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Role of ubiquitination in the regulation of plant defence against pathogens
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Role of ubiquitination in the regulation of plant defence against pathogens Alessandra Devoto, Paul R Muskettz and Ken Shirasuy Ubiquitination is emerging as a common regulatory mechanism that controls a range of cellular processes in plants. Recent exciting discoveries from several laboratories suggest that ubiquitination may also play an important role in plant disease resistance. Several putative ubiquitin ligases have been identified as defence regulators. In addition, a combination of genetic screens and gene-silencing technologies has identified subunits and proposed regulators of SCF ubiquitin ligases as essential components of resistance (R)-gene-mediated resistance. Although no ubiquitin ligase targets that are associated with disease resistance have yet been identified in plants, there is evidence that this well-known protein-modification system may regulate plant defences against pathogens. Addresses The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK y e-mail: [email protected] z Max-Plank-Institute for Plant Breeding Research, Department of Plant–Microbe Interactions, Carl-von-Linne´-Weg 10, D-50829 Cologne, Germany
Current Opinion in Plant Biology 2003, 6:307–311 This review comes from a themed issue on Biotic interactions Edited by Barbara Baker and Jane Parker 1369-5266/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S1369-5266(03)00060-8
Abbreviations CHORD cysteine- and histidine-rich domain COI1 CORONATINE INSENSITIVE1 COP9 CONSTITUTIVELY PHOTOMORPHOGENIC9 CUL1 CULLIN1 E1 ubiquitin-activating enzyme E2 ubiquitin-conjugating enzyme E3 ubiquitin-ligating enzyme HSP90 heat shock protein90 JA jasmonic acid LRR leucine-rich-repeat RAR1 REQUIRED FOR MLA RESISTANCE1 RBX1 RING-box protein1 R gene resistance gene SAR systemic acquired resistance SCF SKP1, CDC53p/CUL1 F-box SGT1 SUPPRESSOR OF THE G2 ALLELE OF skp1-4 SKP1 S-PHASE KINASE-ASSOCIATED PROTEIN1 SON1 SUPPRESSOR OF nim1-1
Introduction During their life cycles, plants routinely encounter a range of biotic challenges, including attack by viruses, www.current-opinion.com
bacteria, fungi, nematodes and insects. Like animals, plants have the capacity to recognise potential pathogens and to mount efficient defence responses. Plants have evolved resistance (R) genes that specifically recognise corresponding pathogen avirulence (avr) genes to trigger plant defences in a mechanism known as gene-for-gene resistance [1,2]. R–Avr recognition triggers signal transduction cascades that lead to rapid defence mobilisation. Once triggered, defence signalling pathways commonly promote local responses including the hypersensitive response (HR), an oxidative burst that produces reactive oxygen intermediates, and the accumulation of salicylic acid, a phenolic molecule that is necessary for the induction of systemic immunity (known as ‘systemic acquired resistance’ [SAR] [3]). In this review, we detail the findings that implicate components of the ubiquitin pathway in plant defence signalling.
Relaying signals via E3 ubiquitin ligases in plant defence Ubiquitin-mediated proteolysis is a central regulatory mechanism in the control of several cellular processes in yeast and animals. Ubiquitination has also been implicated in a growing number of plant signalling pathways, including those mediating responses to hormones, light, sucrose, developmental cues and pathogens [4]. The biochemical process of ubiquitination is operated by a multienzymatic system that consists of ubiquitin-activating (E1), -conjugating (E2), and -ligating (E3) enzymes [5]. Carboxy-terminal activation at a cysteine residue of an ubiquitin monomer is catalysed by E1 and E2 enzymes. E3 ubiquitin ligases are the specificity determinants that mediate the final transfer of ubiquitin to the e-aminogroup of target proteins. In some cases, additional ubiquitin moieties are transferred to the target protein by a multiubiquitin chain assembly factor, E4 [6]. Polyubiquitinated proteins are often escorted to the 26S proteasome to undergo degradation. However, recent advances have extended the function of the ubiquitin (and ubiquitinlike) system from simply providing a degradation signal to having more dynamic roles, such as the activation of kinases, provision of a localisation signal, and protein lipidation [7,8]. SCF (SKP1, CDC53p/CUL1 F-box) complexes belong to a family of RING-type E3 ubiquitin ligases that are characterised by the presence of a RING-finger domain [9]. This domain acts as a docking site for E2 enzymes [9]. The RING-finger protein RBX1 (also called REGULATOR OF CULLINS1 [ROC1]) is a core component of the SCF complex, and interacts directly with CULLIN1 Current Opinion in Plant Biology 2003, 6:307–311
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(CUL1). SKP1 links CUL1 to a F-box protein, which acts as an adaptor molecule that recruits specific substrates to the SCF complex for ubiquitination [10]. Structural analysis of the RBX1–CUL1–SKP1–F-box complex revealed that CUL1 serves as a scaffold that orientates the other subunits to allow transfer of the ubiquitin moiety from E2 to the target protein [11]. Arabidopsis contains 694 potential F-box proteins, but functions have been assigned only to a very limited number of these [12]. A role in plant defence has been demonstrated only for two F-box proteins, CORONATINE INSENSITIVE1 (COI1) [13] and SUPPRESSOR OF nim1-1 (SON1) [14]. COI1 controls defence pathways that are regulated by jasmonic acids (JAs), which are signalling molecules that coordinate plant responses to numerous biotic and abiotic stresses [15]. The coi1 mutants fail to express the JAinducible gene PLANT DEFENSIN1.2 (PDF1.2) and are susceptible to insect herbivores and to fungal and bacterial pathogens [16,17]. The COI1 gene encodes a protein that contains an amino-terminal F-box motif and a leucine-richrepeat (LRR) domain at its carboxy-terminal end [13]. Immunoprecipitation studies have demonstrated that COI1 associates with SKP1, CUL1 and RBX1, providing evidence that an SCFCOI1 complex functions to regulate JA-mediated responses in vivo [18,19]. This hypothesis has been further supported by experiments in which the RBX1 gene was silenced in Arabidopsis. This gave rise to plants whose phenotypes were similar to that of the coi1 mutant [19]. The histone deacetylase RPD3b (REDUCED POTASSIUM DEPENDANCY3B) is a potential substrate for COI1-mediated ubiquitination and belongs to a class of transcriptional repressors [18]. Interestingly, some of these repressors have already been linked to the ubiquitination process in mammals [20]. The Arabidopsis F-box protein SON1 regulates a novel induced defence response that is independent of both salicylic acid and SAR [14]. The son1 mutant was isolated as a genetic suppressor of the SAR-compromised mutant non-inducible immunity1 (nim1), and exhibits constitutive resistance against virulent fungal and bacterial pathogens in which SAR-defence-related genes are not induced. Thus, in contrast to COI1, SON1 acts negatively to regulate plant defence responses, indicating that some SCF complexes may exert a negative effect on disease resistance. Several genes encoding other RING-type E3 ubiquitin ligases have been identified as candidates for involvement in defence signalling pathways. For example, the ATL2 and ATL6 genes encode putative RING-finger proteins that are induced rapidly in Arabidopsis after elicitor treatment [21]. Other members of the ATL family, such as EL5 (ELICITOR-RESPONSIVE5) in rice and ACRE-132 (Avr9/Cf-9 RAPIDLY ELICITED-132) in tomato, are also induced very soon after elicitor treatment [22,23]. Several putative E3 ubiquitin ligases that contain a U-box, which is a modified RING-finger motif, can also Current Opinion in Plant Biology 2003, 6:307–311
be induced by elicitors [24,25–27]. Potentially, these RING-type E3 ubiquitin ligases can target defence regulatory proteins for ubiquitination to control downstream events. Loss-of-function studies will be crucial to assess the importance of these proteins in defence signalling in the near future.
Potential influence of ubiquitination in R-gene-mediated disease resistance Five classes of R proteins have been identified to date and the majority are characterised by the presence of either extra- or intra-cellular LRRs, a modular structure that determines protein–protein interaction [28]. For specific details on R proteins and the importance of intramolecular interactions in plant disease resistance see the review by Rathjen and Moffett in this issue. Forward and reverse genetic approaches (combining mutant analyses and gene-silencing tools) have been employed to dissect R-gene-mediated resistance, revealing a complex network of defence pathways. These analyses have identified essential defence signalling components, such as ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), NONRACE-SPECIFIC DISEASE RESISTANCE1 (NDR1) and REQUIRED FOR MLA RESISTANCE1 (RAR1), that are required for resistance mediated by many different R proteins [29]. RAR1 may provide a possible link between ubiquitination and R-gene-mediated resistance. RAR1 is required for R-gene-mediated resistance against a range of pathogens in monocot and dicot species, indicating that its defence function has been conserved over about 150 million years of evolution [30,31,32,33]. RAR1 encodes a predicted cytosolic protein that contains two highly similar but distinct cysteine- and histidine-rich (CHORD) Zn2þbinding domains [30]. These domains are conserved in all eukaryotes except yeast [30]. Metazoan homologues of RAR1 contain a carboxy-terminal extension that has significant homology to a domain found in the yeast protein SGT1(SUPPRESSOR OF THEG2 ALLELEOF skp1-4), raising the possibility that these two proteins may interact in plants [30]. In yeast, one function of SGT1 is to regulate the activity of SCF complexes, with which it associates through interaction with SKP1 [34]. Recent breakthroughs in several laboratories have identified SGT1 as a RAR1interacting protein and, more crucially, as a component of R-gene-mediated resistance [35,36]. Mutant analyses in Arabidopsis and silencing experiments in barley and N. benthamiana have revealed that SGT1 is required for responses that are mediated by a diverse range of R-gene structural types, which induce resistance against a variety of pathogens [35,36,37,38,39]. The conserved function of SGT1 in regulating SCF activity in plants is supported by complementation of yeast sgt1 mutations by the two highly related Arabidopsis SGT1 genes (SGT1a and SGT1b), and by the observation that SGT1 co-immunoprecipitates with core SCF subunits in barley and in www.current-opinion.com
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N. benthamiana extracts [35,36]. Strong evidence to confirm this function has come from the recent discovery that SGT1b is required for SCFTIR1-mediated auxin responses in Arabidopsis [40]. The work by Gray et al. [40] also provides evidence that sgt1b mutants exhibit a reduced response to JA (which is regulated by SCFCOI1; see above), suggesting that SGT1b is a key component of multiple SCF-regulated pathways. These results, together with the finding that silencingof the core SCF subunit SKP1 resulted in loss of N-mediated resistance against tobacco mosaic virus [36], have strengthened the notion that SCFmediated ubiquitination may be an important regulator of R-gene-mediated resistance. The importance of ubiquitination in plant defence is further supported by the finding that subunits of the CONSTITUTIVELY PHOTOMORPHOGENIC9 (COP9) signalosome are essential for N-gene-dependent resistance [36]. The COP9 signalosome is a multiprotein complex that is involved in protein degradation by the 26S proteasome [41]. COP9 mediates several physiological responses that are controlled by SCF-type and non-SCF-type E3 ligases [41]. The structure of the COP9 signalosome is similar to that of the 19S regulatory lid of the proteasome [42]. It functions as a metalloprotease to cleave a ubiquitinlike protein, NEDD8 (for NEURALLY EXPRESSED DEVELOPMENTALLY DOWNREGULATED PROTEIN), from CUL1 in the SCF complex, a process thought to be important in regulating of SCF activity [43,44]. RAR1 and SGT1 interact with the COP9 signalosome in plants, consistent with the idea that these multi-protein complexes may play an important role in establishing disease resistance.
some chaperone-like function in the assembly or regulation of the R protein complex. Excitingly, SGT1 associates with RAR1 and HSP90 in the yeast two-hybrid assay and in plants, supporting this idea (A Takahashi, K Shirasu, unpublished data).
Conclusions As ever, key questions remain to be addressed. How many of the identified components involved in diseaseresistance signalling pathways do actually form and/or activate functional E3-type ubiquitin ligases in plants? What substrates are recruited for ubiquitination? Does ubiquitination mediate the degradation or the activation of substrates to regulate defence responses? At which level(s) of the resistance response does the ubiquitination process operate? We could easily conclude that the adaptability of protein ubiquitination as a cellular regulatory mechanism is now widely recognised and appears to be comparable to that of phosphorylation, another wellstudied protein-modification process (see review by SC Peck, this issue). Many cellular processes are regulated by concerted protein phosphorylation and ubiquitination [5]. To determine the general impact of ubiquitination on plant defence, it will be necessary to identify and determine E3 ubiquitin ligases and their targets, and to define the precise roles of SGT1, SCF, and the COP9 signalosome in the various signalling pathways of R-genemediated resistance. The use of in vivo binding assays, affinity purification of protein complexes and gene-silencing approaches will further contribute to identifying the function of these proteins, and will clarify the role of ubiquitination in plant disease resistance signalling.
Acknowledgements Recent studies in yeast have revealed that SGT1 has multiple functions and associates with several distinct protein complexes. This molecular promiscuity may be explained by the observation that SGT1 has features of the heat shock protein90 (HSP90) co-chaperones. The SGT1 amino-terminal tetratricopeptide repeat (TPR) and central CS (CHORD and SGT1) motifs resemble, respectively, the three-dimensional folds of the hsp70and hsp90-organizing protein (HOP) and P23 families of HSP70 and HSP90 co-chaperones [45,46,47]. Thus, it is tempting to speculate that SGT1 may have a co-chaperone-like function [48]. If so, this is not inconsistent with its role as a regulator of SCF complexes. Supporting this notion, the mouse SCFSKP2 complex contains both SGT1 and HSP90 [43], and small HSPs have been implicated in promoting SCF-mediated ubiquitination [49]. Furthermore, this has raised the possibility that SGT1 may have further roles in R-protein-triggered resistance. The formation and/or activation of R protein complexes may also need such chaperone functions [50]. The Arabidopsis R protein RPM1 (RESISTANCE TO POWDERY MILDEW1) appears to be unstable in rar1 mutant plants [32], suggesting that RAR1 may also play www.current-opinion.com
Research work in KS’s laboratory is supported by grants from the Gatsby Foundation and the Biotechnology and Biological Sciences Research Council (BBSRC). PM’s research is carried out in Jane Parker’s laboratory, and has been funded by The Max-Planck Society, the Alexander von Humboldt Foundation and the BBSRC.
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36. Liu Y, Schiff M, Serino G, Deng XW, Dinesh-Kumar SP: Role of SCF ubiquitin-ligase and the COP9 signalosome in the N genemediated resistance response to Tobacco mosaic virus. Plant Cell 2002, 14:1483-1496. The authors confirm the binding between RAR1 and SGT1 in N. bentamiana. Silencing of SGT1, SKP1 and genes that encode components of the COP9 signalosome compromise the N-dependent resistance response. 37. Austin MJ, Muskett P, Kahn K, Feys BJ, Jones JD, Parker JE: Regulatory role of SGT1 in early R gene-mediated plant defenses. Science 2002, 295:2077-2080. The authors demonstrate that R genes differ in their requirements for SGT1b in Arabidopsis. 38. Tor M, Gordon P, Cuzick A, Eulgem T, Sinapidou E, Mert-Turk F, Can C, Dangl JL, Holub EB: Arabidopsis SGT1b is required for defense signaling conferred by several downy mildew resistance genes. Plant Cell 2002, 14:993-1003. 39. Peart JR, Lui R, Sadanandom A, Malcuit I, Moffett P, Brice DC, Schauser L, Jaggard DAW, Xiao S, Coleman MJ et al.: The ubiquitin ligase-associated protein SGT1 is required for host and nonhost disease resistance in plants. Proc Natl Acad Sci USA 2002, 99:10865-10869. Silencing in N. benthamiana shows that SGT1 functions not only in host but also in non-host resistance. 40. Gray WM, Muskett PR, Chuang H-W, Parker JE: The Arabidopsis SGT1b protein is required for SCFTIR1-mediated auxin response. Plant Cell 2003, in press. 41. Schwechheimer C, Serino G, Deng XW: Multiple ubiquitin ligasemediated processes require COP9 signalosome and AXR1 function. Plant Cell 2002, 14:2553-2563. 42. Schwechheimer C, Deng X-W: COP9 signalosome revisited: a novel mediator of protein degradation. Trends Cell Biol 2001, 11:420-426. 43. Lyapina S, Cope G, Shevchenko A, Serino G, Tsuge T, Zhou CS, Wolf DA, Wei N, Deshaies RJ: Promotion of NEDD8–CUL1 conjugate cleavage by COP9 signalosome. Science 2001, 292:1382-1385.
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The authors demonstrate the in vivo and in vitro association of COP9 with multiple cullins. COP9 promotes cleavage of the ubiquitin-like protein NEDD8 from CUL1. It is possible that cycles of neddylation, in analogy to cycles of protein phosphorylation, may provide different functions in various contexts. 44. Cope GA, Suh GSB, Aravind L, Schwarz SE, Zipursky SL, Koonin EV, Deshaies RJ: Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1. Science 2002, 298:608-611. Using in silico analysis, the authors identified the isopeptidase motif in COP9 signalosome components. The COP9 signalosome possesses the metalloprotease activity that cleaves NEDD8 from CUL1. This is an important function regulating SCF activity. 45. Dubacq C, Guerois R, Courbeyrette R, Kitagawa K, Mann C: Sgt1p contributes to cyclic AMP pathway activity and physically interacts with the adenylyl cyclase Cyr1p/Cdc35p in budding yeast. Eukaryot Cell 2002, 1:568-582. The authors identify SGT1 as an activator of adenylyl cyclase in yeast. SGT1 appears to interact with many other LRR-containing proteins. Structural similarity between the CHORD and SGT1 (CS) domain and p23 is suggested by computer modelling. 46. Garcia-Ranea JA, Mirey G, Camonis J, Valencia A: p23 and HSP20/ alpha-crystallin proteins define a conserved sequence domain present in other eukaryotic protein families. FEBS Lett 2002, 529:162-167. 47. Stemmann O, Neidig A, Kocher T, Wilm M, Lechner J: Hsp90 enables Ctf13p/Skp1p to nucleate the budding yeast kinetochore. Proc Natl Acad Sci USA 2002, 99:8585-8590. 48. Peart JR, Shirasu K: To degrade or not to degrade: the emerging question in plant disease resistance. Physiol Mol Plant Pathol 2002, 61:73-76. 49. den Engelsman J, Keijsers V, de Jong WW, Boelens WC: The small heat-shock protein alpha B-crystallin promotes FBX4dependent ubiquitination. J Biol Chem 2003, 278:4699-4704. 50. Shirasu K, Schulze-Lefert P: Complex formation, promiscuity, and multi-functionality: protein interactions in disease resistance pathways. Trends Plant Sci 2003, in press.
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Role of ubiquitination in the regulation of plant defence against pathogens Alessandra Devoto, Paul R Muskettz and Ken Shirasuy Ubiquitination is emerging as a common regulatory mechanism that controls a range of cellular processes in plants. Recent exciting discoveries from several laboratories suggest that ubiquitination may also play an important role in plant disease resistance. Several putative ubiquitin ligases have been identified as defence regulators. In addition, a combination of genetic screens and gene-silencing technologies has identified subunits and proposed regulators of SCF ubiquitin ligases as essential components of resistance (R)-gene-mediated resistance. Although no ubiquitin ligase targets that are associated with disease resistance have yet been identified in plants, there is evidence that this well-known protein-modification system may regulate plant defences against pathogens. Addresses The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK y e-mail: [email protected] z Max-Plank-Institute for Plant Breeding Research, Department of Plant–Microbe Interactions, Carl-von-Linne´-Weg 10, D-50829 Cologne, Germany
Current Opinion in Plant Biology 2003, 6:307–311 This review comes from a themed issue on Biotic interactions Edited by Barbara Baker and Jane Parker 1369-5266/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S1369-5266(03)00060-8
Abbreviations CHORD cysteine- and histidine-rich domain COI1 CORONATINE INSENSITIVE1 COP9 CONSTITUTIVELY PHOTOMORPHOGENIC9 CUL1 CULLIN1 E1 ubiquitin-activating enzyme E2 ubiquitin-conjugating enzyme E3 ubiquitin-ligating enzyme HSP90 heat shock protein90 JA jasmonic acid LRR leucine-rich-repeat RAR1 REQUIRED FOR MLA RESISTANCE1 RBX1 RING-box protein1 R gene resistance gene SAR systemic acquired resistance SCF SKP1, CDC53p/CUL1 F-box SGT1 SUPPRESSOR OF THE G2 ALLELE OF skp1-4 SKP1 S-PHASE KINASE-ASSOCIATED PROTEIN1 SON1 SUPPRESSOR OF nim1-1
Introduction During their life cycles, plants routinely encounter a range of biotic challenges, including attack by viruses, www.current-opinion.com
bacteria, fungi, nematodes and insects. Like animals, plants have the capacity to recognise potential pathogens and to mount efficient defence responses. Plants have evolved resistance (R) genes that specifically recognise corresponding pathogen avirulence (avr) genes to trigger plant defences in a mechanism known as gene-for-gene resistance [1,2]. R–Avr recognition triggers signal transduction cascades that lead to rapid defence mobilisation. Once triggered, defence signalling pathways commonly promote local responses including the hypersensitive response (HR), an oxidative burst that produces reactive oxygen intermediates, and the accumulation of salicylic acid, a phenolic molecule that is necessary for the induction of systemic immunity (known as ‘systemic acquired resistance’ [SAR] [3]). In this review, we detail the findings that implicate components of the ubiquitin pathway in plant defence signalling.
Relaying signals via E3 ubiquitin ligases in plant defence Ubiquitin-mediated proteolysis is a central regulatory mechanism in the control of several cellular processes in yeast and animals. Ubiquitination has also been implicated in a growing number of plant signalling pathways, including those mediating responses to hormones, light, sucrose, developmental cues and pathogens [4]. The biochemical process of ubiquitination is operated by a multienzymatic system that consists of ubiquitin-activating (E1), -conjugating (E2), and -ligating (E3) enzymes [5]. Carboxy-terminal activation at a cysteine residue of an ubiquitin monomer is catalysed by E1 and E2 enzymes. E3 ubiquitin ligases are the specificity determinants that mediate the final transfer of ubiquitin to the e-aminogroup of target proteins. In some cases, additional ubiquitin moieties are transferred to the target protein by a multiubiquitin chain assembly factor, E4 [6]. Polyubiquitinated proteins are often escorted to the 26S proteasome to undergo degradation. However, recent advances have extended the function of the ubiquitin (and ubiquitinlike) system from simply providing a degradation signal to having more dynamic roles, such as the activation of kinases, provision of a localisation signal, and protein lipidation [7,8]. SCF (SKP1, CDC53p/CUL1 F-box) complexes belong to a family of RING-type E3 ubiquitin ligases that are characterised by the presence of a RING-finger domain [9]. This domain acts as a docking site for E2 enzymes [9]. The RING-finger protein RBX1 (also called REGULATOR OF CULLINS1 [ROC1]) is a core component of the SCF complex, and interacts directly with CULLIN1 Current Opinion in Plant Biology 2003, 6:307–311
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(CUL1). SKP1 links CUL1 to a F-box protein, which acts as an adaptor molecule that recruits specific substrates to the SCF complex for ubiquitination [10]. Structural analysis of the RBX1–CUL1–SKP1–F-box complex revealed that CUL1 serves as a scaffold that orientates the other subunits to allow transfer of the ubiquitin moiety from E2 to the target protein [11]. Arabidopsis contains 694 potential F-box proteins, but functions have been assigned only to a very limited number of these [12]. A role in plant defence has been demonstrated only for two F-box proteins, CORONATINE INSENSITIVE1 (COI1) [13] and SUPPRESSOR OF nim1-1 (SON1) [14]. COI1 controls defence pathways that are regulated by jasmonic acids (JAs), which are signalling molecules that coordinate plant responses to numerous biotic and abiotic stresses [15]. The coi1 mutants fail to express the JAinducible gene PLANT DEFENSIN1.2 (PDF1.2) and are susceptible to insect herbivores and to fungal and bacterial pathogens [16,17]. The COI1 gene encodes a protein that contains an amino-terminal F-box motif and a leucine-richrepeat (LRR) domain at its carboxy-terminal end [13]. Immunoprecipitation studies have demonstrated that COI1 associates with SKP1, CUL1 and RBX1, providing evidence that an SCFCOI1 complex functions to regulate JA-mediated responses in vivo [18,19]. This hypothesis has been further supported by experiments in which the RBX1 gene was silenced in Arabidopsis. This gave rise to plants whose phenotypes were similar to that of the coi1 mutant [19]. The histone deacetylase RPD3b (REDUCED POTASSIUM DEPENDANCY3B) is a potential substrate for COI1-mediated ubiquitination and belongs to a class of transcriptional repressors [18]. Interestingly, some of these repressors have already been linked to the ubiquitination process in mammals [20]. The Arabidopsis F-box protein SON1 regulates a novel induced defence response that is independent of both salicylic acid and SAR [14]. The son1 mutant was isolated as a genetic suppressor of the SAR-compromised mutant non-inducible immunity1 (nim1), and exhibits constitutive resistance against virulent fungal and bacterial pathogens in which SAR-defence-related genes are not induced. Thus, in contrast to COI1, SON1 acts negatively to regulate plant defence responses, indicating that some SCF complexes may exert a negative effect on disease resistance. Several genes encoding other RING-type E3 ubiquitin ligases have been identified as candidates for involvement in defence signalling pathways. For example, the ATL2 and ATL6 genes encode putative RING-finger proteins that are induced rapidly in Arabidopsis after elicitor treatment [21]. Other members of the ATL family, such as EL5 (ELICITOR-RESPONSIVE5) in rice and ACRE-132 (Avr9/Cf-9 RAPIDLY ELICITED-132) in tomato, are also induced very soon after elicitor treatment [22,23]. Several putative E3 ubiquitin ligases that contain a U-box, which is a modified RING-finger motif, can also Current Opinion in Plant Biology 2003, 6:307–311
be induced by elicitors [24,25–27]. Potentially, these RING-type E3 ubiquitin ligases can target defence regulatory proteins for ubiquitination to control downstream events. Loss-of-function studies will be crucial to assess the importance of these proteins in defence signalling in the near future.
Potential influence of ubiquitination in R-gene-mediated disease resistance Five classes of R proteins have been identified to date and the majority are characterised by the presence of either extra- or intra-cellular LRRs, a modular structure that determines protein–protein interaction [28]. For specific details on R proteins and the importance of intramolecular interactions in plant disease resistance see the review by Rathjen and Moffett in this issue. Forward and reverse genetic approaches (combining mutant analyses and gene-silencing tools) have been employed to dissect R-gene-mediated resistance, revealing a complex network of defence pathways. These analyses have identified essential defence signalling components, such as ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), NONRACE-SPECIFIC DISEASE RESISTANCE1 (NDR1) and REQUIRED FOR MLA RESISTANCE1 (RAR1), that are required for resistance mediated by many different R proteins [29]. RAR1 may provide a possible link between ubiquitination and R-gene-mediated resistance. RAR1 is required for R-gene-mediated resistance against a range of pathogens in monocot and dicot species, indicating that its defence function has been conserved over about 150 million years of evolution [30,31,32,33]. RAR1 encodes a predicted cytosolic protein that contains two highly similar but distinct cysteine- and histidine-rich (CHORD) Zn2þbinding domains [30]. These domains are conserved in all eukaryotes except yeast [30]. Metazoan homologues of RAR1 contain a carboxy-terminal extension that has significant homology to a domain found in the yeast protein SGT1(SUPPRESSOR OF THEG2 ALLELEOF skp1-4), raising the possibility that these two proteins may interact in plants [30]. In yeast, one function of SGT1 is to regulate the activity of SCF complexes, with which it associates through interaction with SKP1 [34]. Recent breakthroughs in several laboratories have identified SGT1 as a RAR1interacting protein and, more crucially, as a component of R-gene-mediated resistance [35,36]. Mutant analyses in Arabidopsis and silencing experiments in barley and N. benthamiana have revealed that SGT1 is required for responses that are mediated by a diverse range of R-gene structural types, which induce resistance against a variety of pathogens [35,36,37,38,39]. The conserved function of SGT1 in regulating SCF activity in plants is supported by complementation of yeast sgt1 mutations by the two highly related Arabidopsis SGT1 genes (SGT1a and SGT1b), and by the observation that SGT1 co-immunoprecipitates with core SCF subunits in barley and in www.current-opinion.com
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N. benthamiana extracts [35,36]. Strong evidence to confirm this function has come from the recent discovery that SGT1b is required for SCFTIR1-mediated auxin responses in Arabidopsis [40]. The work by Gray et al. [40] also provides evidence that sgt1b mutants exhibit a reduced response to JA (which is regulated by SCFCOI1; see above), suggesting that SGT1b is a key component of multiple SCF-regulated pathways. These results, together with the finding that silencingof the core SCF subunit SKP1 resulted in loss of N-mediated resistance against tobacco mosaic virus [36], have strengthened the notion that SCFmediated ubiquitination may be an important regulator of R-gene-mediated resistance. The importance of ubiquitination in plant defence is further supported by the finding that subunits of the CONSTITUTIVELY PHOTOMORPHOGENIC9 (COP9) signalosome are essential for N-gene-dependent resistance [36]. The COP9 signalosome is a multiprotein complex that is involved in protein degradation by the 26S proteasome [41]. COP9 mediates several physiological responses that are controlled by SCF-type and non-SCF-type E3 ligases [41]. The structure of the COP9 signalosome is similar to that of the 19S regulatory lid of the proteasome [42]. It functions as a metalloprotease to cleave a ubiquitinlike protein, NEDD8 (for NEURALLY EXPRESSED DEVELOPMENTALLY DOWNREGULATED PROTEIN), from CUL1 in the SCF complex, a process thought to be important in regulating of SCF activity [43,44]. RAR1 and SGT1 interact with the COP9 signalosome in plants, consistent with the idea that these multi-protein complexes may play an important role in establishing disease resistance.
some chaperone-like function in the assembly or regulation of the R protein complex. Excitingly, SGT1 associates with RAR1 and HSP90 in the yeast two-hybrid assay and in plants, supporting this idea (A Takahashi, K Shirasu, unpublished data).
Conclusions As ever, key questions remain to be addressed. How many of the identified components involved in diseaseresistance signalling pathways do actually form and/or activate functional E3-type ubiquitin ligases in plants? What substrates are recruited for ubiquitination? Does ubiquitination mediate the degradation or the activation of substrates to regulate defence responses? At which level(s) of the resistance response does the ubiquitination process operate? We could easily conclude that the adaptability of protein ubiquitination as a cellular regulatory mechanism is now widely recognised and appears to be comparable to that of phosphorylation, another wellstudied protein-modification process (see review by SC Peck, this issue). Many cellular processes are regulated by concerted protein phosphorylation and ubiquitination [5]. To determine the general impact of ubiquitination on plant defence, it will be necessary to identify and determine E3 ubiquitin ligases and their targets, and to define the precise roles of SGT1, SCF, and the COP9 signalosome in the various signalling pathways of R-genemediated resistance. The use of in vivo binding assays, affinity purification of protein complexes and gene-silencing approaches will further contribute to identifying the function of these proteins, and will clarify the role of ubiquitination in plant disease resistance signalling.
Acknowledgements Recent studies in yeast have revealed that SGT1 has multiple functions and associates with several distinct protein complexes. This molecular promiscuity may be explained by the observation that SGT1 has features of the heat shock protein90 (HSP90) co-chaperones. The SGT1 amino-terminal tetratricopeptide repeat (TPR) and central CS (CHORD and SGT1) motifs resemble, respectively, the three-dimensional folds of the hsp70and hsp90-organizing protein (HOP) and P23 families of HSP70 and HSP90 co-chaperones [45,46,47]. Thus, it is tempting to speculate that SGT1 may have a co-chaperone-like function [48]. If so, this is not inconsistent with its role as a regulator of SCF complexes. Supporting this notion, the mouse SCFSKP2 complex contains both SGT1 and HSP90 [43], and small HSPs have been implicated in promoting SCF-mediated ubiquitination [49]. Furthermore, this has raised the possibility that SGT1 may have further roles in R-protein-triggered resistance. The formation and/or activation of R protein complexes may also need such chaperone functions [50]. The Arabidopsis R protein RPM1 (RESISTANCE TO POWDERY MILDEW1) appears to be unstable in rar1 mutant plants [32], suggesting that RAR1 may also play www.current-opinion.com
Research work in KS’s laboratory is supported by grants from the Gatsby Foundation and the Biotechnology and Biological Sciences Research Council (BBSRC). PM’s research is carried out in Jane Parker’s laboratory, and has been funded by The Max-Planck Society, the Alexander von Humboldt Foundation and the BBSRC.
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