Classification, Expression Pattern, and E3 Ligase Activity Assay of Rice U-Box-Containing Proteins
a Department of Plant Pathology, Plant Molecular Biology and Biotechnology Program, The Ohio State University, 2021 Coffey Rd, 201 Kottman Hall, Columbus, OH 43210, USA
b Present address: Boyce Thompson Institute for Plant Research, Tower Rd, Ithaca, NY 14853, USA
c Department of Computer Science, University of Bristol, Woodland Rd, Bristol BS8 1UB, UK
d Crop Gene Engineering Key Laboratory of Hunan Province, Hunan Agricultural University, Changsha, Hunan 410128, China
1 To whom correspondence should be addressed. E-mail wang.620{at}osu.edu, fax 614 292 4455, tel. 614 292 9280.
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Abstract |
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Ubiquitin ligases play a central role in determining the specificity of the ubiquitination system by selecting a myriad of appropriate candidate proteins for modification. The U-box is a recently identified, ubiquitin ligase activity-related protein domain that shows greater presence in plants than in other organisms. In this study, we identified 77 putative U-box proteins from the rice genome using a battery of whole genome analysis algorithms. Most of the U-box protein genes are expressed, as supported by the identification of their corresponding expressed sequence tags (ESTs), full-length cDNAs, or massively parallel signature sequencing (MPSS) tags. Using the same algorithms, we identified 61 U-box proteins from the Arabidopsis genome. The rice and Arabidopsis U-box proteins were classified into nine major classes based on their domain compositions. Comparison between rice and Arabidopsis U-box proteins indicates that the majority of rice and Arabidopsis U-box proteins have the same domain organizations. The inferred phylogeny established the homology between rice and Arabidopsis U-box/ARM proteins. Cell death assay using the rice protoplast system suggests that one rice U-box gene, OsPUB51, might act as a negative regulator of cell death signaling. In addition, the selected U-box proteins were found to be functional E3 ubiquitin ligases. The identification and analysis of rice U-box proteins hereby at the genomic level will help functionally characterize this class of E3 ubiquitin ligase in the future.
Received for publication May 12, 2008. Accepted for publication June 23, 2008.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Ubiquitination is a major type of post-translational modification of proteins that occurs in eukaryotic cells (Ciechanover, 2005). The ubiquitination process involves in-concert catalytic activities of three types of enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). The ubiquitin molecule—a highly conserved 76-amino acid protein—is initially activated by E1 in an ATP-dependent manner, and the activated ubiquitin is then transferred to an E2. With the help of an E3, the ubiquitin molecule is covalently attached to the target protein through the
-amino group of a lysine residue (Ciechanover and Schwartz, 1998). The attachment of the first ubiquitin moiety to the substrate protein is usually followed by the addition of more ubiquitin molecules to form a polyubiquitin chain. A polyubiquitin chain with at least four sequentially attached ubiquitins linked through the lysine 48 of the ubiquitin molecules will be sufficient to allow target protein to be recognized and degraded by the 26S proteasome (Thrower et al., 2000). Importantly, polyubiquitination that links through other lysine residues of the ubiquitin molecules as well as mono-ubiquitination also regulate non-proteolytic processes in the cell (Bray et al., 2005; Huang et al., 2006; Li et al., 2003; Terrell et al., 1998). Of the three enzymes that are implicated in the ubiquitination process, E3 ubiquitin ligase plays a central role in determining specificity of the ubiquitination system by selecting appropriate candidate proteins (Ciechanover, 1998). Not surprisingly, given the myriad of different substrates, E3s are the most diverse components in the ubiquitination pathway. For example, over 1300 distinct E3 components are encoded by the Arabidopsis genome (Smalle and Vierstra, 2004). During the transfer of activated ubiquitin molecule from E2 to the substrate, the ubiquitin molecule may either be or not be covalently attached to the E3 ligase before it is attached to the substrate. Accordingly, E3 ligases can be largely classified into two classes—the HECT type and the family of RING/U-box-type ligases—based on the occurrence of such covalent linkage. The HECT-type ligases are the only known E3 ubiquitin ligases that form an E3-ubiquitin thiolester intermediate before the final attachment of ubiquitin to the substrate (Huibregtse et al., 1995; Scheffner et al., 1993). They contain an approximately 350-amino-acid in length HECT (homologous to E6-AP COOH terminus) domain that was initially identified in the viral E6-associated protein (E6-AP). A conserved active cysteine residue that is required for the ligase function is presented at the C-termini of these ligases (Scheffner et al., 1993). Depending on their subunit composition and mechanism of actions, members of the RING/U-box family can be further classified into three sub-classes—the CRL (Cullin-RING-Ligase) subgroup, including SCF type (Skp1-Cullin (CDC53)-F-box protein complex) (Bai et al., 1996; Skowyra et al., 1997) and other cullin-based types (such as ElonginC-CUL2-SOCS box type, CUL3-BTB type, etc.) (Pintard et al., 2003; Stebbins et al., 1999); RING (really interesting new gene) (Freemont et al., 1991)/U-box (Koegl et al., 1999) subgroup; and APC (anaphase promoting complex) subgroup (Irniger et al., 1995; King et al., 1995; Sudakin et al., 1995). The family of RING/U-box-type E3 ligases can be either single-subunit proteins or multi-subunit complexes. The RING-type E3 ligases belong to the class of single-polypeptide (subunit) E3s that contain a motif known as RING. The RING motif is maintained by the arrangement of eight cysteines and histidines in a cross-brace manner that chelate two zinc ions, which is distinct from other zinc finger domains (Borden, 2000). It is proposed that the RING-type E3 ligases serve as scaffolds to bring together the activated ubiquitin-E2 intermediate and the substrate protein to promote the transfer of the ubiquitin molecule to the substrate protein (Borden, 2000; Lorick et al., 1999).
The U-box is a more recently identified E3 ligase activity-related protein domain that was first shown in yeast to be involved in polyubiquitin chain assembly (Koegl et al., 1999). The U-box contains 
70 amino acids and possesses a tertiary structure resembling that of the RING domain (Aravind and Koonin, 2000; Ohi et al., 2003). The major difference between U-box and RING domains is that the U-box lacks the characteristic zinc-chelating cysteine and histidine residues. Consequently, the U-box E3s use intramolecular interactions other than zinc chelation to maintain the RING finger motif (Ohi et al., 2003). U-box proteins are present in yeast, plants, and animals (Azevedo et al., 2001; Koegl et al., 1999; Meacham et al., 2001; Stone et al., 2003). Nevertheless, only a few U-box proteins have been functionally studied to date. UFD2 (ubiquitin fusion degradation protein-2) is responsible for the assembly of polyubiquitin chains in yeast (Koegl et al., 1999). The Prp19p (precursor RNA processing 19p) is a subunit of the spliceosome and is involved in pre-mRNA splicing (Ohi and Gould, 2002). The human homolog of Prp19p, SNEV (senescence evasion factor), has been demonstrated to be a multifaceted protein playing a role in cellular senescence, cellular lifespan, pre-mRNA splicing, DNA double-strand break repair and the transport of ubiquitinated substrate to the proteasome (Loscher et al., 2005; Mahajan and Mitchell, 2003; Voglauer et al., 2006). The CHIP (C-terminal of Hsc70-interacting protein) protein acts as chaperone or co-chaperone in the cell to ensure protein homeostasis and has been implicated in stress responses and several neurodegenerative diseases (Ballinger et al., 1999; Dickey et al., 2007; Imai et al., 2002; Jiang et al., 2001; Miller et al., 2005; Rosser et al., 2007; Sahara et al., 2005). The plant proteins CMPG1 and ACRE276 are implicated in defense against pathogen attack (Gonzalez-Lamothe et al., 2006; Kirsch et al., 2001; Yang et al., 2006). SPL11 is related to cell death and other cellular processes in rice, such as flowering time control (Zeng et al., 2004) (L. Zeng and GL Wang, unpublished data). PHOR1 (photoperiod-responsive 1) from potato is a general component of the gibberellins signaling pathway (Amador et al., 2001). The protein ARC1 (arm repeat containing 1) was shown interacting with the S-locus kinase and is involved in self-incompatibility of Brassica (Gu et al., 1998; Stone et al., 2003).
The completion of rice genome sequencing allows us to study the function of the rice U-box proteins at the genome scale (Goff et al., 2002; Yu et al., 2002). By using a battery of extensive whole genome analysis algorithms, we identify 77 genes encoding U-box domain-containing proteins from the rice genome in this study. Expressed sequence tags (ESTs) or full-length cDNAs are found for most of the identified U-box proteins. These proteins are classified into eight major groups, based on their domain organizations. Phylogenetic analysis establishes the orthologies and paralogies of rice and Arabidopsis U-box proteins, which will help functionally characterize plant U-box protein genes in the future. In-vitro ubiquitination assay indicates that five randomly picked rice U-box proteins display E3 ligase activity in an E2-dependent manner. Interestingly, silencing one of the U-box genes causes cell death in rice protoplasts, suggesting that it might be involved in rice cell death control or defense responses.
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RESULTS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Identification of U-Box-Containing Proteins from the Rice Genome
To have a comprehensive assessment of the rice U-box proteins, genome sequences of two rice subspecies were explored in our survey (Goff et al., 2002; Yu et al., 2002). To have the latest genome information, rice genome pseudomolecules release five sequences from The Institute of Genome Research (TIGR; the same database is now moved to Michigan State University) were used in the identification of U-box genes in japonica subspecies (http://rice.plantbiology.msu.edu). U-box proteins from the two annotated genomes were identified with high confidence using the same battery of algorithms (see Methods). The two sets of identified U-box proteins were then compared and combined. Feng et al. (2002) and Han and Xue (2003) reported that there is, on average, approximately 1% polymorphism in amino acid sequence of the same protein encoded by the japonica and indica genomes. However, proteins from the same loci of the japonica and indica genome may have lower protein sequence identity due to the outcome of different annotation procedures. Therefore we considered U-box proteins from the two genomes that show at least 95% protein sequence identity are likely encoded by the same loci and the corresponding genomic DNA sequences of these proteins were then identified and compared manually. The putative U-box proteins identified in the indica genome (see Supplemental Doc. 1) were eliminated from the combined list after their counterparts are confirmed to exist in the annotated japonica genome. Such analyses indicate that all U-box proteins identified from the indica genome have their corresponding counterparts in the japonica genome. Seventy-seven rice U-box proteins were identified after such extensive comparisons (Table 1, Supplemental Figure 1, and Supplemental Doc. 2). Using the same algorithms, we identified 61 U-box proteins from the Arabidopsis genome. Among them, sixty are common to those identified by other groups (http://www.arabidopsis.org/info/genefamily/pub.html), corroborating the efficacy of the algorithms we used in the survey (see Supplemental Table 1 and Supplemental Doc. 3).
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Domain Organization of the Rice U-Box Proteins
The amino acid sequences of rice U-box proteins were used to search against the PFAM database (Bateman et al., 2004) and the National Center for Biotechnology Information (NCBI) protein database to identify other domains and motifs. Besides the U-box, there are various other protein domains/motifs present in these proteins (Table 2). The rice U-box proteins are grouped into eight classes, based on the presence of common motifs/domains other than the U-box. The numbering of these classes is mandated so that the previously described classes in Arabidopsis are incorporated (Azevedo et al., 2001). It is noteworthy that, in addition to U-box and the domain used for classifying, proteins in the same class could also possess other domains. For example, the U-box/ARM-type protein SPL11 was shown containing a coiled-coil motif in the central region of the protein (Zeng et al., 2004). In general, E3 ubiquitin ligases have a protein–protein interaction domain to interact with their substrates for ubiquitination (Patterson, 2002). Consistently, most rice U-box proteins contain domains or motifs that were implicated in protein–protein interactions.
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Similarly to Arabidopsis, class I consists of one rice gene, OsUFD2. OsUFD2 is highly homologous to AtUFD2 (Azevedo et al., 2001), with 62% identity at amino acid level (data not shown). Previous study indicated that yeast UFD2 interacted with CDC48, a member of AAA-type ATPase family that is implicated in multiple cellular processes (Koegl et al., 1999). The region of yeast UFD2 that is proposed to interact with CDC48 and its human ortholog p97 is conserved in OsUFD2.
The largest class of rice U-box proteins contains the armadillo (ARM)/HEAT repeats. The ARM repeat is an approximately 40-amino-acid-long tandemly repeated sequence motif first identified in the Drosophila melanogaster segment polarity gene armadillo (Riggleman et al., 1989). It was shown to be involved in protein–protein interactions (Huber and Weis, 2001). HEAT repeats derive their name from four diverse eukaryotic proteins in which they were first identified: huntingtin, elongation factor 3, PR65/A subunit of protein phosphatase A, and the TOR (target of rapamycin) (Andrade and Bork, 1995). ARM and HEAT repeats are grouped into the same class in this study due to their structural similarity (Andrade et al., 2001). It is noteworthy that the ARM/HEAT repeats found in these proteins are quite divergent, similar to what was described previously (Mudgil et al., 2004).
The second largest class of U-box proteins in rice and Arabidopsis show no significant domain or motif hits other than U-box in the PFAM or NCBI databases. Nevertheless, sequence alignments do detect a conserved domain containing 100 amino acid residues that is located close to the C-terminus of these proteins (Figure 1). In addition to the high percentage of leucine residues described before (Azevedo et al., 2001), a high percentage of homology and several highly conserved residues were detected. We named this putative domain GKL domain after the conserved Glycine (G), Lysine (K)/Arginine (R) residues and its leucine-rich feature. Sequence alignments of the Arabidopsis proteins from the same class gave a highly similar pattern (see Supplemental Figure 2). NtCMPG1, the Nicotiana benthamiana homolog to the Arabidopsis U-box proteins AtPUB20 and AtPUB21 from this class, was shown to be essential for plant defense and disease resistance (Gonzalez-Lamothe et al., 2006). It remains unknown, however, if members of the corresponding rice class have similar functions.
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The third largest class of rice U-box-containing proteins has a kinase domain at the N-terminal region of the proteins. Given the involvement of phosphorylation modification in most, if not all, cellular processes, we speculate that members of this class of rice U-box proteins could play a broad range of roles in the cell.
In addition to the three classes mentioned above, there are another two classes that have more than one protein from each genome. These two classes contain tetratrico peptide repeat (TPR) motif and WD40 repeats domain, respectively. Both TPR and WD40 domains have been shown to be involved in protein–protein interactions (Das et al., 1998; Li and Roberts, 2001). The rice and Arabidopsis U-box proteins that contain the WD40 repeats are homologous to the animal and human Prp19p protein, which has been shown to be involved in pre-mRNA splicing and other biological processes (Loscher et al., 2005; Mahajan and Mitchell, 2003; Ohi and Gould, 2002; Voglauer et al., 2006).
Difference of Rice and Arabidopsis U-Box Proteins in Domain Contents
The domain contents of Arabidopsis U-box proteins were also investigated using the same method as described above (Table 2). One significant difference between rice and Arabidopsis U-box proteins is that there is only one TPR-U-box-type protein in Arabidopsis, while six are identified in the rice genome. The TPR is a motif present in a wide range of proteins (Lamb et al., 1995). It mediates protein–protein interactions and the assembly of multi-protein complexes (D'Andrea and Regan, 2003). The only Arabidopsis TPR-U-box protein is homologous to the mammalian carboxyl terminus of Hsc70-interacting protein (CHIP) (AtCHIP) and it is involved in abiotic stress responses and in the control of chloroplast protein turnover (Luo et al., 2006; Shen et al., 2007a, 2007b; Yan et al., 2003). In human and animals, CHIP interacts with the protein chaperones such as Hsp70 and Hsp90 and acts as chaperone or co-chaperone in the cell to ensure protein homeostasis. CHIP has been implicated in stress responses and several neurodegenerative diseases (Ballinger et al., 1999; Dickey et al., 2007; Imai et al., 2002; Jiang et al., 2001; Miller et al., 2005; Rosser et al., 2007; Sahara et al., 2005). Nevertheless, no study regarding the biological functions of rice TPR-U-box-type protein has been reported. Interestingly, we identified an expressed protein (OsPUB70) that contains both TPR and kinase domains in addition to the U-box domain. It will be intriguing to find out whether this protein is a newly evolved protein that acquired either the TPR or kinase domain relatively recently.
Another difference of domain contents in rice and Arabidopsis U-box proteins is that two MIF4G-type U-box proteins exist in Arabidopsis but the same type of protein is absent in rice. The MIF4G domain is named after the middle domain of eukaryotic initiation factor 4G (eIF4G) and it is implicated in RNA metabolic processes (Ponting, 2000). The absence of such a protein in the rice genome suggests that the arising of the corresponding genes in Arabidopsis occurred after speciation of the two species.
Relationship between Rice U-Box/ARM Proteins
The U-box/ARM-type proteins constitute the largest family of U-box-containing proteins in both rice and Arabidopsis genomes. Moreover, most plant U-box-containing proteins that have been functionally characterized belong to this class (Amador et al., 2001; Gu et al., 1998; Stone et al., 2003; Yang et al., 2006; Zeng et al., 2004). This prompted us to further study the relationship of members in this class. Phylogenetic analysis using full-length protein sequences indicates that rice U-box/ARM proteins largely fall into four clusters in the phylogenetic tree. Consistently, members that are evolutionally closer in the phylogenetic map (Figure 2A) also show higher similarity in their number and arrangement of ARM repeats (Figure 2B). Unlike clusters I, II, and III, in which the ARM repeats in most members occur successively, most of the ARM repeats of cluster IV members are distributed discretely. The phylogeny of rice U-box/ARM proteins are slightly different when only the U-box sequences of each protein are used to construct the phylogenetic tree (data not shown), suggesting that the U-box domain might have evolved differently from other parts of those proteins. Similar to what have been observed in Arabidopsis (Mudgil et al., 2004), the domain organizations (Figure 2B) indicate that the ARM repeats of rice Ubox/ARM proteins are divergent in both number and arrangement.
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Relationship between Rice and Arabidopsis U-Box/ARM Proteins
The identification of the corresponding set of U-box-containing proteins from both rice and Arabidopsis genomes allowed us to evaluate the evolutionary relationships within the U-box gene families of both species. Therefore, we performed a combined phylogenetic analysis of both rice and Arabidopsis U-box/ARM proteins to obtain a joint tree. As shown in Figure 3, most rice U-box/ARM proteins have their corresponding putative orthologs in Arabidopsis. Moreover, most rice U-box/ARM proteins show closer phylogenetic distance to their putative rice homologs than to their corresponding putative Arabidopsis orthologs. Nevertheless, rice proteins OsPUB4, OsPUB12, OsPUB29, and OsPUB30 show closer phylogenetic relationship to the Arabidopsis proteins than to their rice paralogs, suggesting that these rice proteins and their corresponding Arabidopsis orthologs have evolved from a common ancestor before the speciation of the two species.
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In-Silico Analysis of Rice and Arabidopsis U-Box Protein Gene Expressions
Expression of the identified U-box protein genes was investigated by searching the rice cDNA (http://cdna01.dna.affrc.go.jp/cDNA/), EST (http://compbio.dfci.harvard.edu/tgi/ and www.ncbi.nlm.nih.gov/), and massively parallel signature sequencing (MPSS) (http://mpss.udel.edu/rice/) databases. Evidence for expression was found for the majority of rice U-box protein genes (Table 1). Corresponding ESTs or full-length cDNAs or both could be identified for 61 out of the 77 rice U-box genes, amongst which 55 have full-length cDNAs (Table 1). Based on the origin of the ESTs, the rice U-box protein genes were expressed in a variety of tissues, such as root, leaf, flower, calli, etc., suggesting that they are likely involved in a broad range of biological functions.
To have a comprehensive idea on the expression pattern of rice U-box genes, we used the same method as described previously (Nobuta et al., 2007) to analyze their MPSS tags in 61 MPSS libraries that were prepared with different rice tissues or rice leaves challenged by various abiotic or biotic stresses (see Supplemental Table 2). Of the 77 rice U-box genes, 64 of them showed significant expression in one or more libraries and only four of the rice U-box genes do not have corresponding MPSS tags presented in the libraries (see Supplemental Table 3). Some U-box genes only expressed at low level under specific conditions. For example, OsPUB58 has low abundance of transcript only at salt-challenged young root and OsPUB65 only express in calli and leaves challenged by incompatible fungal pathogen (see Supplemental Table 3). This may explain why no EST was identified for these genes.
By utilizing the collection of microarray data available on the Genevestigator database (Zimmermann et al., 2004), we also investigate the expression of 52 Arabidopsis U-box genes on the Affymetrix array chips under various experimental conditions (see Supplemental Table 4). Interestingly, lots of the Arabidopsis U-box genes have over two-fold change in their expression when the plant is challenged by various abiotic (such as chitin treatment or salt stress) or biotic stresses (such as Pseudomonas syringae infection).
Role of Rice U-Box Gene in Disease Resistance and Cell-Death Signaling
To date, a few plant U-box proteins have been implicated in defense against pathogen attack (Gonzalez-Lamothe et al., 2006; Kirsch et al., 2001; Yang et al., 2006; Zeng et al., 2004). To assess the expression of rice U-box genes during pathogen infection, we chose nine rice U-box genes that showed differential expression in our microarray hybridizations (C.H. Park and G.L. Wang, unpublished) and examined their expression in rice leaf tissue challenged by the rice fungal pathogen Magnaporthe oryzae. As shown in Figure 4, seven of them showed differential expression patterns after inoculation with the fungal isolate PO6-6. Specifically, OsPUB4, 12, and 23 had a similar expression in both resistant and susceptible plants. OsPUB 51, 64, and 73 showed stronger expression in the susceptible plants. Nevertheless, OsPUB57 showed a stronger expression only in the resistant plants carrying the Pi9-resistant gene (Qu et al., 2006). These results suggest some of the genes might play a role in the signaling or regulation of rice defense against M. grisea.
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To further confirm their function in defense responses, a rice protoplast system was used (Chen et al., 2006). The rice protoplasts were transiently co-transformed with a U-box gene-silencing construct and a beta-glucuronidase (GUS) gene expression construct. The strength of GUS activity was monitored as an indicator of viability of the protoplasts. Among the seven genes tested, OsPUB57 causes cell death when it is knocked down in the cell, which suggests that it might act as a negative regulator in rice cell death signaling (Figure 5).
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E3 Ubiquitin Ligase Activities of Rice U-Box Proteins
The U-box-containing proteins are predicted to possess E3 ubiquitin ligase activity. To determine if rice U-box proteins do encode functional E3 ligases, in-vitro ubiquitination assays were performed. Five rice U-box proteins (OsPUB4, OsPUB12, OsPUB57, OsPUB73, and OsPUB77) were randomly picked for test. These proteins were expressed with GST-tags in Escherichia coli and tested for E3 ligase activity using an in-vitro ubiquitination assay as described (Zeng et al., 2004). The self-ubiquitination of the U-box proteins presented in the reaction, as shown by a ladder of higher molecular bands, was used as an indicator of E3 liagse activity (Hatakeyama et al., 2001).
As shown in Figure 6, the five rice U-box proteins were found to possess E3 ligase activity. Importantly, like the observation with mammalian U-box proteins (Hatakeyama et al., 2001), the ligase activity of the rice U-box proteins also depends on the E2 enzyme used for the assay. The preference for a particular E2 enzyme did not show any correlation with the presence of a particular domain in the rice U-box proteins. In the control experiment, with the omission of either E1, E2, or ubiquitin, no ubiquitination was observed (data not shown). Therefore, the ubiquitination observed was due to the E2-dependent E3 ligase activity of the rice U-box proteins.
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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The distribution of U-box proteins among species of different kingdoms is uneven. There are a larger number of genes encoding such proteins in the plant genomes. Three U-box genes were identified among the 6300 annotated genes in the yeast genome. Similarly, fewer than 20 U-box proteins were predicted in the human genome (Patterson, 2002). Only two putative U-box proteins were identified in the rice fungal pathogen Magnaporthe oryzae’s genome (L. Zeng, unpublished). In contrast, 61 U-box proteins were identified in Arabidopsis when a series of analyses were performed (Azevedo et al., 2001) (www.Arabidopsis.org/info/genefamily/pub.html). The U-box domain is structurally analogous to the RING domain. The large number of RING-type proteins constitutes a super-family in plants (Stone et al., 2005). The high similarity between U-box and other sub-family members of RING-type proteins makes it a daunting challenge to correctly identify U-box sequences from the RING-type proteins pool. In this study, 77 U-box-containing proteins were identified from the annotated rice genome by a six-step prodedure that involves a series of algorithms. Of the 77 U-box protein genes, either cDNA or EST were detected for 61 of them and only four of them do not have matching MPSS tags, corroborating the effectiveness of the algorithms used in the survey of this study. Sixty-one U-box proteins were identified from Arabidopsis when data from this study and other resources (www.Arabidopsis.org/info/genefamily/pub.html) were combined. The identification of two nearly complete sets of U-box-containing proteins from the genomes of the model monocot and dicot plants provided the basis for studying the phylogeny of U-box proteins in plants.
Sequence alignment indicates that there exists a class of U-box proteins with highly conserved sequences at their C-termini in both rice and Arabidopsis genomes (Figure 1 and supplementary Figure 2). We named this region as GKL domain, based on the presence of highly conserved Glycine, Lysine/Arginine residues and leucine-rich features. To date, however, no biological function has been associated with such a domain. It is generally considered that the U-box domain serves as the E2 recruitment domain, while another distinct domain in U-box proteins, such as ARM repeats or TPR domain, serve to recognize and recruit the substrate. However, no other known domain/motif was identified in these proteins. Therefore, we propose that the GKL domain might be involved in protein–protein interaction. Recently, one tobacco homolog of U-box/GKL-type protein, NtCMPG1, was implicated in plant immunity (Gonzalez-Lamothe et al., 2006). Identification of the substrate for NtCMPG1 and testing whether mutation in the GKL domain will compromise the interaction of NtCMPG1 to the substrate will help to verify our hypothesis.
The ARM repeat was first identified in the Drosophila melanogaster segment polarity gene armadillo that is involved in signal transduction (Riggleman et al., 1989). Structural characteristics of the ARM motif suggest its involvement in protein–protein interactions, which has been demonstrated in several cases (Huber et al., 1997). In a few cases, HEAT repeats were detected in proximity to the ARM repeats. Phylogenetic analysis indicated most rice U-box proteins are evolutionally closer to their homologs from the rice genome than those from the Arabidopsis genome (Figure 3). The establishment of such a relationship between rice and Arabidopsis U-box proteins could help in characterizing these genes. For example, the identification of the putative Arabidopsis ortholog of rice Spl11 gene (AtSpl11) based on the combined phylogenetic tree led us to the identification of a loss-of-function mutant of AtSpl11 that displayed similar cell death and altered flowering phenotype to the rice spl11 mutant (L. Zeng and G.L. Wang, unpublished). Given the advantage of Arabidopsis over rice in terms of plant growth and genetic resources, we expect that an in-depth characterization of the Atsp11 mutant will help to elucidate the connection of immunity and reproductive development in rice. Similarly, it would be interesting to investigate whether the putative rice ortholog of AtPUB17 (ACRE276), OsPUB4, has similar functions in rice cell death and defense (Yang et al., 2006).
Why plants require more U-box proteins is unclear at present. One speculation, given the lack of animal-like immune system and the static life of plants, is that U-box proteins may play a role in plants’ responding to various environmental stresses (Patterson, 2002). Domain organization analysis indicates that the U-box family could be divided into at least nine classes. A variety of other motifs/domains, such as protein–protein interaction domains, protein phosphorylation-related domain, and transcription initiation-related domain, were found to be associated with the U-box domain in rice and Arabidopsis U-box proteins (Table 2). U-box proteins with similar domain organization to other organisms have been demonstrated to function in different cellular processes (Amador et al., 2001; Kirsch et al., 2001; Stone et al., 2003). These data suggest that the biological functions of U-box proteins might be diverse rather than limited to stress response signaling. Of the rice and Arabidopsis U-box proteins identified hereby, only a few of them have been functionally characterized. In addition, the existence of multiple members in each class of U-box proteins raised the possibility of function redundancy among the members. Such function redundancy could make the study of biological roles for plant U-box genes a daunting challenge. Among the seven rice U-box genes analyzed for their role in cell death signaling, only one was found to cause cell death when it is silenced in rice cells (Figure 5). The failure of the other six genes to display cell death phenotype could be due to such function redundancy as well. Therefore, a systemic approach, using knockout mutants or transgenic RNAi lines, to modify the expression of most or all members in the same class might be essential to functionally characterize these genes on a large scale.
U-box-containing proteins are predicted to possess E3 ubiquitin ligase activity. In Arabidopsis, several U-box proteins were revealed to possess E3 ligase activity (Mudgil et al., 2004; Yan et al., 2003; Yang et al., 2006). Of the 77 rice U-box proteins, only SPL11 has been demonstrated to possess E3 activity in previous studies. In this study, we tested five randomly chosen rice U-box proteins for their E3 ligase activity in vitro. Such ubiquitination assay indicates that all the five proteins have E3 ligase activity and they demonstrate different preferences for E2 enzymes in these assays (Figure 6). Unlike the E3s in the HECT family, RING finger and U-box E3s facilitate the transfer of ubiquitin molecule by precise spatial orientation of the E2 and the substrate (Wu et al., 2003). The U-box or RING-finger domain serves as the E2 recruitment domain, while another distinct domain, such as ARM repeats or TPR domain, serve to recognize and recruit the substrate. The crystal structure of the U-box protein Prp19 indicated that, for those U-box E3s that oligmerize to function, the U-box and the substrate-recruiting domain are arranged in such an architecture that it brings the E2 recruitment domain into close proximity with a substrate recognition domain. Therefore, such E2 preference may reflect the minor difference in the U-box domains of these proteins. Nevertheless, such preference could make it difficult to test their in-vitro E3 ligase activity for some U-box proteins due to the availability of the best E2s they required for the assay.
Although the majority of the genes that encode the 77 identified rice U-box proteins are expressed, only the function of Spl11 has been reported (Zeng et al., 2006). The diverse motifs/domains presented in the rice U-box proteins support the notion that the rice U-box family may target a myriad of substrates. Biochemical assay of the ligase activity of the U-box proteins, identification of their substrates, and elucidation of the mechanisms under which they work in the cell will be the next study in order to fully understand the biological roles of these genes.
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METHODS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Identification of U-Box-Containing Proteins in the Rice Genome
The U-box proteins in the genomes of rice japonica and indica subspecies were identified using a battery of algorithms. The complete results are available as supplementary materials at the following address: http://supfam.org/ubox/ubox.html. Taxonomically, we place the U-box family as a member of the RING domain superfamily. The U-box family shows some distant sequence homology to other member families under the RING domain superfamily. To identify them correctly, the U-box proteins must be distinguished from other members in the same superfamily. A crucial difference between the U-box proteins and other superfamily members is the presence of fewer cysteine/histidine residues. The following seven procedures were used in the identification process: (1) WU-BLAST of known U-box proteins against the genome, (2) searching PFAM models of non-U-box families within the RING domain superfamily (Bateman et al., 2004), (3) scoring a SAM hidden Markov model (HMM) built from the PFAM seed alignment of U-box domains (Sonnhammer et al., 1998), (4) a SAM T99 ‘family’-level model was built for U-box proteins (Karplus and Hu, 2001), (5) scoring an HMM built from the PFAM ‘full’ alignment, (6) a SAM T99 ‘superfamily’-level model was built and scored. The final results of the above seven sequential analyses were further examined with respect to the number of aligned cysteine residues and multiple sequence alignments annotated with the structural key residues highlighting the differences between U-box family members and other RING domain superfamily members. Additional evidence such as structural information was consulted in some specific cases.
Phylogenetic Analysis
Rice and Arabidopsis U-box proteins were used as queries to search against the PFAM or GenBank database and were classified into different classes based on their domain contents. Rice and Arabidopsis proteins from the ARM class were then aligned using the clustal_X program (Thompson et al., 1997). The aligned sequence data were then input into the MEGA4 program (Kumar et al., 2001; Tamura et al., 2007) and a neighbor-joining algorithm was used to build the phylogenetic tree.
Preparation of MPSS Libraries and Data Analysis
The growth of rice plants, collection of rice tissues, preparation of MPSS libraries, and data mining are as described (Nobuta et al., 2007).
Plant Materials and Rice Blast Inoculation
Rice seeds were first sterilized by treatment in 75% ethanol for 1 min, followed by immersion in 2% sodium hypochlorite for 15–20 min. After several washes with sterile distilled water, seeds were germinated in half-MS medium for 7–10 d, and then transferred to soil. Growth chamber conditions were 12 h light, 26°C, 80% relative humidity (RH), followed by 12 h dark, 20°C, 60% RH, unless otherwise specified. Three-week-old plants of rice japonica variety TP309 and its isogenic transgenic lines containing the Pi9 resistance gene were inoculated with Magnaporthe oryzae isolate PO6-6 at spore suspension of rice blast at 1 x 105 spores ml–1. The inoculated plants were placed in a the dark for 24 h at 26°C, and leaf tissues were collected every 24 h after inoculation until 96 h.
Cell Death Analysis of the Candidate Genes in Rice Protoplasts
RNAi constructs containing the candidate gene fragments and the construct containing the beta-glucuronidase (GUS) gene under the control of maize ubiquitin promoter were transiently expressed in rice protoplasts as described (Chen et al., 2006). The assay of GUS activity was carried out according to the protocol described by Chen et al. (2006).
In-Vitro Ubiquination Assay
The in-vitro ubiquitination assays were performed as described (Zeng et al., 2004) with some modifications. In brief, full-length OsPUB proteins were expressed as N-terminal GST-tagged in BL21 (DE3) cells (Stratagene) and affinity-purified with glutathione matrix (Sigma, USA). The in-vitro ubiquitination reactions were performed by adding 1 µg of protein of interest, 40 ng of wheat E1, 100 ng of Arabidopsis E2, 10 µg of ubiqutin and 1.5 µL of 20X reaction buffer (1 M Tris HCl, pH 7.5, 40 mM ATP, 100 mM MgCl2, 40 mM DTT, 600 mM creatine phosephate and 1 mg ml–1 creatine phosphokinase). The reaction was incubated at 30°C for 1.5 h in 30 µl volume before being stopped with SDS sample loading buffer and heated to 100°C for 5 min. Samples of the reactions were then separated by 10% SDS–PAGE. Ubiquitinated substrates were detected by Western blotting and detected by ubiquitin antibody (Biomol, USA) followed by detection by chemiluminescence with the ECL kit.
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Supplementary Data are available at Molecular Plant Online.
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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This project is supported by the National Research Initiative (NRI) Plant Genome Program of the USDA Cooperative State Research, Education and Extension Service (CSREES) to G.L.W. (#: 2004–35604–14226 and 2007–35301–18093). No conflict of interest declared.
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2 These authors contributed equally to the project.
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