1. UPS is a prerequisite for hormone-mediated plant growth and development
Coordinating external and internal cues with developmental physiology requires a rapid and mobile mechanism that can be highly regulated in space and time in every cell. The mobile nature of plant hormones and the mechanistic potency of the UPS fit this purpose. It is therefore not surprising that many of the signalling pathways associated with hormones rely on the UPS-mediated degradation of downstream targets to elicit their effect. Examples described later underline the intimate relationship between the UPS and plant growth and development mediated by phytohormones.
Mutants of components of the multisubunit E3 ligases show broad pleiotropic effects on development. Although the ASK component of SCF E3 ligases contain 21 family members, ask1/ask2 double mutants show severe developmental defects in all stages of plant development (Liu et al., 2004), with a similar effect seen in mutants of the RING component RBX1 (Gray et al., 2002). In addition, the nuclear localized CUL1 component of SCFs is essential for embryogenesis, while CUL3A CUL3B act redundantly in embryo pattern formation and endosperm development (Thomann et al., 2005).
Gibberellins comprise a family of diterpene hormones and are the prototypical growth promoting hormones in plants, regulating a large number of growth related responses (de Lucas et al., 2008). The response to GA is elicited through targeted degradation of DELLA growth repressors, mediated by GID1 which binds to the DELLA repressors in a GA-dependent manner resulting in a conformational change in the DELLA protein and degradation by the 26S proteasome via SCF(SLY) (Murase et al., 2008). Jasmonates (JA) play a role in regulation of development and defence, and notably in development by responding to light-coordinated daily growth cycles and shade-avoidance behaviours (Kazan & Manners, 2011). Jasmonate, in a similar manner to GA, leads to the degradation of the jasmonate ZIM-domain (JAZ) family of proteins. The F-box component COI1 (Xie et al., 1998) binds to a JA–amino acid conjugate (Thines et al., 2007) and once bound, the SCF(COI1) complex directs the degradation of the JAZ repressors. Auxin signalling follows a similar pattern with SCF(TIR1/AFB) (Gray et al., 2001; Dharmasiri et al., 2005b) degrading the transcriptional repressor AUX/IAA family of proteins in the presence of auxin, with the F-box again being the auxin receptor (Dharmasiri et al., 2005a; Fig. 5).
Figure 5. Involvement of Skp1-Cullin-F-box (SCF) E3 ligase in hormone perception. (a) Auxin binds to the F-box TIR1 forming an active complex that targets the transcriptional repressors Aux and IAA for degradation. (b) In an identical paradigm, jasmonate (JA) as an amino acid conjugate binds to COI1 leading to degradation of the JAZ family of transcriptional repressors. (c) Gibberellic acid (GA) binds to GID1 leading to a conformational change allowing binding of DELLA, this complex is then detected by SCFSLY which directs the DELLA protein for degradation, releasing downstream transcription factors from repression. (d) The ethylene pathway is dissimilar to the other three hormones in that ethylene protects the transcriptional regulators EIN3 and EIL1 from degradation by SCFEBF1/2 through degrading the EBF1 and EBF2 F-box proteins components by directing their degradation by an as of yet undiscovered E3 ligase.
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Ethylene signal transduction is also regulated by the ubiquitin proteasome system, but instead of the hormone leading to degradation of transcriptional repressors, ethylene leads to the stabilization of the positive transcriptional regulators EIN3 and EIL1 by inhibiting the activity of SCF(EBF1/2) (Gagne et al., 2004) by degrading the F-box components EBF1 and EBF2, although the E3 ligase(s) responsible have yet to be confirmed (An et al., 2010). In addition to the direct involvement of E3s in hormone perception, ethylene production is regulated by the BTB protein Ethylene-Overproducer 1 (ETO1) which targets ACS5 – an enzyme acting at the rate-limiting step in ethylene production (Christians et al., 2009).
From the hormones mentioned so far there is a consistent paradigm of direct involvement of E3 ligases in hormone perception. So far, in contrast to this trend, perception of the other major plant hormone, ABA, does not rely on an E3 ligase, rather, perception is via a range of receptors (Raab et al., 2009). See the section on Abiotic stress for further discussion of ABA.
Downstream of ABA, the UPS is emerging as a complex regulator of release from dormancy and germination mostly through a diverse range of E3 ligases. The germination-related E3 ligases also appear to have a significant overlap with early development processes. The SCF component, ASK1 is important for ABA signalling as overexpression of a wheat homologue of ASK1 in Arabidopsis shows hypersensitive ABA responses in germination and growth (Li et al., 2011a); thus it is likely that ABA regulates physiological processes through altering the stability of various proteins. While the influence of ASK1 is far ranging, recently many E3 ligase components involved in ABA-mediated germination have been discovered. The RING E3 ligases AIRP1 and AIRP2 (Cho et al., 2011) and the F-box protein EDL3 (Koops et al., 2011), are responsible for reducing root growth rate in response to ABA but show divergence in other developmental processes. AIRP1 and AIRP2 are responsible for regulating stomatal closure and H2O2 production in response to ABA while ELD3 plays a vital role in early development. EDL3 transcripts are induced by ABA and stress conditions and in turn EDL3 causes chlorophyll production in etiolated plants and inhibits germination. CULBPM E3 ligases have been shown to reduce plant sensitivity under stress conditions by degrading the ABA-induced transcription factor HB6 (Lechner et al., 2011). The RING E3s RHA2a, RHA2b (Li et al., 2011b) and the BRIZ1/BRIZ2 dimer (Hsia & Callis, 2010) and the ARM E3s PUB18, PUB19 (Bergler & Hoth, 2011), PUB43 and PUB44 (Salt et al., 2011) are also responsible for inhibition of germination in response to ABA.
2. Circadian rhythm to floral development: it's clockwork for the UPS
Post-translational regulation of protein stability is emerging as a vital mechanism of regulation in the Arabidopsis circadian clock. As the protein components of the clock are in a continual state of flux, there is a need for these components to be promptly removed once transcription has ceased. The F-box proteins ZTL (Han et al., 2004), FKF1 and LKP2 play a vital role in circadian regulation and share a functional overlap (Baudry et al., 2010). These F-box proteins all contain a distinguishing Light–Oxygen–Voltage domain that allows them to detect blue light (Kiyosue & Wada, 2000; Imaizumi et al., 2003) and translate this into a response by inhibiting their ability to ubiqutinate targets. ZTL, FKF1 and LKP2 appear to act redundantly as single mutants show only limited disruption to the circadian rhythm while triple knockout shows severe disruption (Baudry et al., 2010). As part of an SCF complex, ZTL causes the degradation of the clock component TOC1, beginning in the evening and resulting a gradual decrease over the night (Más et al., 2003). ZTL also degrades the regulator PRR5 in a stringent dark-dependent manner (Kiba et al., 2007). PRR5, in turn, has regulatory effects on the circadian clock by influencing TOC1 phosphorylation and localization (Wang et al., 2010) as well regulating flowering time (Nakamichi et al., 2007; Fig. 6).
Figure 6. Ubiquitin–proteasome system (UPS) in the plant clock. The Arabidopsis circadian clock consists of a large number of regulatory proteins forming three oscillating protein abundance loops (a, b and c) focusing around the central hub of CCA1 and LHY which integrate external stimuli to entrain the clock. LHY abundance is regulated by the Really Interesting New Gene (RING) E3 SINAT5. The regulator PRR5 and clock component TOC1 are negatively regulated by the F-box proteins ZTL, FKF1 and LPK2 in the absence of blue light.
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In another branch of the circadian clock, the single subunit RING E3 ligase, SINAT5, facilitates the degradation of LHY and appears to play an integral role in flowering. In addition, SINAT5 protein levels are induced by the hormone auxin, so it serves as a branch to integrate hormonal signals into the circadian clock (Xie et al., 2002). Another protein, DET1, adds an additional layer of complexity to this relationship by protecting LHY from degradation by SINAT5 (Park et al., 2010); however, the role of DET1 is not fully understood. The interplay between these three proteins appears to be important in determining flowering time as both sinat5 (Park et al., 2010) mutants and LHY (Mizoguchi et al., 2002) over-expressers show delayed flowering.
Floral development is a complex process governed by complex regulation that has attracted much research over the last few decades. Ubiquitination appears to play a role in most if not all phases of floral development. The first phase of floral development involves determination of flowering time, which depends on a large number of factors, including input from the circadian clock via the floral regulator CONSTANS (CO). The cyclic peaks in CO appear to determine transition into flowering depending on daylength. The RING-like E3 DAY NEUTRAL FLOWERING (DNF) plays a role in regulating the sensitivity of the flowering response to daylength. Although direct evidence is lacking, it seems likely that CO is a target for DNF-mediated degradation (Morris et al., 2010). The stability of CO is also regulated by blue light via the COP1, with blue light preventing the ubiquitination by COP1. The cryptochrome CRY2 is the detector of blue light, entering an excited state in its presence. The excited CRY2 protein then binds to SPA1, an interactor of COP1 and blocks the action COP1 (Zuo et al., 2011). COP1 also targets photomorphogenic transcription factors, including HY5, HYH and LAF, for degradation (Seo et al., 2003). Thus COP1 is a hub for integrating light and circadian signals to direct initiation of photomorphogenesis at an optimal developmental stage. It appears that the light/dark localization of COP1 is regulated by the E2 COP10 through nonK48-linked polyubiquitin as well as by derubylation by the CSN that is emerging as a regulator of SCF E3 ligase activity (Smalle & Vierstra, 2004).
After ignition, development of meristem identity is initiated by the homoeotic regulator LFY, which leads to the development of floral organs. An interesting regulator of LFY has been identified, the F-box UFO. While UFO has typical E3 domains it also has DNA-binding domains and, with LFY, regulates AP3 expression, which plays a role in correct floral organ assignment. It may also regulate LFY through targeted degradation, although no direct evidence for this remains has been shown (Chae et al., 2008).
The correct development of male structures in plants depends on the action of SCFSAF, which leads to the essential thinning of the endothelial secondary wall (Kim et al., 2012). In pollen the F-box FBL17 plays a role in regulating the development of sperm by directing second mitosis of the generative microspore during pollen development. The targets of SCFFBL17 are not known, but it has been suggested that the second mitosis is achieved through degradation of cell cycle components (Gusti et al., 2009), which remains to be tested. In addition, self incompatibility in Brassica napus has been shown to be regulated by the action of the U-box E3 ARC1, which is activated by self-incompatible pollen, leading to an increase in ubiquitinated proteins in the pistil (Stone et al., 2003).
3. UPS coordinates plants responses to abiotic stress
Plants face constant stresses from the abiotic conditions of their environment. These stresses come from a variety of factors, encompassing all aspects of their life cycle, with a wide range of possible effects. Changes to the physical conditions, which cause the stress, can be just for the short term, such as the differences in light and temperature intensity in normal diurnal cycles or, alternatively, they can take place or be sustained over a much longer time, as seen with seasonal effects or long-term climate changes. Abiotic stresses can also be very quick to develop, such as mechanical damage and be more troublesome for the plant to predict, such as prolonged periods of drought, or anywhere in-between. As such, it is clear that all plants must possess very diverse systems to combat all forms of abiotic stress. Some will need to be flicked on and off like a switch, while others will need to lead to long-term cellular changes. However, one constant among all of these defensive strategies is the presence of the ubiquitin system.
There are a large number of examples of the UPS affecting plant's ability to cope with abiotic stresses. These can vary across numerous signalling pathways involved in responding to all types of abiotic stresses. Drought, cold and salt are three very closely related stresses, which are common and widespread among plants in many environments. Here we will focus mainly on the action of the UPS in these areas.
Abscisic acid-mediated signalling is involved in all three stresses and allows us to map out the role of the UPS in perceiving and responding to drought, cold and salt stress via this signalling pathway. Perception of these environmental stresses can lead to the biosynthesis of ABA, which, in turn, can result in changes to the expression of hundreds of ABA-regulated genes. The promoter region of these genes contains the ABA-regulatory element (ABRE), to which certain ABA-regulated transcription factors bind (Hattori et al., 2002). Abscisic acid acts in a variety of different cell types to produce differing actions. Therefore, it is possible that there are a number of different mechanisms by which it can function. Nonetheless, the ubiquitin proteasome system is still key to controlling this pathway and bringing about abiotic stress responses.
One gene, thought to be involved in this process, is the Arabidopsis RING E3 ligase XERICO, which is upregulated in response to osmotic stress. Transgenic over-expressers were hypersensitive to osmotic stress, as well as ABA treatment and produced significantly greater amounts of ABA in the cells (Ko et al., 2006). These results suggest that XERICO is involved in the biosynthesis of ABA in response to stress. However, the major level of ubiquitin-related control in this system appears to involve the regulation of stability of the ABRE-binding transcription factors. One such transcription factor is Abscisic acid insensitive 3 (ABI3), which is regulated by the ABI3-interacting E3 ligase AIP2. Abscisic acid has been shown to induce expression of AIP2, therefore leading to a reduction in the presence of ABI3.
As mentioned previously, the ABA-induced transcription factor HB6, a negative regulator of ABA responses (Himmelbach et al., 2002), is also controlled by the UPS. The concentrations of HB6 protein increase in response to a reduction in expression of CUL3BPM E3 ligases, which have been shown to target the transcription factor for proteasomal degradation (Lechner et al., 2011). The stability of another such transcription factor, ABI5, the accumulation of which is induced by osmotic stress, is controlled by E3 ligases in an ABA-dependent manner (Lopez-Molina et al., 2001). While the mechanism for this is less well known, it has been shown that the E3 ligase KEG (Liu & Stone, 2010) as well as the E3 complex of DWA1 and DWA2 (Lee et al., 2010) are important in ABI5 regulation. The transcription factor is shown to accumulate at higher levels when either is missing, and is shown to interact directly with both, suggesting that it is targeted for proteasomal breakdown.
Drought, cold and salt stresses can all be harmful to the plant by causing cellular desiccation, and plants combat this particular aspect of all three stresses in a similar way. This is mainly achieved through ABA- and UPS-mediated signalling. It seems, therefore, that a key aspect of ABA in stress responses is in tying together of all these stresses, while also producing individual responses.
Perception of each of these stresses generates the same response of ABA, and yet not all ABA genes are upregulated in response to all stresses, suggesting that it is the action of the ABA that is producing different results. This could be related to the possible different mechanisms for ABA action, which was suggested earlier. The availability of several modes of action would allow production of the single hormone to result in different responses (Fig. 7).
Figure 7. Action of the ubiquitin–proteasome system (UPS) on selected components of the drought, cold- and salt-response pathways. (a) The abscisic acid (ABA) dependent pathway affects gene expression via ABA-regulated transcription factors, including ABI3 and ABI5 interacting with the ABA-regulatory element (ABRE) promoter region. (b) The ABA independent pathway affects gene expression via stress-induced transcription factors, DREB1 and DREB2A, binding the Drought Response Element (DRE) promoter region.
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While the ABA pathways mentioned play an important role in coping with cold, drought and salt stress, the plant must also rely on many ABA-independent pathways. These are also reliant on the UPS as a control system. One such example is HOS1, which was originally identified by a genetic screen (Ishitani et al., 1998) as a negative regulator of the CBF cold-response pathway, and mutation of the gene produced increased levels of a number of cold-responsive genes. More recently, HOS1 was shown to be a functional ubiquitin ligase enzyme (Dong et al., 2006). Yeast two-hybrid assays also showed a probable interaction between HOS1 and ICE1, a transcription factor, which also regulates cold responsive genes in the CBF pathway (Chinnusamy et al., 2003). Dong et al. (2006) went on to show that ICE1 could be targeted for ubiquitination by HOS1. hos1 mutants not only prevented this ubiquitination but also produced higher expression of CBF pathway components downstream of ICE1.
DREB2A is a transcription factor that binds to the Drought Response Element (DRE), a motif present in the promoter of many cold- and drought-responsive genes (Chinnusamy et al., 2003). DREB2A is thought to be proteasomally degraded by ubiquitination by DREB2A Interacting Protein (DRIP) 1 and 2, both of which are RING E3 ligases (Qin et al., 2008). DRIP double knockouts plants show increased drought tolerance, brought about by an increase in production of DREB2A-regulated stress response genes.
Another functional RING E3 ligase, Salt and Drought Induced Ring finger (SDIR1) has been shown to be involved in the response to salt, specifically in the prevention of germination under high salt conditions, although the mechanism behind this is not known (Zhang et al., 2007a,b). Interestingly, SDIR1 is also involved in ABA signalling, acting as a positive regulator of ABI5, suggesting a possibility of crossover between the two pathways.
This evidence does show clear differences in the signals used for these slightly differing stresses, which is to be expected as they do require different action, and so the plant must be able to respond to each individually. However, once again the systems are linked to some extent, both by the shared use of the DRE and, by the presence of the UPS. The intricate control possible with ubiquitination appears to be key in the crosslinking of these responses, while also keeping them, to some extent, separate and distinguishable (Fig. 7).
4. Plant pathogens target the UPS: proof of its importance in plant immunity
Without a circulatory system to carry dedicated immune surveillance cells, plants have evolved a global single-cell immune system (Spoel & Dong, 2012). Detection through ‘pattern recognition receptors’ (PRRs) of widely conserved pathogen associated molecular patterns (PAMPs) induces ‘PAMP triggered immunity’ (PTI). Whereas strain-specific recognition of effector molecules (virulence factors), or their effects on the host, leads to effector triggered immunity (ETI; Jones & Dangl, 2006). These recognition events activate defence responses such as a burst of reactive oxygen species (ROS) and hypersensitive response associated programmed cell death (HR PCD), acting to limit or inhibit pathogen spread through the host. The plant UPS is heavily implicated in plant immunity with a plethora of studies identifying ubiquitin ligases that are upregulated in response to PAMP elicitors, effector treatments or indeed pathogens themselves. These genetic upregulations are just a fraction of the vast transcriptional reprogramming that occurs in response to challenge by pathogens (reviewed by Katagiri, 2004) but indicates a clear link between the UPS and host–pathogen interactions. A simple survey of the data landscape on plant–pathogen interaction indicates that nearly every component of the ubiquitin system is implicated in plant immunity (Fig. 8). We highlight a few below with special emphasis on E3 ligases.
Figure 8. Pathogen-associated molecular pattern (PAMP) and effector-triggered immune pathways are targeted by pathogens to overcome host defence responses. FLS2 flagellin perception is targeted by the AvrPto effector with E3 ligase activity. Pathogen encoded F-box motifs are present in GALA and PO effectors which bind to host Skp1-Cullin-F-box (SCF) ubiquitin ligase subunits. SCFPO has been shown to target host component ARGONAUTE to proteasomal degradation blocking post-transcriptional gene silencing of viral factors. Bacterial coronatine mimics active jasmonic acid (JA) and promotes the degradation of the JAZ transcriptional repressors, activating the JA pathway which antagonizes the salicylic acid (SA) mediated systemic acquired resistance (SAR) response. AvrB targets RAR1 blocking PAMP triggered immune responses in susceptible interactions Oomycete effector Avr3a binds to CMPG1 suppressing effector-triggered hypersensitive response programmed cell death (HR PCD). For further details of components included in this figure see section III, subsection 4., ‘Plant pathogens target the UPS: proof of its importance in plant immunity’ of this review. For details of the RAR1-SGT1-HSP90 complex in plant immunity see the recent review by Shirasu (2009).
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A triple knockout of Arabidopsis Plant U-Box genes Pub22, Pub23, and Pub24 results in enhanced resistance to bacterial and oomycete pathogens. Oxidative burst elicited by flg22 and chitin was shown to accumulate to a much greater extent in the triple knockout and enzymes involved in ROS production were also found to be upregulated. These results suggest these three U-box E3 ligases act redundantly as negative regulators of PTI in response to flg22 and chitin PAMPs (Trujillo et al., 2008).
The perception of bacterial flagellin by FLAGELLIN SENSING 2 (FLS2), a transmembrane PRR implicates two essential Plant U-Box E3 enzymes, PUB12 and PUB13. These are recruited and phosphorylated by BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) and this activity appears to be enhanced by another kinase BOTRYTIS-INDUCED KINASE1 (BIK1). BAK1–PUB12–PUB13 protein complex associates with FLS2 upon its binding of the conserved flagellin peptide flg22. This interaction results in PUB12- and PUB13-mediated polyubiquitination of FLS2 and its apparent internalization into the plant cytosol (Fig. 8; Robatzek, 2006; Lu et al., 2011).
ACRE74, ACRE276 and ACRE189 are E3 ligases identified as positive regulators of HR-PCD using the heterologous tobacco Cf-9/Avr9 experimental system (González-Lamothe et al., 2006; Yang et al., 2006; van den Burg et al., 2008). Silencing these E3 ligases, through virus-induced gene silencing (VIG) in tomato, reduces HR PCD induced by avirulent fungal strains of Cladosporium fulvum, leading to susceptibility.
The rice SPL11 lesion mimic mutant produces a spontaneous cell death phenotype that enhances defense against avirulent and virulent bacterial and fungal pathogens. Spl11 encodes a plant U-Box E3 with in vitro ligase activity (Zeng et al., 2004). SPL11 has been shown to interact with SPL11-interacting protein1 (SPIN1) a Signal Transduction and Activation of RNA (STAR) protein involved in regulation of flowering time. The Arabidopsis SPL11 homologue has also been shown to have a role in development underlining the apparent parallels between plant immunity and the regulation of plant development (González-Lamothe et al., 2006; Yang et al., 2006; van den Burg et al., 2008; Vega-Sánchez et al., 2008; Shikata et al., 2009).
Arabidopsis Botrytis Susceptible1 Interactor (BOI) encodes a RING E3 ligase, which interacts with the transcription factor MYB108. BOI is capable of ubiquitinating MYB108 in vitro. Knockdown boi lines displayed increased susceptibility to Botrytis cinerea. Overexpressing and RNAi lines showed no alteration in disease susceptibility to avirulent and virulent Pseudomonas strains. Using electrolyte leakage as a proxy for cell death the authors found elevated levels of cell death in virulent pathogen interactions whereas avirulent-induced HR PCD was unchanged (Lin et al., 2008; Luo et al., 2010). This could suggest that BOI is manipulated by the pathogen to degrade MYB108 in compatible interactions and perturbing BOI levels is not significant enough to disrupt this activity.
These studies clearly point to the E3 ligase group as important players in plant–pathogen interactions, This is not surprising considering the presence of a large number of Plant U-box and RING E3 encoding genes in the plant genome; however, relatively few F-boxs proteins have been implicated in plant stress responses even though they form the largest subclass within the E3s.
The messages carried by hormones are a vital part of plant immune signalling. Salicylic acid (SA), JAs and ethylene (ET) have established roles in plant defence, which include crosstalk between these pathways (Leon-Reyes et al., 2009; Salt et al., 2011). Recent findings have also implicated the classical growth and development hormones auxin and gibberellins as well as the brassinosteroids, demonstrating the integration of multiple signalling pathways (hormones in immunity reviewed by Pieterse et al. (2009). We highlight the role of SA as a typical example of how the UPS is interlinked with immunity.
Salicylic acid is a key signaller in systemic-acquired resistance (SAR; reviewed by Durrant & Dong, 2004). NONEXPRESSOR OF PR GENES1 (NPR1) is a coactivator of pathogenesis-related (PR) gene expression and is essential to SAR transduction. NPR1 contains a BTB protein–protein interaction domain that mediates formation of a CRL ubiquitin ligase. NPR1 E3 mediates the continual turnover of itself in the nucleus by the UPS (Spoel et al., 2009). NPR1 interacts with TGA2 a transcriptional activator of SA-responsive genes in SAR. Binding of NPR1 to TGA2 by its repressor domain is thought to prevent TGA2 oligomers and thus promote gene expression in the SA pathway (Boyle et al., 2009).
Systemic-acquired resistance compromised npr1 mutants have been exploited in a number of studies looking for mutants capable of recovering resistance. Li et al. (2009) discovered a suppressor of npr1-1 susceptibility, snc1, which constitutively activated defence-related responses. snc1 encodes an R protein that activates defence entirely independent of SA signalling (Zhang et al., 2003). A mutant screen for suppressors of the double mutants enhanced resistance, identified 11 MOS (modifier of snc1) components involved in this immune pathway (Monaghan et al., 2009). These include the Arabidopsis E1 enzyme UBA1 and a protein complex that is conserved in animals, although it has no known role in immunity and associates with two Plant U-box E3s (Goritschnig et al., 2007; Palma et al., 2007; Monaghan et al., 2009).
Similarly Kim & Delaney (2002) have isolated the Arabidopsis suppressor of Nim 1-1 (SON1) from a mutant screen for silencers of noninducible immunity1 NIM1 (NPR1). son1 encodes a protein containing an F-box domain. The son1 mutant similarly displays SAR-independent, constitutive resistance against both the virulent oomycete Hyaloperonospora arabidopsidis and the bacterial pathogen Pseudomonas syringae.
Screening of mutants sensitive to a putative SA precursor led to the isolation of BAH1 benzoic acid (BA) hypersensitive. Bah1 encodes a RING E3 ligase previously implicated in nitrogen limitation adaptation (Peng et al., 2007; Yaeno & Iba, 2008). The Bah1 mutant accumulated SA under BA treatment and Pseudomonas inoculation. The increase in SA was accompanied by higher resistance against P. syringae DC3000 (Yaeno & Iba, 2008).
In order to combat the immune system of host plants pathogens secrete effector molecules. The dual roles of UPS components in defence and development make them ideal targets for exploitation during infection. A number of effectors have been found to hijack the host UPS and steer it toward the degradation of host defence molecules.
Pseudomonas syringae injects effectors into host cells through Type III secretion, these include a hormone mimic (coronatine), a protease (AvrRpt2) a proteasomal inhibitor (syringolin A) and a U-box-like E3 ligase (AvrPtoB; Kim et al., 2005; Rosebrock et al., 2007; Groll et al., 2008; Katsir et al., 2008). AvrPto targets host kinases, including FLS2 and FEN, which mediates activation of basal defences through association with Prf R protein, leading to its degradation (Xiang et al., 2008). Pto, a host kinase involved in R gene AvrPto-mediated resistance has been shown to evade AvrPto ubiqitination activity via phosphorylation and hence inactivation of AvrPto E3 ligase activity (Ntoukakis et al., 2009). Coronatine mimics the active form of JA, binding to COI F-box causing repressor degradation and activation of the JA pathway antagonizing SA-mediated defence (Katsir et al., 2008). RAR1, required for RPM1-mediated ETI, is targeted by P. syringae effector AvrB in susceptible Arabidopsis leading to suppression of flg22 PAMP-triggered immunity (Shang et al., 2006).
A number of phytopathogens possess F-box effectors such as coronatine, capable of forming SCF complexes with host subunits to form a functional CRL E3 ligase. Agrobacterium tumefaciens VirF F-box interacts with the plant SKP1 proteins to degrade VIP1 and VirE2 for t-DNA integration into the host genome. The proteasomal inhibitor MG132 hinders the transformation process (Tzfira et al., 2004). The Ralstonia solanacearum effector GALA contains an F-box like domain that has been shown to interact with Arabidopsis SKP1-like ASK proteins, although activity and host targets are, as yet, undetermined (Angot et al., 2006). Similarly polerovirus PO F-box effector interacts with ASK1,2 and is essential to pathogenicity, blocking host post-transcriptional gene silencing (PTGS) defence (Pazhouhandeh et al., 2006). The virally hijacked SCF complex degrades ARGONAUTE proteins, which usually target host siRNA to viral RNA for silencing (Baumberger et al., 2007; Bortolamiol et al., 2007).
The oomycete RXLR effectors have attracted a large amount of research effort over the past decade. The Phytophthora infestans Avr3a effector has been shown to interact and stabilize U-box CMPG1 (Bos et al., 2010). This interaction was shown to suppress Avr-triggered HR PCD using an Agrobacterium transient expression system of R genes treated with Avr peptides (Gilroy et al., 2011).
A recent study dubbed ‘The effector interactome’ indicates that there may be 165 possible effector–host interactions in Arabidopsis (Mukhtar et al. (2011). A significant number of these interactions impinge on host ubiquitin machinery, indicating the high value pathogens put on the UPS as a target for defence suppression. However, despite clear evidence that the UPS is important for plant immunity our understanding of the mechanistic connections is still fragmented.