RD. The ubiquitin-26S proteasome system at the nexus of plant biology. Nat Rev Mol Cell Biol. 2009;10(6):385–97.View ArticlePubMedGoogle Scholar
- Aravind L, Koonin EV. The U box is a modified RING finger - a common domain in ubiquitination. Curr Biol. 2000;10(4):R132–4.View ArticlePubMedGoogle Scholar
- Koegl M, Hoppe T, Schlenker S, Ulrich HD, Mayer TU, Jentsch S. A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell. 1999;96(5):635–44.View ArticlePubMedGoogle Scholar
- Harper JW, Tan MK. Understanding cullin-RING E3 biology through proteomics-based substrate identification. Mol Cell Proteomics. 2012;11(12):1541–50.View ArticlePubMedPubMed CentralGoogle Scholar
- Hua Z, Vierstra RD. The cullin-RING ubiquitin-protein ligases. Annu Rev Plant Biol. 2011;62:299–334.View ArticlePubMedGoogle Scholar
- Skaar JR, Pagan JK, Pagano M. Mechanisms and function of substrate recruitment by F-box proteins. Nat Rev Mol Cell Biol. 2013;14(6):369–81.View ArticlePubMedGoogle Scholar
- Angot A, Peeters N, Lechner E, Vailleau F, Baud C, Gentzbittel L, Sartorel E, Genschik P, Boucher C, Genin S. Ralstonia solanacearum requires F-box-like domain-containing type III effectors to promote disease on several host plants. Proc Natl Acad Sci U S A. 2006;103(39):14620–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Lechner E, Achard P, Vansiri A, Potuschak T, Genschik P. F-box proteins everywhere. Curr Opin Plant Biol. 2006;9(6):631–8.View ArticlePubMedGoogle Scholar
- Xu G, Ma H, Nei M, Kong H. Evolution of F-box genes in plants: different modes of sequence divergence and their relationships with functional diversification. Proc Natl Acad Sci U S A. 2009;106(3):835–40.View ArticlePubMedPubMed CentralGoogle Scholar
- Kerscher O, Felberbaum R, Hochstrasser M. Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu Rev Cell Dev Biol. 2006;22:159–80.View ArticlePubMedGoogle Scholar
- Koenning SR, Wrather JA: Suppression of soybean yield potential in the continental United States by plant diseases from 2006 to 2009. Plant Health Progress 2010:online, doi:https://doi.org/10.1094/PHP-2010-1122-1001-RS.
- Tian B, Wang S, Todd TC, Johnson CD, Tang G, Trick HN. Genome-wide identification of soybean microRNA responsive to soybean cyst nematodes infection by deep sequencing. BMC Genomics. 2017;18(1):572.View ArticlePubMedPubMed CentralGoogle Scholar
- Jia Q, Xiao ZX, Wong FL, Sun S, Liang KJ, Lam HM. Genome-wide analyses of the soybean F-box gene family in response to salt stress. Int J Mol Sci. 2017;18(4):818.Google Scholar
- Wang N, Liu Y, Cong Y, Wang T, Zhong X, Yang S, Li Y, Gai J. Genome-wide identification of soybean U-box E3 ubiquitin ligases and roles of GmPUB8 in negative regulation of drought stress response in Arabidopsis. Plant Cell Physiol. 2016;57(6):1189–209.View ArticlePubMedGoogle Scholar
- Meng X, Wang C, Rahman SU, Wang Y, Wang A, Tao S. Genome-wide identification and evolution of HECT genes in soybean. Int J Mol Sci. 2015;16(4):8517–35.View ArticlePubMedPubMed CentralGoogle Scholar
- Chen P, Zhang X, Zhao T, Li Y, Gai J. Genome-wide identification and characterization of RBR ubiquitin ligase genes in soybean. PLoS One. 2014;9(1):e87282.View ArticlePubMedPubMed CentralGoogle Scholar
- Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, Nelson W, Hyten DL, Song Q, Thelen JJ, Cheng J, et al. Genome sequence of the palaeopolyploid soybean. Nature. 2010;463(7278):178–83.View ArticlePubMedGoogle Scholar
- Schulman BA, Harper JW. Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways. Nat Rev Mol Cell Biol. 2009;10(5):319–31.View ArticlePubMedPubMed CentralGoogle Scholar
- Hochstrasser M. Evolution and function of ubiquitin-like protein-conjugation systems. Nat Cell Biol. 2000;2(8):E153–7.View ArticlePubMedGoogle Scholar
- Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, Heger A, Hetherington K, Holm L, Mistry J, et al. Pfam: the protein families database. Nucleic Acids Res. 2014;42(Database issue):D222–30.View ArticlePubMedGoogle Scholar
- Finn RD, Clements J, Arndt W, Miller BL, Wheeler TJ, Schreiber F, Bateman A, Eddy SR. HMMER web server: 2015 update. Nucleic Acids Res. 2015;43(W1):W30–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Aravind L, Iyer LM, Koonin EV. Comparative genomics and structural biology of the molecular innovations of eukaryotes. Curr Opin Struct Biol. 2006;16(3):409–19.View ArticlePubMedGoogle Scholar
- Burroughs AM, Jaffee M, Iyer LM, Aravind L. Anatomy of the E2 ligase fold: implications for enzymology and evolution of ubiquitin/Ub-like protein conjugation. J Struct Biol. 2008;162(2):205–18.View ArticlePubMedPubMed CentralGoogle Scholar
- Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: a sequence logo generator. Genome Res. 2004;14(6):1188–90.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhao Q, Tian M, Li Q, Cui F, Liu L, Yin B, Xie Q. A plant-specific in vitro ubiquitination analysis system. Plant J. 2013;74(3):524–33.View ArticlePubMedGoogle Scholar
- Dikic I, Wakatsuki S, Walters KJ. Ubiquitin-binding domains - from structures to functions. Nat Rev Mol Cell Biol. 2009;10(10):659–71.View ArticlePubMedGoogle Scholar
- Borden KL, Freemont PS. The RING finger domain: a recent example of a sequence-structure family. Curr Opin Struct Biol. 1996;6(3):395–401.View ArticlePubMedGoogle Scholar
- Freemont PS. The RING finger. A novel protein sequence motif related to the zinc finger. Ann N Y Acad Sci. 1993;684:174–92.View ArticlePubMedGoogle Scholar
- Hanzawa H, de Ruwe MJ, Albert TK, van Der Vliet PC, Timmers HT, Boelens R. The structure of the C4C4 ring finger of human NOT4 reveals features distinct from those of C3HC4 RING fingers. J Biol Chem. 2001;276(13):10185–90.View ArticlePubMedGoogle Scholar
- Andersen P, Kragelund BB, Olsen AN, Larsen FH, Chua NH, Poulsen FM, Skriver K. Structure and biochemical function of a prototypical Arabidopsis U-box domain. J Biol Chem. 2004;279(38):40053–61.View ArticlePubMedGoogle Scholar
- Schulman BA, Carrano AC, Jeffrey PD, Bowen Z, Kinnucan ER, Finnin MS, Elledge SJ, Harper JW, Pagano M, Pavletich NP. Insights into SCF ubiquitin ligases from the structure of the Skp1-Skp2 complex. Nature. 2000;408(6810):381–6.View ArticlePubMedGoogle Scholar
- Bai C, Sen P, Hofmann K, Ma L, Goebl M, Harper JW, Elledge SJ. SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell. 1996;86(2):263–74.View ArticlePubMedGoogle Scholar
- Gagne JM, Downes BP, Shiu SH, Durski AM, Vierstra RD. The F-box subunit of the SCF E3 complex is encoded by a diverse superfamily of genes in Arabidopsis. Proc Natl Acad Sci U S A. 2002;99(17):11519–24.View ArticlePubMedPubMed CentralGoogle Scholar
- Jia F, Wu B, Li H, Huang J, Zheng C. Genome-wide identification and characterisation of F-box family in maize. Mol Gen Genomics. 2013;288(11):559–77.View ArticleGoogle Scholar
- Cui HR, Zhang ZR, Lv W, Xu JN, Wang XY. Genome-wide characterization and analysis of F-box protein-encoding genes in the Malus domestica genome. Mol Gen Genomics. 2015;290(4):1435–46.View ArticleGoogle Scholar
- Wang Y, Tang H, Debarry JD, Tan X, Li J, Wang X, Lee TH, Jin H, Marler B, Guo H, et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012;40(7):e49.View ArticlePubMedPubMed CentralGoogle Scholar
- Mudgil Y, Shiu SH, Stone SL, Salt JN, Goring DR. A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box E3 ubiquitin ligase family. Plant Physiol. 2004;134(1):59–66.View ArticlePubMedPubMed CentralGoogle Scholar
- Jung C, Zhao P, Seo JS, Mitsuda N, Deng S, Chua NH. PLANT U-BOX PROTEIN10 regulates MYC2 stability in Arabidopsis. Plant Cell. 2015;27(7):2016–31.View ArticlePubMedPubMed CentralGoogle Scholar
- Kong L, Cheng J, Zhu Y, Ding Y, Meng J, Chen Z, Xie Q, Guo Y, Li J, Yang S, et al. Degradation of the ABA co-receptor ABI1 by PUB12/13 U-box E3 ligases. Nat Commun. 2015;6:8630.View ArticlePubMedPubMed CentralGoogle Scholar
- Furlan G, Nakagami H, Eschen-Lippold L, Jiang X, Majovsky P, Kowarschik K, Hoehenwarter W, Lee J, Trujillo M. Changes in PUB22 ubiquitination modes triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 dampen the immune response. Plant Cell. 2017;29(4):726–45.View ArticlePubMedPubMed CentralGoogle Scholar
- Grant D, Nelson RT, Cannon SB, Shoemaker RC. SoyBase, the USDA-ARS soybean genetics and genomics database. Nucleic Acids Res. 2010;38(Database issue):D843–6.View ArticlePubMedGoogle Scholar
- Belamkar V, Weeks NT, Bharti AK, Farmer AD, Graham MA, Cannon SB. Comprehensive characterization and RNA-Seq profiling of the HD-zip transcription factor family in soybean (Glycine max) during dehydration and salt stress. BMC Genomics. 2014;15:950.View ArticlePubMedPubMed CentralGoogle Scholar
- Lanubile A, Muppirala UK, Severin AJ, Marocco A, Munkvold GP. Transcriptome profiling of soybean (Glycine max) roots challenged with pathogenic and non-pathogenic isolates of fusarium oxysporum. BMC Genomics. 2015;16:1089.View ArticlePubMedPubMed CentralGoogle Scholar
- Yuan S, Li R, Chen S, Chen H, Zhang C, Chen L, Hao Q, Shan Z, Yang Z, Qiu D, et al. RNA-Seq analysis of differential gene expression responding to different rhizobium strains in soybean (Glycine max) roots. Front Plant Sci. 2016;7:721.PubMedPubMed CentralGoogle Scholar
- Cheng YT, Li X. Ubiquitination in NB-LRR-mediated immunity. Curr Opin Plant Biol. 2012;15(4):392–9.View ArticlePubMedGoogle Scholar
- Mural RV, Liu Y, Rosebrock TR, Brady JJ, Hamera S, Connor RA, Martin GB, Zeng L. The tomato Fni3 lysine-63-specific ubiquitin-conjugating enzyme and suv ubiquitin E2 variant positively regulate plant immunity. Plant Cell. 2013;25(9):3615–31.View ArticlePubMedPubMed CentralGoogle Scholar
- Park CH, Chen S, Shirsekar G, Zhou B, Khang CH, Songkumarn P, Afzal AJ, Ning Y, Wang R, Bellizzi M, et al. The Magnaporthe oryzae effector AvrPiz-t targets the RING E3 ubiquitin ligase APIP6 to suppress pathogen-associated molecular pattern-triggered immunity in rice. Plant Cell. 2012;24(11):4748–62.View ArticlePubMedPubMed CentralGoogle Scholar
- Hosseini P, Matthews BF. Regulatory interplay between soybean root and soybean cyst nematode during a resistant and susceptible reaction. BMC Plant Biol. 2014;14:300.View ArticlePubMedPubMed CentralGoogle Scholar
- Lee I, Schindelin H. Structural insights into E1-catalyzed ubiquitin activation and transfer to conjugating enzymes. Cell. 2008;134(2):268–78.View ArticlePubMedGoogle Scholar
- Stone SL, Hauksdottir H, Troy A, Herschleb J, Kraft E, Callis J. Functional analysis of the RING-type ubiquitin ligase family of Arabidopsis. Plant Physiol. 2005;137(1):13–30.View ArticlePubMedPubMed CentralGoogle Scholar
- Semple CA, Group RG, Members GSL. The comparative proteomics of ubiquitination in mouse. Genome Res. 2003;13(6B):1389–94.View ArticlePubMedPubMed CentralGoogle Scholar
- Zeng LR, Park CH, Venu RC, Gough J, Wang GL. Classification, expression pattern, and E3 ligase activity assay of rice U-box-containing proteins. Mol Plant. 2008;1(5):800–15.View ArticlePubMedGoogle Scholar
- Yang X, Kalluri UC, Jawdy S, Gunter LE, Yin T, Tschaplinski TJ, Weston DJ, Ranjan P, Tuskan GA. The F-box gene family is expanded in herbaceous annual plants relative to woody perennial plants. Plant Physiol. 2008;148(3):1189–200.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang X, Wang N, Chen P, Gao M, Liu J, Wang Y, Zhao T, Li Y, Gai J. Overexpression of a soybean ariadne-like ubiquitin ligase gene GmARI1 enhances aluminum tolerance in Arabidopsis. PLoS One. 2014;9(11):e111120.View ArticlePubMedPubMed CentralGoogle Scholar
- Du QL, Cui WZ, Zhang CH, Yu DY. GmRFP1 encodes a previously unknown RING-type E3 ubiquitin ligase in soybean (Glycine max). Mol Biol Rep. 2010;37(2):685–93.View ArticlePubMedGoogle Scholar
- Zhang H, Song BH. RNA-seq data comparisons of wild soybean genotypes in response to soybean cyst nematode (Heterodera glycines). Genom Data. 2017;14:36–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Eddy SR. Accelerated profile HMM searches. PLoS Comput Biol. 2011;7(10):e1002195.View ArticlePubMedPubMed CentralGoogle Scholar
- Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30(12):2725–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23(21):2947–8.View ArticlePubMedGoogle Scholar
- Robert X, Gouet P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 2014;42(Web Server issue):W320–4.View ArticlePubMedPubMed CentralGoogle Scholar
- Artimo P, Jonnalagedda M, Arnold K, Baratin D, Csardi G, de Castro E, Duvaud S, Flegel V, Fortier A, Gasteiger E, et al. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res. 2012;40(Web Server issue):W597–603.View ArticlePubMedPubMed CentralGoogle Scholar
- Ariani P, Regaiolo A, Lovato A, Giorgetti A, Porceddu A, Camiolo S, Wong D, Castellarin S, Vandelle E, Polverari A. Genome-wide characterisation and expression profile of the grapevine ATL ubiquitin ligase family reveal biotic and abiotic stress-responsive and development-related members. Sci Rep. 2016;6:38260.View ArticlePubMedPubMed CentralGoogle Scholar