Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world's population. Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here we report the sequence of the P. infestans genome, which at approximately 240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for approximately 74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.

Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analyzing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAMM DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs.

Contents Summary13I.Brief history13II.Components of the ubiquitin–proteasome system14III.Ubiquitin‐mediated degradation: a recurrent theme in the plant life cycle18IV.Conclusion and future prospects25Acknowledgements25References25 Summary Ubiquitin is well established as a major modifier of signalling in eukaryotes. However, the extent to which plants rely on ubiquitin for regulating their lifecycle is only recently becoming apparent. This is underlined by the over‐representation of genes encoding ubiquitin‐metabolizing enzymes in Arabidopsis when compared with other model eukaryotes. The main characteristic of ubiquitination is the conjugation of ubiquitin onto lysine residues of acceptor proteins. In most cases the targeted protein is rapidly degraded by the 26S proteasome, the major proteolysis machinery in eukaryotic cells. The ubiquitin–proteasome system is responsible for removing most abnormal peptides and short‐lived cellular regulators, which, in turn, control many processes. This allows cells to respond rapidly to intracellular signals and changing environmental conditions. This review maps out the roles of the components of the ubiquitin–proteasome system with emphasis on areas where future research is urgently needed. We provide a flavour of the diverse aspects of plant lifecycle where the ubiquitin–proteasome system is implicated. We aim to highlight common themes using key examples that reiterate the importance of the ubiquitin–proteasome system to plants. The future challenge in plant biology is to define the targets for ubiquitination, their interactors and their molecular function within the regulatory context.

Previous analysis of transcriptional changes after elicitation of Cf-9 transgenic tobacco (Nicotiana tabacum) by Avr9 peptide revealed a rapidly upregulated gene, ACRE276. We show that ACRE276 is transiently induced in wounded leaves within 15 min, but upon Avr9 elicitor treatment, this upregulation is enhanced and maintained until cell death onset in Cf-9 tobacco. ACRE276 RNA interference (RNAi) silencing in tobacco results in loss of hypersensitive response (HR) specified by Cf resistance genes. ACRE276 RNAi plants are also compromised for HR mediated by the tobacco mosaic virus defense elicitor p50. Silencing tomato (Lycopersicon esculentum) ACRE276 leads to breakdown of Cf-9-specified resistance against Cladosporium fulvum leaf mold. We confirmed that tobacco ACRE276 is an E3 ubiquitin ligase requiring an intact U-box domain. Bioinformatic analyses revealed Arabidopsis thaliana PLANT U-BOX17 (PUB17) and Brassica napus ARC1 as the closest homologs of tobacco ACRE276. Transiently expressing PUB17 in Cf-9 tobacco silenced for ACRE276 restores HR, while mutant PUB17 lacking E3 ligase activity fails to do so, demonstrating that PUB17 ligase activity is crucial for defense signaling. Arabidopsis PUB17 knockout plants are compromised in RPM1-and RPS4-mediated resistance against Pseudomonas syringae pv tomato containing avirulence genes AvrB and AvrRPS4, respectively. We identify a conserved class of U-box ARMADILLO repeat E3 ligases that are positive regulators of cell death and defense across the Solanaceae and Brassicaceae.

In yeast and in animals the ubiquitin-proteasome system (UPS) is responsible for removing or modifying most abnormal peptides and also short-lived cellular regulators. The UPS therefore influences many processes such as the cell cycle, signal transduction, transcription, and stress responses including defence. In recent years, similar regulatory roles have been identified in plants. In Arabidopsis, mutations in the ubiquitin-proteasome pathway block development, circadian rhythms, photomorphogenesis, floral homeosis, hormone responses, senescence, and pathogen invasion. Plants have evolved an armoury of defence mechanisms that allow them to counter infection. These encompass both basal responses, triggered by recognition of conserved pathogen-associated molecular patterns, and pathogen-specific responses, mediated via pathogen- and plant-specific gene-for-gene recognition events. The role of E3 ubiquitin ligases in mediating plant defence signalling is reviewed and examples where pathogens impinge on the host's ubiquitination machinery acting as molecular mimics to undermine defence are also highlighted.

Summary• Signalling by ubiquitination is implicated in diverse aspects of the plant lifecycle, and enzymes of ubiquitin metabolism are overrepresented in the Arabidopsis genome compared with other model eukaryotes. Despite the importance of ubiquitination in the regulation of signalling, little is known about deubiquitinating enzymes, which reverse the process of ubiquitination.• Transgenic RNA interference-based cosuppression and the isolation of Atubp12 ⁄ 13 double mutants collectively provides the first report that AtUBP12 and AtUBP13 are functionally redundant and are required for immunity against virulent Pseudomonas syringae pv tomato in Arabidopsis. The Solanaceous AtUBP12 orthologue NtUBP12 was identified. Viral-induced gene silencing and transient gain-of-function assays were employed to establish that the NtUBP12 protein functions as a negative regulator of the Cf-9-triggered hypersensitive response.• Here, we demonstrate that NtUBP12 and AtUBP12 are bona fide deubiquitinating enzymes capable of cleaving lysine-48-linked ubiquitin chains. AtUBP12 and NtUBP12 are functionally interchangeable and their deubiquitinating activity is required to suppress plant cell death.• Overall, our data implicate AtUBP12-and NtUBP12-dependent deubiquitination in the stabilization of common substrates across Solanaceae and Brassicaceae which regulate disease resistance.

Plant pathogens establish infection by secretion of effector proteins that may be delivered inside host cells to manipulate innate immunity. It is increasingly apparent that the ubiquitin proteasome system (UPS) contributes significantly to the regulation of plant defences and, as such, is a target for pathogen effectors. Bacterial effectors delivered by the type III and IV secretion systems have been shown to interact with components of the host UPS. Some of these effectors possess functional domains that are conserved in UPS enzymes, whilst others contain novel domains with ubiquitination activities. Relatively little is known about effector activities in eukaryotic microbial plant pathogens. Nevertheless, effectors from oomycetes that contain an RXLR motif for translocation to the inside of plant cells have been shown to suppress host defences. Annotation of the genome of one such oomycete, the potato late blight pathogen Phytophthora infestans, and protein-protein interaction assays to discover host proteins targeted by the RXLR effector AVR3a, have revealed that this eukaryotic plant pathogen also has the potential to manipulate host plant UPS functions.

Dishevelled relays Wnt signals from the plasma membrane to different cytoplasmic effectors. Its signalling activity depends on its DIX domain, which undergoes head-to-tail polymerization to assemble signalosomes. The DIX domain is ubiquitinated in vivo at multiple lysines, which can be antagonized by various deubiquitinases (DUBs) including the CYLD tumour suppressor that attenuates Wnt signalling. Here, we generate milligram quantities of pure human Dvl2 DIX domain mono-ubiquitinated at two lysines (K54 and K58) by genetically encoded orthogonal protection with activated ligation (GOPAL), to investigate their effect on DIX polymerization. We show that the ubiquitination of DIX at K54 blocks its polymerization in solution, whereas DIX58-Ub remains oligomerization-competent. DUB profiling identified 28 DUBs that cleave DIX-ubiquitin conjugates, half of which prefer, or are specific for, DIX54-Ub, including Cezanne and CYLD. These DUBs thus have the potential to promote Dvl polymerization and signalosome formation, rather than antagonize it as previously thought for CYLD.