The U-Box E3 Ligase SPL11/PUB13 Is a Convergence Point of Defense and Flowering Signaling in Plants

The ubiquitin-proteasome system (UPS) is involved in the selective degradation of proteins in the cells of eukaryotic organisms and consists of three main enzymes: ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). In a typical ubiquitination reaction, E1, E2, and E3 enzymes participate in the transfer of the ubiquitin monomers to the ε-amino group of a Lys residue in the target protein. In the first step of the reaction, E1 catalyzes the activation of ubiquitin via ATP, creating a high-energy thiol intermediate. After activation, ubiquitin is transferred to the E2 and then to the E3 ligase for the final step in the process, which involves the creation of an isopeptide bond between the C-terminal Gly of ubiquitin and a Lys residue in the substrate protein (for review, see Vierstra, 2009; Ye and Rape, 2009). The UPS in plants has a hierarchical structure for the three enzymes: there are few E1s, about 50 E2s, but over 1,000 E3s. For example, the rice (Oryza sativa) genome encodes six E1 genes, 49 E2 and E2-like genes, and over 1,300 E3 genes (Du et al., 2009). The abundance of the E3 proteins in the UPS system allows plants to target substrates for many biological processes, because each E3 ubiquitin ligase acts for the ubiquitination of only one or a few target proteins. Based on the subunit component and action modes, E3 ligases can be divided into two major types: the single-subunit type (Homology to E6 carboxyl terminus [HECT] and RING/U-box) and the multisubunit type (Skp1 [for S-phase kinase-associated protein1]-CUL1 [for Cullin1]-F-box [SCF], Anaphase promoting complex [APC], CUL3-BTB [for Bric-a-Brac, tramtrack broad complex], CUL4-DDB [for damage-specific DNA binding protein] complex, and others; Vierstra, 2009; Fig. 1). Two types of E3 ligases, their interaction components, and their modes of action. Depending on the number (single or multiple) of ubiquitins attached to the substrates and on the manner in which multiple ubiquitins are attached to the substrates, the ubiquitin-mediated protein modifications can be classified as monoubiquitination, multiubiquitination, and polyubiquitination (for review, see Hochstrasser, 2006; Mukhopadhyay and Riezman, 2007; Vierstra, 2009). Monoubiquitination and multiubiquitination usually alter substrate protein localizations, affect protein-protein interactions, and modulate protein activities. The polyubiquitination can differ in structure and function depending on how the seven Lys residues in ubiquitin (Lys-6, Lys-11, Lys-27, Lys-29, Lys-33, Lys-48, and Lys-63) are connected to each other and to the substrates. For example, Lys-48-mediated and Lys-11-mediated polyubiquitination target substrates for degradation by the 26S proteasome, while Lys-63-mediated polyubiquitination usually mediates DNA repair, membrane trafficking, and chromatin remodeling (for review, see Ye and Rape, 2009; Liu and Chen, 2011). The function of polyubiquitination mediated by the other four Lys residues is not clear. The UPS functions in nearly all aspects of plant life, including the cell cycle, embryogenesis, photomorphogenesis/flowering, hormone signaling, and abiotic and biotic stress responses (for review, see Stone and Callis, 2007; Vierstra, 2009; Santner and Estelle, 2010). In this paper, we focus on the function of the rice U-box protein SPOTTED LEAF11 (SPL11) and its Arabidopsis (Arabidopsis thaliana) ortholog protein PLANT U-BOX13 (PUB13) in programmed cell death (PCD), defense, and flowering (Table I). During the course of coevolution, plants have developed two layers of innate immune systems that rely on the pattern-recognition receptors (PRRs) and resistance (R) proteins to defend against pathogen attack (for review, see Jones and Dangl, 2006; Dodds and Rathjen, 2010). The first line of defense depends on the activation of PRRs by the perception of pathogen-associated molecular patterns (PAMPs). The PAMP-triggered immunity (PTI) causes the accumulation of reactive oxygen species (ROS) and the deposition of phenolic compounds. To suppress PTI, pathogens have evolved effector proteins that can be secreted into the cytoplasm of host cells. In response, plants employ R proteins, such as nucleotide-binding site leucine-rich repeat (LRR) proteins (or NLRs), to monitor the entry of these effector proteins directly or indirectly, resulting in the second layer of defense, which is called effector-triggered immunity (ETI). ETI is more robust and effective than PTI and is often associated with a hypersensitive response (HR), which is characterized by the rapid death of cells in the local region surrounding an infection. Several recent studies, however, have challenged the distinction between PTI and ETI and have provided evidence for a continuum between the two types of immunities (Lee et al., 2009; Thomma et al., 2011). Lesion-mimic (LM) mutants displaying spontaneous HR-like cell death under normal growth conditions represent a unique kind of PCD in plants. Many LM mutants have been identified and characterized in maize (Zea mays), Arabidopsis, rice, barley (Hordeum vulgare), and tobacco (Nicotiana tabacum). One of the common characteristics of numerous LM mutants is that they exhibit enhanced resistance to biotrophic or hemibiotrophic pathogens. Cloning and molecular characterization have revealed that the mutated genes encode various proteins involved in defense-related signaling pathways, including ion channels, ROS generation, sphingolipid metabolism, porphyrin/phenolics/chorophyll biosynthesis and metabolism, ubiquitination, and other signaling transduction (for review, see Love et al., 2008; Moeder and Yoshioka, 2008). An intimate relationship between LM mutation and ETI was indicated by the genetic analysis of the Arabidopsis LM mutant lesion simulating disease1 (lsd1). This analysis revealed that the phenotypes of the mutant were nullified by mutation in EHANCED DISEASE SUSCEPTIBILITY1 and PHYTOALEXIN DEFICIENT4 (PAD4), two important genes required for the Toll-IL-1 receptor (TIR)-type NLR R gene-mediated resistance and also for basal resistance (Rustérucci et al., 2001; Wiermer et al., 2005). Both genes are also essential for cell death regulation in Arabidopsis under intense light and for the ROS- and salicylic acid (SA)-dependent defense signal amplification loop (Glazebrook, 2005; Mühlenbock et al., 2008). Recently, a suppressor screening of the lsd1 mutant revealed that PHOENIX21 is a positive regulator of lsd1 runaway cell death and is a member of the ACTIVATED DISEASE RESISTANCE1 family of the coiled coil (CC)-type NLR proteins (Bonardi et al., 2011). Thus, these results suggested that the HR and cell death in some LM mutants share common signaling pathways. The role of UPS in regulating apoptosis in animals has been well established (for review, see Broemer and Meier, 2009). Recently, the role of UPS in plant PCD and defense responses has become clearer based on many studies with dicot plants. One of the first studies reported that Arabidopsis SGT1, a homolog of yeast SUPPRESSOR OF G2 ALLELE OF SKP1 (SGT1), interacts with SUPPRESSOR OF KINETOCHORE PROTEIN1 (SKP1) and CULLIN1 (CUL1), subunits of the Skp1-Cullin-F-box (SCF) E3 ligase, and is required for defense signaling mediated by multiple NLR-type R genes (Kitagawa et al., 1999; Austin et al., 2002; Azevedo et al., 2002; Tör et al., 2002; Moon et al., 2004). In tobacco, a set of E3 ligase genes induced by the fungal avirulence protein Avr9 are required for the HR of Cf9-mediated resistance (González-Lamothe et al., 2006; Yang et al., 2006; van den Burg et al., 2008). In Arabidopsis, two RING finger E3 ligases, RPM1-INTERACTING PROTEIN2 (RIN2) and RIN3, have also been implicated in the HR of both CC-type NLR R proteins RPM1- and RPS2-mediated resistance (Kawasaki et al., 2005), whereas a PUB E3 ligase, PUB17, is required for CC-type NLR R protein RPM1- and TIR-type NLR R protein RPS4-mediated resistance (Yang et al., 2006), indicating the critical role of UPS in ETI. Similarly, a homologous triplet of PUBs (PUB22, PUB23, and PUB24) and PUB12/13 in Arabidopsis have been demonstrated to negatively regulate PTI (Trujillo et al., 2008; Li et al., 2012a), suggesting the involvement of the UPS in plant PTI signaling. The role of UPS in the regulation of PCD and defense of the monocot rice is also becoming clearer. Our laboratory’s identification and characterization of SPL11 in rice as a U-box protein with E3 ligase activity provided, to our knowledge, the first direct evidence that ubiquitination controls resistance and PCD in rice (Zeng et al., 2004). In addition, a RING finger E3 ligase, BLAST AND BTH-INDUCED1, was found to positively regulate resistance against Magnaporthe oryzae by modifying the rice cell wall (Li et al., 2011). Another interesting protein is the RING finger E3 ligase XA21-BINDING PROTEIN3, which is required for the accumulation of the rice bacterial blight R protein XA21 and XA21-mediated defense signaling against Xanthomonas oryzae pv oryzae (Wang et al., 2006). Surprisingly, XA21 was identified to be a PRR that binds a PAMP-like type I-secreted sulfated peptide, AxYS22, derived from the Ax21 protein (Lee et al., 2009). Further characterization of these PCD- and defense response-related UPS components will shed light on the molecular mechanisms underlying HR cell death through the UPS in rice. Flowering is a well-defined developmental process that is controlled by environmental cues and intrinsic biological rhythms (for review, see Amasino, 2010). Extensive investigations in Arabidopsis have identified four major pathways that perceive and process different signals. The autonomous and GA pathways perceive and transduce internal signals to promote flowering. The external stimuli of variations in daylength and temperature mediate signal transduction in the photoperiodic and vernalization pathways, respectively. Ultimately, signaling in all pathways converges at the floral pathway integrators, a group of genes that are turned on or off in a manner that is consistent with the decision to flower. Among these integrators, the best characterized are FLOWERING LOCUS T (FT; Kardailsky et al., 1999) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1; Borner et al., 2000; Lee et al., 2000; Moon et al., 2003). Both FT and SOC1 are activated by the photoperiodic protein CONSTANS (CO) and are repressed by FLOWERING LOCUS C (FLC), a negative regulator of the autonomous and vernalization pathways (for review, see Lee and Lee, 2010). In recent years, the UPS has been found to contribute to the regulation of flowering time mainly by regulating the accumulation and stability of CO, GIGANTEA (GI), and FLC. The evening stability and morning instability of CO are affected by the blue light receptor CRYPTOCHROME2 and the light receptor PHYTOCHROME B, respectively (Valverde et al., 2004; Liu et al., 2008). Furthermore, the stability of the CO protein is controlled by the 26S proteasome, because the degradation of CO in the morning and in the dark is inhibited by proteasome inhibitors (Valverde et al., 2004). Intriguingly, the photomorphogenesis-related RING finger protein CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) was identified as the E3 ligase responsible for the degradation of CO in the dark (Liu et al., 2008), while another RING finger E3 ligase, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1, was recently determined to interact synergistically with COP1 and to negatively regulate CO abundance, especially during the day (Lazaro et al., 2012). The stability of GI, a circadian-associated protein and promoter of CO, is also mediated by COP1, which acts with the clock-associated protein EARLY FLOWERING3 (ELF3) to regulate circadian function and photoperiodic flowering by degrading GI (Yu et al., 2008). GI stability is also mediated by COP1, which acts with the clock-associated protein ELF3 to regulate circadian function and photoperiodic flowering by degrading GI (Yu et al., 2008). In addition, two E2 ubiquitin-conjugating enzymes (UBC), AtUBC1 and AtUBC2, contribute to flowering time regulation by working with the E3 ligases HISTONE and to the protein of is required for the activation of in Arabidopsis et al., 2008; et al., 2009). these studies have revealed a role for the UPS in flowering time defense and flowering are two signaling pathways in plants. evidence that the two pathways are connected through the is an essential hormone for plant resistance (for review, see et al., and plant growth and et al., 2004; et al., and 2011). which encodes a E3 ligase in Arabidopsis, and signaling to regulate innate immunity (Lee et al., The mutant under conditions with Further indicated that flowering by a floral pathway and activity through to promote and 2008; et al., 2008). Recently, was also found to regulate both plant innate immunity and flowering time in Arabidopsis (Wang et al., 2011). Among its multiple resistance to biotrophic and cell death in the signaling mutant cell and to PTI (Wang et al., 2011). negatively flowering under conditions via the regulation of and FT (Wang et al., 2011). indicated that be one of the for plant defense and flowering signaling pathways. The rice LM mutant which was identified from an of is controlled by a et al., et al., resistance indicated that enhanced resistance to both oryzae pv oryzae and the the resistance is with a activation of defense-related genes, including genes genes involved in the of ROS and genes et al., of the between the and plants in three types of with different phenotypes revealed that the mutation causes in the rice (Zeng et al., 2006). genes are induced in the of and nearly are classified as or defense-related results suggested a between the resistance and the activation of defense signaling in the In addition, et that the ROS accumulation induced by is by the in suggesting that SPL11 is important for the regulation of ROS The was on (Zeng et al., and by a (Zeng et al., 2004). analysis revealed that encodes a U-box and repeat E3 ligase which is a member of the family (Zeng et al., 2008). The protein is in both the and An E3 ligase activity indicated that the U-box is essential for SPL11 E3 ligase activity (Zeng et al., 2004). To for other components in the rice signaling we the in SPL11 as the in a yeast and identified proteins et al., 2008). Among is a protein and interacts with SPL11 in Liu and the protein to the which to the activation of the of and signaling and In rice, a protein has been implicated in ROS signaling et al., 2010). the mutant is in the regulation of the activity of et al., 2006). also found that is induced in the and mutant plants and that is in the mutant Liu and results suggested that regulate ROS accumulation through will be interesting to see there is a direct interaction between the complex and the between the protein and the in regulating ROS and immunity signaling. In addition, six of lesion have been mutants or lesion in the and have been and The or of in is to the resistance and defense The is in the than in the indicating that a role in the regulation of cell death and defense in rice. Cloning of these genes will into the defense signaling Among the PUB genes in Arabidopsis (Zeng et al., 2008), is the ortholog of the rice U-box ligase has in with SPL11 and also a structure with E3 ligase recently characterized the and found that of the by results in spontaneous cell death and accumulation of and with the phenotypes of the mutant plants resistance to biotrophic or hemibiotrophic pathogens but to pathogens (Li et al., also found that cell death is in the mutant with the in under and another is with the cell death in the mutant is also under both results demonstrated that the in cell death and accumulation in depends on the In addition, the LM of is to the the of under and indicating that and are in regulating cell death and et recently reported that the PRR a is a target of the E3 ligases and via the induced by the bacterial is and by the is required for a PUB12/13 by the an important component in the plant hormone receptor signaling. results revealed a role for E3 ubiquitin ligase protein degradation in plant PTI signaling. such as and affect the lesion of LM mutants and 2009; et al., and 2011). In rice mutant LM is under conditions than under conditions phenotypes are enhanced in under conditions C and suggesting that lesion in both and depends on light or the circadian in cell and resistance to biotrophic and hemibiotrophic pathogens with (Li et al., Similarly, LM is the mutant is in a growth with and under in a in results suggested that both SPL11 and are involved in responses to multiple environmental of the and phenotypes of under of light and of and of light and of conditions in growth B, Flowering of rice and plants under conditions of light and of C and death in under conditions of light and of in a growth is the blue was to the cell death of and in Flowering of under conditions in a growth Both and were In to its function in cell death and defense SPL11 is also involved in regulating flowering time et al., 2008). The mutant a only under In the the flowering under the suggesting that SPL11 is involved in flowering time evidence for SPL11 involvement in flowering from the analysis of the which encodes a protein that is a member of the (for signal transduction and activation of SPL11 interacts with in yeast and in and but not et al., 2008). SPL11 negatively during the light under both and conditions et al., 2008). of causes flowering under both and conditions in the growth and flowering time via of which is daylength et al., 2008). results demonstrated that SPL11 controls flowering time through the of the flowering suppressor and that ubiquitination and are the interaction between SPL11 and in the regulation of flowering To the of in the regulation of flowering we a yeast as the and found another (for and and analysis of the and of revealed that also positively flowering time and Based on this and we that SPL11 into the to a protein complex with the proteins and and that the of these two proteins by SPL11 can alter the flowering signaling pathway in rice. recently found that is also involved in the regulation of flowering time in Arabidopsis (Li et al., the rice mutant which has a under conditions et al., 2008), the Arabidopsis mutant an under or conditions (Li et al., The functions of and SPL11 in flowering time are by in the of Arabidopsis and rice or by the in molecular mechanisms flowering in these two types of photoperiodic plants. For GI is a promoter of flowering time in Arabidopsis, while its ortholog in rice, is a suppressor of flowering time under both and conditions et al., 1999; et al., 2003). In addition, CO is a positive regulator of flowering time under or conditions in Arabidopsis, but its ortholog in rice, functions as a negative regulator of flowering time under conditions but as a positive regulator under conditions et al., Intriguingly, of in can the of (Li et al., suggesting that the function of these two E3 ligases, and in flowering time is and that the E3 ligase activity of the two homologous proteins be in dicot and monocot plants. many biological processes, such as and responses to environmental (for review, see and 2011). The hormone is not only in plant responses to biotic stress but is also involved in flowering (for review, see et al., 2004). controls flowering time through the and vernalization flowering pathways that are of CO, and The is in but the of in is by of the two important components and in the the in is to the (Li et al., results suggested that flowering time mainly through an The function of in flowering in is consistent with that the from the to the by the floral genes and the positive regulator of flowering et al., 2004). is that SPL11 flowering time through in rice et al., 2008). To a ortholog in Arabidopsis, we a yeast the mutant protein an E3 as the because we not the was (Li et al., One of the proteins identified in the is which encodes a et al., 2004). The interaction between and was by a (Li et al., and is and by the E3 ligase COP1 in a protein that is involved in plant and flowering time et al., 2008). to flowering time and the regulation is associated with the COP1 protein complex are In the years, we have on the function of SPL11 and in the regulation of defense, and flowering in monocot and dicot plants. Based on our results and from other studies, we have working to the function of SPL11 and and their proteins in the regulation of defense and flowering in both the biological functions of SPL11 and are the components in and signaling have become during the course of (Table I). In rice, a negative regulator of cell with the protein to suppress ROS and activation and to the of defense responses the activity of proteins. The protein is a signal of PTI and ETI signaling pathways (for review, see et al., 2010). Thus, we that to suppress the signaling and to cell death and defense activation pathogen is The SPL11 defense signaling be to protein and the complex by The SPL11 also negatively because mutation of the in the mutant In Arabidopsis, negatively the defense pathway and PTI signaling is also by a type regulator of the pathway (Wang et al., 2011). interacts with and is by The to with an that is required for the of by causes After is activated by the signal is to the to regulate ROS and and to cell death and defense of the function of SPL11 and in defense, some PRRs be by SPL11 in rice or some proteins from be by in interesting to be in working of cell death and defense signaling mediated by rice SPL11 and Arabidopsis in the working of flowering time regulation by rice SPL11 and Arabidopsis in the For flowering time regulation in rice, SPL11 and negatively its activity the of by SPL11 the pathway by and to flowering but of SPL11 the day in to flowering can also interact with to regulate flowering but how the interaction flowering In Arabidopsis, however, positively the flowering suppressor to flowering under is a negative regulator of the flowering FT and SOC1 also with a positive regulator of plant but its role in flowering Furthermore, flowering also the positively signaling and the flowering pathway under conditions (Wang et al., 2011). The relationship between and to be has been in the of the SPL11 and pathways, critical the E3 ligases SPL11 and regulate their substrates to modulate both defense and there an intimate between the complex and the PTI and ETI genes are by the complex in the regulation of flowering is the relationship between and other hormone signaling pathways in regulate flowering through and other associated with resistance proteins, as has recently been demonstrated for et al., to these will a of the functions of SPL11 and in rice and for of the ubiquitin-proteasome system programmed cell death pattern-recognition receptor pathogen-associated molecular PAMP-triggered immunity reactive oxygen species effector-triggered immunity leucine-rich repeat hypersensitive response salicylic acid coiled coil protein