Changes in PUB22 Ubiquitination Modes Triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 Dampen the Immune Response

Furlan, Giulia; Nakagami, Hirofumi; Eschen‐Lippold, Lennart; Jiang, Xiyuan; Majovsky, Petra; Kowarschik, Kathrin; Hoehenwarter, Wolfgang; Lee, Justin; Trujillo, Marco

doi:10.1105/tpc.16.00654

Cited by 67 publications

(64 citation statements)

References 73 publications

(124 reference statements)

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“…E3 ligases generally carry out functions in determining the substrate by acting as a scaffold, bringing the E2–Ub conjugate into close proximity to the target, and activating the E2–Ub conjugate by inducing a ‘closed conformation’ that primes Ub for aminolysis (Stewart et al ., ). Notably, most E3 ligases display an inherent autoubiquitination activity in vitro , which often results in their degradation in vivo (Stegmann et al ., ; Furlan & Trujillo, ; Furlan et al ., ). This feature is a straightforward read‐out, which allows the determination of the activity of an E3 Ub ligase.…”

Section: Resultsmentioning

confidence: 97%

“…PUB22, was previously shown to ubiquitinate and mediate the degradation of the Exocyst subunit Exo70B2, which is required for the full activation of the immune response in plants (Stegmann et al ., ; Furlan et al ., ). We inserted a Myc‐tagged Exo70B2 in an operon containing His–Ub, UBA1, HA‐UBC8 and MBP‐PUB22.…”

Section: Resultsmentioning

confidence: 97%

“…Coexpression with PUB20 resulted in a single higher band, suggestive of mono‐ubiquitination. These results further support the fidelity of the system and reflect previous observation of PUB22 activity in vivo (Stegmann et al ., ; Furlan et al ., ).…”

Section: Resultsmentioning

confidence: 97%

“…Proteins were resolved by SDS‐PAGE using tricine or glycine gels, as indicated. Following the separation on SDS‐PAGE and blotting on PVDF membranes (GE Healthcare), samples were analysed by immunoblotting using the indicated antibodies: anti‐cMyc (Sigma Aldrich, St. Louis, MO, USA), anti‐HA (Eurogentech, Liège, Belgium), anti‐ubiquitin P4D1 (Santa Cruz Biotechnology, Dallas, TX, USA), anti‐His (Miltenyi Biotech, Bergisch Gladbach, Germany), anti‐MBP (Sigma‐Aldrich), anti‐GST (GE Healthcare), anti‐GFP Jl‐8 (Takara, Kyoto, Japan) and anti‐PUB22 (Furlan et al ., ).…”

Section: Methodsmentioning

confidence: 97%

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UbiGate: a synthetic biology toolbox to analyse ubiquitination

et al. 2017

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Ubiquitination is mediated by an enzymatic cascade that results in the modification of substrate proteins, redefining their fate. This post-translational modification is involved in most cellular processes, yet its analysis faces manifold obstacles due to its complex and ubiquitous nature. Reconstitution of the ubiquitination cascade in bacterial systems circumvents several of these problems and was shown to faithfully recapitulate the process. Here, we present UbiGate - a synthetic biology toolbox, together with an inducible bacterial expression system - to enable the straightforward reconstitution of the ubiquitination cascades of different organisms in Escherichia coli by 'Golden Gate' cloning. This inclusive toolbox uses a hierarchical modular cloning system to assemble complex DNA molecules encoding the multiple genetic elements of the ubiquitination cascade in a predefined order, to generate polycistronic operons for expression. We demonstrate the efficiency of UbiGate in generating a variety of expression elements to reconstitute autoubiquitination by different E3 ligases and the modification of their substrates, as well as its usefulness for dissecting the process in a time- and cost-effective manner.

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 97%

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UbiGate: a synthetic biology toolbox to analyse ubiquitination

et al. 2017

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“…Preference of MAP kinases towards specific residues within a set of potential phosphosites has been shown previously in other substrates (e.g. Furlan et al ., ). Thus the identity of the kinase(s) phosphorylating T392 and/or T393 remains elusive in light of current kinase analysis tools.…”

Section: Discussionmentioning

confidence: 97%

Characterization of auxin transporter PIN6 plasma membrane targeting reveals a function for PIN6 in plant bolting

et al. 2017

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Auxin gradients are sustained by series of influx and efflux carriers whose subcellular localization is sensitive to both exogenous and endogenous factors. Recently the localization of the Arabidopsis thaliana auxin efflux carrier PIN-FORMED (PIN) 6 was reported to be tissue-specific and regulated through unknown mechanisms. Here, we used genetic, molecular and pharmacological approaches to characterize the molecular mechanism(s) controlling the subcellular localization of PIN6. PIN6 localizes to endomembrane domains in tissues with low PIN6 expression levels such as roots, but localizes at the plasma membrane (PM) in tissues with increased PIN6 expression such as the inflorescence stem and nectary glands. We provide evidence that this dual localization is controlled by PIN6 phosphorylation and demonstrate that PIN6 is phosphorylated by mitogen-activated protein kinases (MAPKs) MPK4 and MPK6. The analysis of transgenic plants expressing PIN6 at PM or in endomembrane domains reveals that PIN6 subcellular localization is critical for Arabidopsis inflorescence stem elongation post-flowering (bolting). In line with a role for PIN6 in plant bolting, inflorescence stems elongate faster in pin6 mutant plants than in wild-type plants. We propose that PIN6 subcellular localization is under the control of developmental signals acting on tissue-specific determinants controlling PIN6-expression levels and PIN6 phosphorylation.

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Ubiquitin/Proteasome System in Plant Pathogen Responses

Sorel

Mooney

Marchi

et al. 2019

Annual Plant Reviews Online

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In eukaryotes, the ubiquitin/proteasome system plays crucial roles in the regulation of protein stability. These functions are largely mediated through the covalent attachment of ubiquitin to substrate proteins and their targeting to the 26S proteasome, a large multi‐subunit protease. In the last three decades, the ubiquitin system has been shown to regulate essentially all processes in eukaryotes. In plants, it appears to play particularly important roles, perhaps as a result of the plants' sessile lifestyle and their need to adapt to rapidly changing environmental conditions. In particular, the ubiquitin system is key to the regulation of plant responses to pathogens, not only during interaction with a pathogen but also in the absence of infection to prevent the constitutive activation of defence responses that would have detrimental effects. In this article, we discuss the roles of the ubiquitin system in the regulation of different cellular pathways involved in plant defence responses, including the regulation of transmembrane and intracellular receptors, and the control of the signalling pathways that are activated downstream of pathogen detection. We also review how pathogens hijack the ubiquitin system to increase their virulence. Finally, we briefly discuss the emerging roles of autophagy in the regulation of plant/pathogen interactions.

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Changes in PUB22 Ubiquitination Modes Triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 Dampen the Immune Response

Cited by 67 publications

References 73 publications

UbiGate: a synthetic biology toolbox to analyse ubiquitination

UbiGate: a synthetic biology toolbox to analyse ubiquitination

Characterization of auxin transporter PIN6 plasma membrane targeting reveals a function for PIN6 in plant bolting

Ubiquitin/Proteasome System in Plant Pathogen Responses

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