Broad-spectrum ubiquitin/ubiquitin-like deconjugation activity of the rhizobial effector NopD from Bradyrhizobium (sp. XS1150).

The post-translational modification of proteins by ubiquitin-like modifiers (UbLs), such as SUMO, ubiquitin, and Nedd8, regulates a vast array of cellular processes. Dedicated UbL deconjugating proteases families reverse these modifications. During bacterial infection, effector proteins, including deconjugating proteases, are released to disrupt host cell defenses and promote bacterial survival. NopD, an effector protein from rhizobia involved in legume nodule symbiosis, exhibits deSUMOylation activity and, unexpectedly, also deubiquitination and deNeddylation activities. Here, we present two crystal structures of Bradyrhizobium (sp. XS1150) NopD complexed with either Arabidopsis SUMO2 or ubiquitin at 1.50 Å and 1.94 Å resolution, respectively. Despite their low sequence similarity, SUMO and ubiquitin bind to a similar NopD interface, employing a unique loop insertion in the NopD sequence. In vitro binding and activity assays reveal specific residues that distinguish between deubiquitination and deSUMOylation. These unique multifaceted deconjugating activities against SUMO, ubiquitin, and Nedd8 exemplify an optimized bacterial protease that disrupts distinct UbL post-translational modifications during host cell infection.

Kinetic Analysis of Plant SUMO Conjugation Machinery.

Plant SUMO conjugation is an essential posttranslational modification involved in plant development and responses to environmental stress. Most likely, this biological diversification is supported by a functional specialization of the different isoforms of the SUMO conjugation machinery. For instance, the two essential Arabidopsis SUMO isoforms, SUMO1/2, display higher conjugation rate than SUMO3 and 5, which are not essential, linking their specific biochemical properties to their biological role. To study the biochemical properties of plant SUMO conjugation systems, quantitative biochemical assays must be performed. We will present a detailed protocol for reconstituting an in vitro SUMO conjugation assay covering all steps from protein preparation to assay development.

(De)Activation (Ir)Reversibly or Degradation: Dynamics of Post-Translational Protein Modifications in Plants.

The increasing dynamic functions of post-translational modifications (PTMs) within protein molecules present outstanding challenges for plant biology even at this present day. Protein PTMs are among the first and fastest plant responses to changes in the environment, indicating that the mechanisms and dynamics of PTMs are an essential area of plant biology. Besides being key players in signaling, PTMs play vital roles in gene expression, gene, and protein localization, protein stability and interactions, as well as enzyme kinetics. In this review, we take a broader but concise approach to capture the current state of events in the field of plant PTMs. We discuss protein modifications including citrullination, glycosylation, phosphorylation, oxidation and disulfide bridges, N-terminal, SUMOylation, and ubiquitination. Further, we outline the complexity of studying PTMs in relation to compartmentalization and function. We conclude by challenging the proteomics community to engage in holistic approaches towards identification and characterizing multiple PTMs on the same protein, their interaction, and mechanism of regulation to bring a deeper understanding of protein function and regulation in plants.

SUMOylation in Phytopathogen Interactions: Balancing Invasion and Resistance.

Plants are constantly confronted by a multitude of biotic stresses involving a myriad of pathogens. In crops, pathogen infections result in significant agronomical losses worldwide posing a threat to food security. In order to enter plant tissues and establish a successful infection, phytopathogens have to surpass several physical, and chemical defense barriers. In recent years, post-translational modification (PTM) mechanisms have emerged as key players in plant defense against pathogens. PTMs allow a highly dynamic and rapid response in front of external challenges, increasing the complexity and precision of cellular responses. In this review, we focus on the role of SUMO conjugation (SUMOylation) in plant immunity against fungi, bacteria, and viruses. In plants, SUMO regulates multiple biological processes, ranging from development to responses arising from environmental challenges. During pathogen attack, SUMO not only modulates the activity of plant defense components, but also serves as a target of pathogen effectors, highlighting its broad role in plant immunity. Here, we summarize known pathogenic strategies targeting plant SUMOylation and, the plant SUMO conjugates involved in host-pathogen interactions. We also provide a catalog of candidate SUMO conjugates according to their role in defense responses. Finally, we discuss the complex role of SUMO in plant defense, focusing on key biological and experimental aspects that contribute to some controversial conclusions, and the opportunities for improving agricultural productivity by engineering SUMOylation in crop species.

Structural insights into SUMO E1-E2 interactions in Arabidopsis uncovers a distinctive platform for securing SUMO conjugation specificity across evolution.

SUMOylation of proteins involves the concerted action of the E1-activating enzyme, E2-conjugating enzyme and E3-ligases. An essential discrimination step in the SUMOylation pathway corresponds to the initial interaction between E1 ubiquitin-fold domain (UFD) and E2 enzymes. Although E2 orthologs possess high sequence identity, the E2 binding region of the UFD domains has diverged across evolution. Moreover, in reciprocal in vitro conjugation reactions Arabidopsis E1 and E2 SCE1 fail to interact efficiently with cognate human E2 Ubc9 and E1 partners, respectively. To gain more insights into the properties of this interface in evolutionary distant organisms, we solved the crystal structure of SUMO E2 SCE1 and its complex with E1 UFD in Arabidopsis. In addition to a few common structural determinants, the interface between the E1 UFD and E2 in Arabidopsis is distinct compared with human and yeast, in particular by the presence of a longer α-helix in the Arabidopsis UFD domain. Despite the variability of E1 UFD domains in these surfaces, they establish specific interactions with highly conserved surfaces of their cognate E2 enzymes. Functional analysis of the different E2 interface residues between human and Arabidopsis revealed Val37 (Met36 in human), as a determinant that provides specificity in the E1-E2 recognition in plants.

Sumoylation in plants: mechanistic insights and its role in drought stress.

Post-translational modification by SUMO is an essential process that has a major role in the regulation of plant development and stress responses. Such diverse biological functions are accompanied by functional diversification among the SUMO conjugation machinery components and regulatory mechanisms that has just started to be identified in plants. In this review, we focus on the current knowledge of the SUMO conjugation system in plants in terms of components, substrate specificity, cognate interactions, enzyme activity, and subcellular localization. In addition, we analyze existing data on the role of SUMOylation in plant drought tolerance in model plants and crop species, paying attention to the genetic approaches used to stimulate or inhibit endogenous SUMO conjugation. The role in drought tolerance of potential SUMO targets identified in proteomic analyses is also discussed. Overall, the complexity of SUMOylation and the multiple genetic and environmental factors that are integrated to confer drought tolerance highlight the need for significant efforts to understand the interplay between SUMO and drought.

SUMOylation Inhibition Mediated by Disruption of SUMO E1-E2 Interactions Confers Plant Susceptibility to Necrotrophic Fungal Pathogens.

Protein modification by SUMO modulates essential biological processes in eukaryotes. SUMOylation is facilitated by sequential action of the E1-activating, E2-conjugating, and E3-ligase enzymes. In plants, SUMO regulates plant development and stress responses, which are key determinants in agricultural productivity. To generate additional tools for advancing our knowledge about the SUMO biology, we have developed a strategy for inhibiting in vivo SUMO conjugation based on disruption of SUMO E1-E2 interactions through expression of E1 SAE2UFDCt domain. Targeted mutagenesis and phylogenetic analyses revealed that this inhibition involves a short motif in SAE2UFDCt highly divergent across kingdoms. Transgenic plants expressing the SAE2UFDCt domain displayed dose-dependent inhibition of SUMO conjugation, and have revealed the existence of a post-transcriptional mechanism that regulates SUMO E2 conjugating enzyme levels. Interestingly, these transgenic plants displayed increased susceptibility to necrotrophic fungal infections by Botrytis cinerea and Plectosphaerella cucumerina. Early after fungal inoculation, host SUMO conjugation was post-transcriptionally downregulated, suggesting that targeting SUMOylation machinery could constitute a novel mechanism for fungal pathogenicity. These findings support the role of SUMOylation as a mechanism involved in plant protection from environmental stresses. In addition, the strategy for inhibiting SUMO conjugation in vivo described in this study might be applicable in important crop plants and other non-plant organisms regardless of their genetic complexity.

Structural analysis and evolution of specificity of the SUMO UFD E1-E2 interactions.

SUMO belongs to the ubiquitin-like family (UbL) of protein modifiers. SUMO is conserved among eukaryotes and is essential for the regulation of processes such as DNA damage repair, transcription, DNA replication and mitosis. UbL modification of proteins occurs via a specific enzymatic cascade formed by the crosstalk between the E1-activating enzyme, the E2-conjugating enzyme and the E3-ligase. An essential discrimination step in all UbL modifiers corresponds to the interaction between E1 and E2 enzymes, which is mediated by the recruitment of the E2 to the UFD domain (Ubiquitin-Fold Domain) of the E1 enzyme. To gain insights in the properties of this interface, we have compared the structures of the complexes between E1 UFD domain and E2 in human and yeast, revealing two alternative UFD platforms that interact with a conserved E2. Comparative sequence analysis of the E1 UFD domain indicates that the E2 binding region has been conserved across phylogenetic closely related species, in which higher sequence conservation can be found in the E2 binding region than in the entire UFD domain. These distinctive strategies for E1-E2 interactions through the UFD domain might be the consequence of a high selective pressure to ensure specificity of each modifier conjugation system.

Kinetic Analysis of Plant SUMO Conjugation Machinery.

Plants display a high diversification degree of the SUMO conjugation machinery, which could confer a biological specialization of the different isoforms. For instance, the two essential Arabidopsis SUMO isoforms, SUMO1/2, display the highest conjugation rate when compared to SUMO3 and 5, suggesting that their specific biochemical properties may be linked to their biological specialization. In order to study the biochemical properties of plant SUMO conjugation systems, quantitative biochemical assays must be performed. We will present a detailed protocol for reconstituting an in vitro SUMO conjugation assay covering all steps from protein preparation to assay development.

Quantitative Analysis of Subcellular Distribution of the SUMO Conjugation System by Confocal Microscopy Imaging.

Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected.

A Simplified and Rapid Method for the Isolation and Transfection of Arabidopsis Leaf Mesophyll Protoplasts for Large-Scale Applications.

Arabidopsis leaf mesophyll protoplasts constitute an important and versatile tool for conducting cell-based experiments to analyze the functions of distinct signaling pathways and cellular machineries using proteomic, biochemical, cellular, genetic, and genomic approaches. Thus, the methods for protoplast isolation and transfection have been gradually improved to achieve efficient expression of genes of interest. Although many well-established protocols have been extensively tested, their successful application is sometimes limited to researchers with a high degree of skill and experience in protoplasts handling. Here we present a detailed method for the isolation and transfection of Arabidopsis mesophyll protoplasts, in which many of the time-consuming and critical steps present in the current protocols have been simplified. The method described is fast, simple, and leads to high yields of competent protoplasts allowing large-scale applications.

Diversification of SUMO-activating enzyme in Arabidopsis: implications in SUMO conjugation.

Sumoylation is an essential posttranslational modification that participates in many biological processes including stress responses. However, little is known about the mechanisms that control Small Ubiquitin-like MOdifier (SUMO) conjugation in vivo. We have evaluated the regulatory role of the heterodimeric E1 activating enzyme, which catalyzes the first step in SUMO conjugation. We have established that the E1 large SAE2 and small SAE1 subunits are encoded by one and three genes, respectively, in the Arabidopsis genome. The three paralogs genes SAE1a, SAE1b1, and SAE1b2 are the result of two independent duplication events. Since SAE1b1 and SAE1b2 correspond to two identical copies, only two E1 small subunit isoforms are present in vivo: SAE1a and SAE1b. The E1 heterodimer nuclear localization is modulated by the C-terminal tail of the SAE2 subunit. In vitro, SUMO conjugation rate is dependent on the SAE1 isoform contained in the E1 holoenzyme and our results suggest that downstream steps to SUMO-E1 thioester bond formation are affected. In vivo, SAE1a isoform deletion in T-DNA insertion mutant plants conferred sumoylation defects upon abiotic stress, consistent with a sumoylation defective phenotype. Our results support previous data pointing to a regulatory role of the E1 activating enzyme during SUMO conjugation and provide a novel mechanism to control sumoylation in vivo by diversification of the E1 small subunit.

Mutations in Escherichia coli aceE and ribB genes allow survival of strains defective in the first step of the isoprenoid biosynthesis pathway.

A functional 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is required for isoprenoid biosynthesis and hence survival in Escherichia coli and most other bacteria. In the first two steps of the pathway, MEP is produced from the central metabolic intermediates pyruvate and glyceraldehyde 3-phosphate via 1-deoxy-D-xylulose 5-phosphate (DXP) by the activity of the enzymes DXP synthase (DXS) and DXP reductoisomerase (DXR). Because the MEP pathway is absent from humans, it was proposed as a promising new target to develop new antibiotics. However, the lethal phenotype caused by the deletion of DXS or DXR was found to be suppressed with a relatively high efficiency by unidentified mutations. Here we report that several mutations in the unrelated genes aceE and ribB rescue growth of DXS-defective mutants because the encoded enzymes allowed the production of sufficient DXP in vivo. Together, this work unveils the diversity of mechanisms that can evolve in bacteria to circumvent a blockage of the first step of the MEP pathway.

Distinctive properties of Arabidopsis SUMO paralogues support the in vivo predominant role of AtSUMO1/2 isoforms.

Protein modification by SUMO (small ubiquitin-related modifier) has emerged as an essential regulatory mechanism in eukaryotes. Even though the molecular mechanisms of SUMO conjugation/deconjugation are conserved, the number of SUMO machinery components and their degree of conservation are specific to each organism. In the present paper, we show data contributing to the notion that the four expressed Arabidopsis SUMO paralogues, AtSUMO1, 2, 3 and 5, have functionally diverged to a higher extent than their human orthologues. We have explored the degree of conservation of these paralogues and found that the surfaces involved in E1-activating enzyme recognition, and E2-conjugating enzyme and SIM (SUMO-interacting motif) non-covalent interactions are well conserved in AtSUMO1/2 isoforms, whereas AtSUMO3 shows a lower degree of conservation, and AtSUMO5 is the most divergent isoform. These differences are functionally relevant, since AtSUMO3 and 5 are deficient in establishing E2 non-covalent interactions, which has not been reported for any naturally occurring SUMO orthologue. In addition, AtSUMO3 is less efficiently conjugated than AtSUMO1/2, and AtSUMO5 shows the lowest conjugation level. A mutagenesis analysis revealed that decreases in conjugation rate and thioester-bond formation are the result of the non-conserved residues involved in E1-activating enzyme recognition that are present in AtSUMO3 and 5. The results of the present study support a role for the E1-activating enzyme in SUMO paralogue discrimination, providing a new mechanism to favour conjugation of the essential AtSUMO1/2 paralogues.

Diversity of the SUMOylation machinery in plants.

In the last decade, SUMOylation has emerged as an essential post-translational modification in eukaryotes. In plants, the biological role of SUMO (small ubiquitin-related modifier) has been studied through genetic approaches that together with recent biochemical studies suggest that the plant SUMOylation system has a high degree of complexity. The present review summarizes our current knowledge on the SUMOylation system in Arabidopsis, focusing on the mechanistic properties of the machinery components identified.