Available online at www.sciencedirect.

com

Ubiquitination in plant immunity

Marco Trujillo1 and Ken Shirasu2
Plant immune responses require the coordination of a myriad of processes that are triggered upon perception of invading pathogens. Ubiquitin, the ubiquitination system (UBS) and the 26S proteasome are key for the regulation of processes such as the oxidative burst, hormone signaling, gene induction, and programmed cell death. E3 ligases, the specicity determinants of ubiquitination, have received by far the most attention. Several single-unit ligases, which are rapidly induced by biotic cues, function as both positive and negative regulators of immune responses, whereas multisubunit ligases are mainly involved in hormone signaling. An increasing body of evidence emphasizes the heavy targeting of the UBS by pathogen virulence effectors, underlining its importance in immunity.
Addresses 1 Julius-von-Sachs Institute, Department of Pharmaceutical Biology, University of Wurzburg, Julius-von-Sachs Platz 2, Wurzburg, Germany 2 Plant Immunity Research Group, RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan Corresponding authors: Trujillo, Marco (mtrujillo@biozentrum.uniwuerzburg.de) and Shirasu, Ken (ken.shirasu@psc.riken.jp)

hormone levels take place that integrate biotic stress cues, and recent studies have demonstrated the extent to which ubiquitin-mediated proteolysis is involved in the regulation of hormone signaling [2,3]. Ubiquitin is a small (8.5 kDa) and highly conserved protein modier, tightly engaged in a wide range of cellular processes [3]. Modication of target proteins by the covalent attachment of ubiquitin, termed ubiquitination (or ubiquitylation), condemns the protein to proteolysis or other fates such as relocalization or endocytosis [4]. Ubiquitination is mediated by a three-step enzymatic cascade that consists of the activating (E1), conjugating (E2), and ligating (E3) enzymes [3]. Attention has centered on ubiquitin ligases (E3s) because they specify the target protein (substrate). On the basis of their domain and subunit composition, as well as mode of action, E3s can be classied into four groups [3]: HECT, RING, U-box, and cullin-RING ligases (CRLs, Figure 1). E3 ligases mediate the attachment of ubiquitin to a lysine (Lys) e-amino group on the target protein either by forming an intermediate, as in the case of HECT-type ligases, or by acting as a scaffold to bring the target and E2 into proximity (Figure 1). Ubiquitin can be conjugated as a monomer or as chains of different lengths linked by any one of its seven Lys residues. The linkage-type of the ubiquitin chain species the function it mediates [4]. The best characterized function of ubiquitin is mediated by the labeling of a protein with a ubiquitin chain linked via its Lys48 residue. Proteins labeled with at least four Lys48-linked ubiquitins are the favored substrate for proteolysis by the 26S proteasome, a large, 2.5 MDa multisubunit protein complex present in the nucleus and cytoplasm. In this review, we summarize and discuss recent insights to the role of the ubiquitination system (UBS) in immunity, focusing on E3 ubiquitin ligases, emerging concepts about their functions, and targeting by pathogen virulence effectors.

Current Opinion in Plant Biology 2010, 13:402408 This review comes from a themed issue on Biotic interactions Edited by Jane E. Parker and Jeffrey G. Ellis Available online 12th May 2010 1369-5266/$ see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2010.04.002

Introduction
From perception to response, plants are in a race against pathogens to react as quickly and effectively as possible. It starts with the perception of conserved pathogenassociated molecular patterns (PAMPs) mediated by plasma membrane-located pattern recognition receptors (PRRs), which relay the signal via mitogen activated protein kinase (MAPK) cascades, leading to the activation of immune responses known as PAMP-triggered immunity (PTI). However, PTI can be suppressed by pathogen-derived virulence effector proteins. In turn, some effector proteins can be perceived by a different subset of receptors that activates a second layer of defense, called effector-triggered immunity (ETI) [1]. Launching of defense responses for these two branches of immunity in plants requires ubiquitination for positive and negative regulation. In addition, profound changes in
Current Opinion in Plant Biology 2010, 13:402408

CRLs and hormone signaling

Jasmonic acid (JA)

The COI1 F-box was the rst component of the UBS shown to play a role in plant immunity [5]. The Arabidopsis coi1 mutant was originally identied because of its insensitivity to the bacterial toxin coronatine, and was later shown to be required for all JA-dependent responses [6]. JA is synthesized in response to pathogen attack, and coi1 mutants are unable to relay the JA-signal, making them more susceptible to necrotrophic pathogens [6]. Two groups recently uncovered the long-sought after
www.sciencedirect.com

Ubiquitination in plant immunity Trujillo and Shirasu 403

Figure 1

Salicylic acid (SA)

E3 ubiquitin ligases categorized by domain. (a) RING and U-box domain E3 ubiquitin ligases are composed of a single subunit. Both domains are structurally related and bind the E2-conjugating enzymeubiquitin complex. The RING motif is stabilized by two zinc ions, while the U-box motif exploits electrostatic interactions. Substrate specificity is conveyed by various proteinprotein interaction domains such as ankyrin repeats in RING type ligases, and prominently by armadillo-like (ARM) repeats in U-box ligases. (b) Cullin-RING ligases (CRLs) are composed of multiple subunits and are composed of a cullin, a RINGbox 1 (RBX1, which binds the E2ubiquitin complex), and different target recognition modules. CRLs include the S phase kinase-associated protein 1 (SKP1)cullin 1 (CUL1)F-box (SCF) and bric-a-brac tramtrackbroad complex (BTB) ligases. The F-box motif mediates interactions with the adaptor protein SKP1 in SCF ligases, whereas additional proteinprotein interaction domains are responsible for target recognition. The modular BTB subunit binds directly to CUL3 via the BTB domain and specifies the substrate through additional motifs, such as ankyrin repeats, as in the case of NPR1. (c) HECT domain ligases are single unit ligases, and in contrast to all other known ubiquitin ligases, the HECT domain itself binds to ubiquitin before mediating ubiquitination of a substrate. Thus far, there is no evidence for the involvement of HECT domain ligases in plant immunity. Numbers in parentheses indicate the number of predicted proteins in Arabidopsis. For a complete and detailed account of E3 ligases, please refer to the outstanding Vierstra review [3].

SA is the key hormone in systemic acquired resistance (SAR), and is also pivotal for defense responses against biotrophic and hemibiotrophic pathogens. Spoel and colleagues recently showed that the master coactivator of the SA pathway, NPR1, is continuously degraded in the nucleus in a proteasome-dependent manner [11]. Intriguingly, SA treatment facilitates recruitment of NPR1 to a CUL3-based ligase, and proteolysis is required for full activation of SA marker genes. This process requires the phosphorylation of NPR1 at residue Ser11/Ser15, showing the importance of phosphorylation to activate, or enhance the recruitment to proteolytical complexes. NPR1 contains a broad-complex, tramtrack, and bric-abrac (BTB) domain, which is present in CRL substrate adaptors [3] (Figure 1). The presence of a BTB domain raises the possibility that NPR1 itself is actively engaged in the ubiquitination process. NPR1 coimmunoprecipitates with CUL3A and the interaction is enhanced by SA [11]; however, an interaction with CUL3 could not be detected in a yeast two-hybrid assay [12]. The lack of post-translational modications in yeast (on target or CRL), which are required to regulate the interaction, may explain these results. CUL3A could possibly recruit NPR1 via its BTB domain to mediate autoubiquitination, thereby regulating its own activity by proteolysis (Figure 2). It may thus also be possible that NPR1 acts a substrate adaptor for a BTBNPR1 CRL. In addition to the BTB domain, NPR1 also contains ankyrin repeats that mediate proteinprotein interactions, as for example, with the TGA2 transcription factor [13]. TGA2 is a repressor of PR genes [14] and an activator of genes of the antagonistic JA pathway [15]. In this scenario, SA would induce a CUL3-based BTBNPR1 ligase that mediates the degradation of transcriptional repressors such as TGA2 or NIMIN1 [14,16]. In addition, phosphorylation of NPR1 could switch autoubiquitination activity of BTBNPR1 to substrate ubiquitination by enhancing its afnity to targets such as TGA2 (Figure 2). This possibility does not contradict the observations that NPR1 restricts TGA2 function by interacting with it [14]. NPR1 is able to sequester TGA2 away from its cognate promoter, negating its repressor activity and at the same time making it more accessible to the proteasome. This model would also agree with the observation that CUL3 and proteasome activity are necessary for full activation of the pathway [11].
Ethylene (ET)

missing link in JA signaling, namely the jasmonate ZIMdomain (JAZ) transcriptional repressors [7,8]. Chini et al. and Thines et al. showed that JAZ proteins are targeted by SCFCOI1 for proteasomal degradation in a JA-dependent manner, and that JA-isoleucine (JA-Ile) is the active form that mediates the recruitment of JAZ proteins to SCFCOI1 [8] (Figure 2). A similar mechanism was observed for SCFTIR1. Auxin binds TIR1, a close homolog of COI1, and prompts the degradation of the AUX/IAA transcriptional repressors [9]. Interestingly, interference with auxin signaling also affects the resistance against necrotrophic pathogens [10].
www.sciencedirect.com

ET biosynthesis is induced in response to PAMPs and infection by several pathogens. ET apparently ne-tunes antagonistic SA and JA responses through NPR1 [17]. Ubiquitin-mediated proteolysis is intricately involved in the regulation of ET perception [18], biosynthesis [19,20], and signaling [21] (Figure 2). The exact mechanism by
Current Opinion in Plant Biology 2010, 13:402408

404 Biotic interactions

Figure 2

Ubiquitination and immunity signaling. Attack by necrotrophic pathogens induces the jasmonic acid (JA) pathway. JA-Ile, the active form of JA, binds to COI1 and induces the interaction with the JAZ transcriptional repressors, leading to ubiquitination by SCFCOI1 and proteasomal degradation followed by the release of MYC2, thus allowing activation of the JA response. Perception of PAMPs and effector proteins induces the salicylic acid pathway. Pathogen perception leads to changes in the redox status of the cell and induces the monomerization of NPR1. NPR1 relocalization to the nucleus is accompanied by transcriptional activation of PR genes. NPR1 is proposed to mediate signaling by sequestering transcriptional repressors (e.g. TGA2). Alternatively, NPR1 could participate in a BTBNPR1 ligase complex that mediates ubiquitination and degradation of repressors. NPR1 function is regulated by phosphorylation, perhaps by affecting substrate affinity and autoubiquitination. Perception of PAMPs or effectors leads to activation of distinct but related immune responses, referred to as PAMP-triggered immunity (PTI) or effector triggered immunity (ETI). Plasma membrane or intracellular receptors mediate signaling, which is positively or negatively regulated by different PUBs and RING ligases. Ethylene biosynthesis is induced by various biotic stresses. The turnover of the ethylene precursor ACC synthases (ACS) 2 and ACS6 is reduced by phosphorylation leading to increased ET synthesis. Proteolysis of the EIN3 transcriptional factor is under the control of antagonistic pathways. CTR1 promotes phosphorylation that renders EIN3 accessible to degradation, probably by the cognate SCFEBF1/EBF2. Conversely, MAPK3/6 mediates the stabilization of EIN3. Arrows and bar-headed lines indicate functional interactions, and double-headed arrows indicate a physical interaction. Dotted forms and arrows denote inferred interactions and components for which data are not available. Light blue arrows indicate ubiquitination, a question mark (?) indicates an unknown target, yellow dots indicate ubiquitin (U), and red dots indicate phosphorylation (P).

which PAMP perception induces ET biosynthesis is not known. However, MPK6, which is activated by PAMP perception, phosphorylates the ET biosynthesis enzymes 1-aminocyclopropane-1-carboxilic acid (ACC) synthase (ACS) 2 and ACS6, protecting them from proteasomemediated degradation [22]. Similarly, protein levels of ACS4, ACS5, and ACS9, which belong to the type 2 ACSs, are also under proteolytic control via the BTB domain E3 subunits ethylene-overproducing 1 (ETO1) and ETO-like 1 (EOL1) and EOL2 [20].
Current Opinion in Plant Biology 2010, 13:402408

The central ethylene signaling hub, ethylene insensitive 3 (EIN3), was recently reported to be under the control of bifurcating and antagonistic MAPK cascades [23]. MPK6 phosphorylates EIN3 at its threonine (Thr) 174 residue, rendering it susceptible to degradation, probably by the cognate SCFEBF1/EBF2 [9] (Figure 2). By contrast, MPK6-independent phosphorylation of the Thr592 residue protects EIN3 from degradation, illustrating the importance of UBS in the ET pathway.
www.sciencedirect.com

Ubiquitination in plant immunity Trujillo and Shirasu 405

Cross-talk between ET and PAMP-triggered signaling was recently shown to involve the stability of the ETresponsive element binding factor 104 (ERF104). Perception of the conserved peptide from the PAMP agellin, g22, induces phosphorylation and release of ERF104 from a complex with MPK6, which results in enhanced turnover [24]. Furthermore, mutation of the phosphorylation site further reduces ERF104 stability, indicating that modication of the target is important for regulating ubiquitination followed by proteasomal degradation.

RINGs and PUBs in early immune responses

RING and Plant U-box type (PUB) E3 ligases involved in immunity have been identied mainly by transcript proling of genes rapidly induced by biotic cues. This circumstantial evidence led to the identication of numerous homologs and orthologs in different plant species [2527]. The gene family Arabidopsis toxicos para levadura (ATL), coding for putative RING-type ligases, has been proposed to play a role in immunity because some of its members are induced by the PAMP chitin [28]. However, a role in immunity for this gene family has only been conrmed for ATL9 (At2g35000) since the atl9 mutants are more susceptible to the biotrophic fungus Erysiphe cichoracearum [27]. A related ATL gene in tomato, RING nger protein 1 (RFP1), is necessary for resistance against the necrotrophic pathogen Phytophthora infestans [29], suggesting a conserved role for the ATL gene family. Notably, transcriptional induction of ATLs is not only restricted to chitin, but also observed for other PAMPs [26,30]. Nevertheless, despite the large number of RINGtype genes in Arabidopsis (477), few have been shown to have a function in immunity. Several PUBs have been documented as positive and negative regulators of immune responses in different plant species. For example, the Avr9/Cf9 rapidly elicited (ACRE) genes ACRE74 (CMPG1) and ACRE276 (PUB17) are required for the hypersensitive response, a programmed cell death-type reaction, in response to perception of the Avr9 peptide by the Cf9 receptor-like protein in tobacco [31,32] (Figure 2). Further supporting their role in resistance, tomato knock-down lines are more susceptible to the biotrophic fungus Cladosporium fulvum. In addition, the two closely related U-box proteins MAC3A and MAC3B are required to mount an effective defense response against several virulent and avirulent biotrophic and hemibiotrophic pathogens [33]. MAC3A and MAC3B are not strongly induced by biotic cues, and show homology to yeast and human Prp19 ubiquitin ligases involved in RNA processing, suggesting a probable role in the regulation of gene expression (Figure 2). By contrast, three closely related E3 ligases, PUB22, PUB23, and PUB24 negatively regulate PAMP-triggered signaling and PTI cumulatively [34] (Figure 2). Interwww.sciencedirect.com

estingly, the pub22/23/24 triple mutant reacts with an enhanced oxidative burst to various PAMPs, indicating that these E3 ligases have a function in a process shared by distinct pattern recognition receptors required for downregulation of signaling. The closest homologs to NtACRE74 in Arabidopsis are PUB20 and PUB21, which, together with PUB22PUB24, are conspicuously induced by many biotic stresses and pathogen attack, suggesting that they also perform a function in immunity. Analysis of pub21 and pub22 mutants has revealed that these two are also negative regulators of PAMP-triggered immunity (our unpublished results), which contrasts to the function of their homolog ACRE74 in tomato and in tobacco [31]. Mutants of the Lotus japonicus CERBERUS, a U-box encoding gene, produce more prenodule structures in response to infection by Rhizobium bacteria, but are unable to support the formation of an infection thread, suggesting a function in the negative regulation of nodulation and possibly of immunity [35]. In comparison to CRLs, little is known about the substrates of PUBs and RING ligases, and therefore the cellular processes they participate in. However, there are a few exceptions that can provide some clues. The rice XA21 binding protein 3 (XB3) is a RING ligase which is phosphorylated by the kinase domain of the PRR XA21 in vitro [36]. Reduced transcript levels of XB3 in transgenic plants lead to an increase in susceptibility to the avirulent Xanthomonas oryzae pv. oryzae [36]. Although unrelated in function, several PUBs in Arabidopsis interact with S-type receptors, which mediate self-incompatibility in Brassica and a chitinase-related receptor-like kinase in tobacco [37,38]. Whether these E3 ligases are able to ubiquitinate the receptors or phosphorylation controls their activity are still open questions. In mammals, RING and U-box proteins, although divergent in domain composition, also function as E3 ligases, and are regulatory components of both innate and adaptive immunity. Most notably, TNF receptor-associated factor (TRAF) 6 catalyses Lys63-linked polyubiquitin chains, leading to the activation of downstream signaling components [39]. TRAF6 is also targeted for Lys63 polyubiquitination by Act1, a U-box-containing ligase required for Interleukin-17 signaling [40]. Importantly, endocytosis of receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), is also regulated by Lys63 ubiquitination by the Cbl RING ligase [41]. EGFR is structurally related to the plant PRRs, and endocytosis of PRRs has been shown [42], opening the possibility that such processes are conserved in plants.

Virulence effectors
The UBS is heavily targeted by pathogen virulence effector proteins and virulence compounds. In many cases, effector proteins co-opt coopt the UBS [43]. The bacterial effector AvrPtoB from Pseudomonas syringe pv.
Current Opinion in Plant Biology 2010, 13:402408

406 Biotic interactions

tomato, is probably the best studied example. The C-terminal domain of AvrPtoB displays structural homology to U-box/RING domains and is an active ubiquitin ligase [44]. In its host tomato, AvrPtoB ubiquitinates the kinase Fen, which is required for resistance, and targets it for degradation [43]. AvrPtoB also ubiquitinates other kinase domains, such as those from the PRRs FLS2 and CERK1 in Arabidopsis [45,46], targeting them for degradation. In the case of FLS2, degradation is sensitive to the proteasomal inhibitor MG132, but in the case of CERK1, degradation is sensitive to balomycin A, which inhibits vacuolar degradation, suggesting different preferences for degradation pathways. Moreover, a whole spectrum of effector proteins can be found in plant pathogens that covers any aspect of ubiquitination. The Agrobacterium tumefaciens VirF was the rst identied effector that functions as an F-box. It hijacks a host CRL, targeting VIP1 and VirE2 which are bound to the transfer-DNA for the degradation and thus liberates it to allow integration into the host genome [47]. Other effectors, such as HopM1, promote degradation of the adenosine diphosphate-ribosylation factor-guanine nucleotide exchange factor AtMIN7, probably by functioning as adaptors of the UBS [48]. Conversely, there are also virulence factors that display ubiquitin or ubiquitinlike modier protease activity such as XopD which belongs to the widely distributed YopJ effector family [49]. Pathogens are also capable of manipulating the UBS by synthesizing virulence molecules that are recognized by the host, such as hormones or hormone analogs [50]. The bacterial toxin coronatine binds to the CRL subunit COI1 and activates the degradation of JAZ transcriptional repressors [7]. By synthesizing coronatine, bacteria are able to suppress PAMP-triggered stomata closure, and induce the JA pathway, which is antagonistic to the SA pathway, required for PTI [51]. In another such example, the necrotrophic fungus Gibberella fujikuroi, the causal agent of the foolish-seedling disease of rice, makes gibberellins. Treatment of plants with g22 results in the stabilization of DELLA proteins, which mediate gibberellin signaling and importantly, g22 also increases resistance against necrotrophs [52,53]. Therefore, gibberellin might act as a virulence factor by counteracting DELLA stabilization induced by other PAMPs such as chitin. In other cases, virulence molecules, such as syringolin A, produced by P. syringae pv. syringae, are able to inhibit proteasomal function [54]. However, how this might be of advantage to the pathogen is yet unknown.

posed by effectors or virulence compounds on UBS and proteasome, especially in light of the fact that plants lack an adaptive immune system. It is conceivable that some ligases have evolved to act as decoys [55]. One important aspect of such a model is that it would assure the stability of the UBS network in the event that one or several of its components were being manipulated by pathogens. Alternatively, it is feasible that some ligases have evolved to recognize effector proteins and to mediate their neutralization by ubiquitination, thus targeting them for degradation. Future work will reveal the targets of PUB and RING single-unit ligases and therefore the cellular processes that they regulate. In contrast to CRLs, U-box and RING domains, which mediate E2 interactions, show variation between the main conserved residues. In fact, PUBs and RING ligases are able to interact with more than one E2 [56,57]. The E2E3 combination determines the type of polyubiquitin linkage, opening the possibility that these ligases mediate noncanonical ubiquitination, such as Lys63 polyubiquitination or monoubiquitination, and thus regulate signaling by proteasome-independent ways. In contrast, hormone signaling often involves the regulation of transcriptional activators or repressors via turnover using CRLs. CRLs use RBX1 as an E2 adapter, indicating interaction with only a small subset of E2s, supporting the concept of a specialized role for CRLs in Lys48 polyubiquitination. Several studies have demonstrated the importance of phosphorylation in regulating ubiquitination. This can take place at the level of the UBS components and at the substrate level. Although the former has not been shown to play a major role for CRLs, which are mainly regulated by other modications, it could be relevant for PUB and RING ligases. PUBs were shown to interact with kinase domains of receptors [37,38], and XA21 phosphorylates the RING ligase OsXB3 [36], possibly indicating that phosphorylation of single unit ligases is implicated in their regulation. Conversely, phosphorylation clearly controls the turnover of proteins, for example, ERF104 [24], ACS2/6 [22], NPR1 [11], and EIN3 [23] (Figure 2). Introduction of negative charges by phosphorylation possibly changes the attributes of the substrate, making it recognizable to the E3, or promoting the interaction. An in-depth understanding of the UBS and target recognition, in addition to the virulence stratagems employed by pathogens to manipulate the system, should prove useful to develop new strategies and tools to improve resistance and other traits of crop plants.

Conclusions and outlook

It is tempting to speculate that the vast expansion of the plant UBS, especially of the E3 ligases, encoded by more than 1400 genes in Arabidopsis, is in part due to the constant attempts of manipulation by pathogens. Expansion of genes coding for proteins targeted by pathogens, presumably by duplication, would relax the constraints
Current Opinion in Plant Biology 2010, 13:402408

Acknowledgements
We thank Rebecca Lyons for critical reading of the manuscript. Research in Ken Shirasus laboratory is funded by KAKENHI 19678001 and in Marco Trujillos lab by the Deutsche Forschungsgemeinschaft SFB 567. www.sciencedirect.com

Ubiquitination in plant immunity Trujillo and Shirasu 407

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1. 2. Jones JD, Dangl JL: The plant immune system. Nature 2006, 444:323-329. Pieterse CMJ, Leon-Reyes A, Van der Ent S, Van Wees SCM: Networking by small-molecule hormones in plant immunity. Nat Chem Biol 2009, 5:308-316.

15. Zander M, La Camera S, Lamotte O, Metraux JP, Gatz C: Arabidopsis thaliana class-II TGA transcription factors are essential activators of jasmonic acid/ethylene-induced defense responses. Plant J 2009, 61:200-210. 16. Weigel RR, Ptzner UM, Gatz C: Interaction of NIMIN1 with NPR1 modulates PR gene expression in Arabidopsis. Plant Cell 2005, 17:1279-1291. 17. Leon-Reyes A, Spoel SH, De Lange ES, Abe H, Kobayashi M, Tsuda S, Millenaar FF, Welschen RA, Ritsema T, Pieterse CM: Ethylene modulates the role of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 in cross talk between salicylate and jasmonate signaling. Plant Physiol 2009, 149:1797-1809. 18. Chen YF, Shakeel SN, Bowers J, Zhao XC, Etheridge N, Schaller GE: Ligand-induced degradation of the ethylene receptor ETR2 through a proteasome-dependent pathway in Arabidopsis. J Biol Chem 2007, 282:24752-24758. 19. Wang KL, Yoshida H, Lurin C, Ecker JR: Regulation of ethylene gas biosynthesis by the Arabidopsis ETO1 protein. Nature 2004, 428:945-950. 20. Christians MJ, Gingerich DJ, Hansen M, Binder BM, Kieber JJ, Vierstra RD: The BTB ubiquitin ligases ETO1, EOL1 and EOL2 act collectively to regulate ethylene biosynthesis in Arabidopsis by controlling type-2 ACC synthase levels. Plant J 2009, 57:332-345. 21. Qiao H, Chang KN, Yazaki J, Ecker JR: Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis. Genes Dev 2009, 23:512-521. 22. Joo S, Liu Y, Lueth A, Zhang S: MAPK phosphorylation-induced stabilization of ACS6 protein is mediated by the non-catalytic C-terminal domain, which also contains the cis-determinant for rapid degradation by the 26S proteasome pathway. Plant J 2008, 54:129-140. 23. Yoo SD, Cho YH, Tena G, Xiong Y, Sheen J: Dual control of  nuclear EIN3 by bifurcate MAPK cascades in C2H4 signalling. Nature 2008, 451:789-795. This article identies the components of two potential MAP kinase cascades that mediate the antagonistic regulation of EIN3 via proteolysis. EIN3 is stabilized when phosphorylated on a particular residue by the MAP kinases MPK3 and MPK6. A second MAPK phosphorylation site on EIN3 promotes EIN3 degradation and CTR1 may play a role in this phosphorylation. 24. Bethke G, Unthan T, Uhrig JF, Poschl Y, Gust AA, Scheel D, Lee J:  Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling. Proc Natl Acad Sci U S A 2009, 106:8067-8072. This paper describes a mechanism by which PAMP perception might by translated into a response via the interruption of a complex between MPK6 and ERF104 by the kinase activation. The release of ERF104 is accompanied by an increase in turnover regulated by phosphorylation. 25. Durrant WE, Rowland O, Piedras P, Hammond-Kosack KE, Jones JD: cDNA-AFLP reveals a striking overlap in racespecic resistance and wound response gene expression proles. Plant Cell 2000, 12:963-977. 26. Navarro L, Zipfel C, Rowland O, Keller I, Robatzek S, Boller T, Jones JD: The transcriptional innate immune response to g22, interplay and overlap with Avr gene-dependent defense responses and bacterial pathogenesis. Plant Physiol 2004, 135:1113-1128. 27. Ramonell K, Berrocal-Lobo M, Koh S, Wan J, Edwards H, Stacey G, Somerville S: Loss-of-function mutations in chitin responsive genes show increased susceptibility to the powdery mildew pathogen Erysiphe cichoracearum. Plant Physiol 2005, 138:1027-1036. 28. Martinez-Garcia M, Garciduenas-Pina C, Guzman P: Gene isolation in Arabidopsis thaliana by conditional overexpression of cDNAs toxic to Saccharomyces cerevisiae: identication of a novel early response zinc-nger gene. Mol Gen Genet 1996, 252:587-596. Current Opinion in Plant Biology 2010, 13:402408

3. Vierstra RD: The ubiquitin26S proteasome system at the  nexus of plant biology. Nat Rev Mol Cell Biol 2009, 10:385-397. An excellent review about the current understanding of the ubiquitination system and the various roles it plays including hormone signaling, morphogenesis, epigenetics, and self-incompatibility. 4. Ikeda F, Dikic I: Atypical ubiquitin chains: new molecular signals, protein modications: beyond the usual suspects review series. EMBO Rep 2008, 9:536-542. Xie DX, Feys BF, James S, Nieto-Rostro M, Turner JG: COI1: an Arabidopsis gene required for jasmonate-regulated defense and fertility. Science 1998, 280:1091-1094. Thomma BPHJ, Eggermont K, Penninckx IAMA, Mauch-Mani B, Vogelsang R, Cammue BPA, Broekaert WF: Separate jasmonate-dependent and salicylate-dependent defenseresponse pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proc Natl Acad Sci U S A 1998, 95:15107-15111.

5.

6.

7. 

Chini A, Fonseca S, Fernandez G, Adie B, Chico JM, Lorenzo O, Garcia-Casado G, Lopez-Vidriero I, Lozano FM, Ponce MR et al.: The JAZ family of repressors is the missing link in jasmonate signalling. Nature 2007, 448:666-671. Through the use of forward genetics the authors identify JAZ proteins as targets of SCFCOI1. The authors demonstrate that JAI3 and other JAZs are direct targets of the SCFCOI1 E3 ubiquitin ligase and JA treatment induces their proteasome degradation.

8. 

Thines B, Katsir L, Melotto M, Niu Y, Mandaokar A, Liu G, Nomura K, He SY, Howe GA, Browse J: JAZ repressor proteins are targets of the SCFCOI1 complex during jasmonate signalling. Nature 2007, 448:661-665. Along with [7], authors identify the family of JAZ genes that encode negative regulators of the JA pathway. In vitro pull-down and yeast-twohybrid assays demonstrate that COI1 interaction with JAZ1 is mediated by JA-Ile but not by nonconjugated JAs. 9. Santner A, Estelle M: Recent advances and emerging trends in plant hormone signalling. Nature 2009, 459:1071-1078.

10. Llorente F, Muskett P, Sanchez-Vallet A, Lopez G, Ramos B, Sanchez-Rodriguez C, Jorda L, Parker J, Molina A: Repression of the auxin response pathway increases Arabidopsis susceptibility to necrotrophic fungi. Mol Plant 2008, 1:496-509. 11. Spoel SH, Mou Z, Tada Y, Spivey NW, Genschik P, Dong X:  Proteasome-mediated turnover of the transcription coactivator NPR1 plays dual roles in regulating plant immunity. Cell 2009, 137:860-872. This article reports the requirement of NPR1 proteasome-mediated degradation for target gene induction. They describe the importance of NPR1 phosphorylation in the recruitment of CUL3 and promotion of degradation. 12. Dieterle M, Thomann A, Renou JP, Parmentier Y, Cognat V, Lemonnier G, Muller R, Shen WH, Kretsch T, Genschik P: Molecular and functional characterization of Arabidopsis Cullin 3A. Plant J 2005, 41:386-399. 13. Despres C, DeLong C, Glaze S, Liu E, Fobert PR: The Arabidopsis NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors. Plant Cell 2000, 12:279-290. 14. Boyle P, Le Su E, Rochon A, Shearer HL, Murmu J, Chu JY, Fobert PR, Despres C: The BTB/POZ domain of the Arabidopsis disease resistance protein NPR1 interacts with the repression domain of TGA2 to negate its function. Plant Cell 2009, 21:3700-3713. www.sciencedirect.com

408 Biotic interactions

29. Ni X, Tian Z, Liu J, Song B, Xie C: Cloning and molecular characterization of the potato RING nger protein gene StRFP1 and its function in potato broad-spectrum resistance against Phytophthora infestans. J Plant Physiol 2010, 167:488-496. 30. Zipfel C, Kunze G, Chinchilla D, Caniard A, Jones JD, Boller T, Felix G: Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 2006, 125:749-760. 31. Gonzalez-Lamothe R, Tsitsigiannis DI, Ludwig AA, Panicot M,  Shirasu K, Jones JD: The U-box protein CMPG1 is required for efcient activation of defense mechanisms triggered by multiple resistance genes in tobacco and tomato. Plant Cell 2006, 18:1067-1083. The authors demonstrate for the rst time the role of a U-box type E3 ligase, namely CMPG1 (ACRE74), as a positive regulator of cell death reactions and resistance against a biotrophic fungus. 32. Yang CW, Gonzalez-Lamothe R, Ewan RA, Rowland O, Yoshioka H,  Shenton M, Ye H, ODonnell E, Jones JD, Sadanandom A: The E3 ubiquitin ligase activity of arabidopsis PLANT U-BOX17 and its functional tobacco homolog ACRE276 are required for cell death and defense. Plant Cell 2006, 18:1084-1098. See annotation to [34]. 33. Monaghan J, Xu F, Gao M, Zhao Q, Palma K, Long C, Chen S, Zhang Y, Li X: Two Prp19-like U-box proteins in the MOS4associated complex play redundant roles in plant innate immunity. PLoS Pathog 2009, 5:e1000526. 34. Trujillo M, Ichimura K, Casais C, Shirasu K: Negative regulation of  PAMP-triggered immunity by an E3 ubiquitin ligase triplet in Arabidopsis. Curr Biol 2008, 18:1396-1401. In this article, a set of highly related PUBs is shown to act as negative regulators of PAMP-triggered signaling and resistance against biotrophic and hemibiotrophic pathogens. 35. Yano K, Shibata S, Chen WL, Sato S, Kaneko T, Jurkiewicz A, Sandal N, Banba M, Imaizumi-Anraku H, Kojima T et al.: CERBERUS, a novel U-box protein containing WD-40 repeats, is required for formation of the infection thread and nodule development in the legumeRhizobium symbiosis. Plant J 2009, 60:168-180. 36. Wang YS, Pi LY, Chen X, Chakrabarty PK, Jiang J, De Leon AL,  Liu GZ, Li L, Benny U, Oard J et al.: Rice XA21 binding protein 3 is a ubiquitin ligase required for full Xa21-mediated disease resistance. Plant Cell 2006, 18:3635-3646. The authors show that the rice XA21 binding protein 3 (XB3) is a RING type ubiquitin ligase, which is able to interact with the XA21 receptor. Furthermore, Xa21 is capable of phosphorylating XB3 suggesting that it might regulate its activity. 37. Samuel MA, Mudgil Y, Salt JN, Delmas F, Ramachandran S, Chilelli A, Goring DR: Interactions between the S-domain receptor kinases and AtPUB-ARM E3 ubiquitin ligases suggest a conserved signaling pathway in Arabidopsis. Plant Physiol 2008, 147:2084-2095. 38. Kim M, Cho HS, Kim DM, Lee JH, Pai HS: CHRK1, a chitinaserelated receptor-like kinase, interacts with NtPUB4, an armadillo repeat protein, in tobacco. Biochim Biophys Acta 2003, 1651:50-59. 39. Bhoj VG, Chen ZJ: Ubiquitylation in innate and adaptive immunity. Nature 2009, 458:430-437. 40. Liu C, Qian W, Qian Y, Giltiay NV, Lu Y, Swaidani S, Misra S, Deng L, Chen ZJ, Li X: Act1, a U-box E3 ubiquitin ligase for IL-17 signaling. Sci Signal 2009, 2:ra63. 41. Marmor MD, Yarden Y: Role of protein ubiquitylation in regulating endocytosis of receptor tyrosine kinases. Oncogene 2004, 23:2057-2070. 42. Robatzek S, Chinchilla D, Boller T: Ligand-induced endocytosis of the pattern recognition receptor FLS2 in Arabidopsis. Genes Dev 2006, 20:537-542.

43. Spallek T, Robatzek S, Gohre V: How microbes utilize host ubiquitination. Cell Microbiol 2009, 11:1425-1434. 44. Janjusevic R, Abramovitch RB, Martin GB, Stebbins CE: A  bacterial inhibitor of host programmed cell death defenses is an E3 ubiquitin ligase. Science 2006, 311:222-226. This study reveals that AvrPtoB is a RING/U-box-type E3 ubiquitin ligase by structural analysis and its function in the suppression of cell death. 45. Gohre V, Spallek T, Haweker H, Mersmann S, Mentzel T, Boller T,  de Torres M, Manseld JW, Robatzek S: Plant patternrecognition receptor FLS2 is directed for degradation by the bacterial ubiquitin ligase AvrPtoB. Curr Biol 2008, 18:1824-1832. In this paper, authors show that the bacterial virulence effector protein AvrPtoB is able to ubiquitinate the agellin receptor FLS2 and target it for proteasome degradation. 46. Gimenez-Ibanez S, Hann DR, Ntoukakis V, Petutschnig E, Lipka V,  Rathjen JP: AvrPtoB targets the LysM receptor kinase CERK1 to promote bacterial virulence on plants. Curr Biol 2009, 19:423-429. Similar as in [45], the authors show that the bacterial virulence effector protein AvrPtoB is able to ubiquitinate the CERK1 receptor and target it for lysosome degradation. 47. Tzra T, Vaidya M, Citovsky V: Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium. Nature 2004, 431:87-92. 48. Nomura K, Debroy S, Lee YH, Pumplin N, Jones J, He SY: A bacterial virulence protein suppresses host innate immunity to cause plant disease. Science 2006, 313:220-223. 49. Hotson A, Chosed R, Shu H, Orth K, Mudgett MB: Xanthomonas type III effector XopD targets SUMO-conjugated proteins in planta. Mol Microbiol 2003, 50:377-389. 50. Robert-Seilaniantz A, Navarro L, Bari R, Jones JD: Pathological hormone imbalances. Curr Opin Plant Biol 2007, 10:372-379. 51. Melotto M, Underwood W, Koczan J, Nomura K, He SY: Plant stomata function in innate immunity against bacterial invasion. Cell 2006, 126:969-980. 52. Navarro L, Bari R, Achard P, Lison P, Nemri A, Harberd NP, Jones JD: DELLAs control plant immune responses by modulating the balance of jasmonic acid and salicylic acid signaling. Curr Biol 2008, 18:650-655. 53. Ferrari S, Galletti R, Denoux C, De Lorenzo G, Ausubel FM, Dewdney J: Resistance to Botrytis cinerea induced in Arabidopsis by elicitors is independent of salicylic acid, ethylene, or jasmonate signaling but requires PHYTOALEXIN DEFICIENT3. Plant Physiol 2007, 144:367-379. 54. Groll M, Schellenberg B, Bachmann AS, Archer CR, Huber R, Powell TK, Lindow S, Kaiser M, Dudler R: A plant pathogen virulence factor inhibits the eukaryotic proteasome by a novel mechanism. Nature 2008, 452:755-758. 55. Shabab M, Shindo T, Gu C, Kaschani F, Pansuriya T, Chintha R, Harzen A, Colby T, Kamoun S, van der Hoorn RA: Fungal effector protein AVR2 targets diversifying defense-related cys proteases of tomato. Plant Cell 2008, 20:1169-1183. 56. Wiborg J, OShea C, Skriver K: Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases. Biochem J 2008, 413:447-457. 57. Kraft E, Stone SL, Ma L, Su N, Gao Y, Lau OS, Deng XW, Callis J:  Genome analysis and functional characterization of the E2 and RING-type E3 ligase ubiquitination enzymes of Arabidopsis. Plant Physiol 2005, 139:1597-1611. A detailed functional analysis of the interaction between E2 conjugating enzymes and RING-type E3 ubiquitin ligases.

Current Opinion in Plant Biology 2010, 13:402408

www.sciencedirect.com