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Publikation

Rausche, J.; Stenzel, I.; Stauder, R.; Fratini, M.; Trujillo, M.; Heilmann, I.; Rosahl, S.; A phosphoinositide 5-phosphatase from Solanum tuberosum is activated by PAMP-treatment and may antagonize phosphatidylinositol 4,5-bisphosphate at Phytophthora infestans infection sites New Phytol. 229, 469-487, (2021) DOI: 10.1111/nph.16853

Potato (Solanum tuberosum) plants susceptible to late blight disease caused by the oomycete Phytophthora infestans display enhanced resistance upon infiltration with the pathogen-associated molecular pattern (PAMP), Pep-13. Here, we characterize a potato gene similar to Arabidopsis 5-phosphatases which was identified in transcript arrays performed to identify Pep-13 regulated genes, and termed StIPP.Recombinant StIPP protein specifically dephosphorylated the D5-position of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in vitro. Other phosphoinositides or soluble inositolpolyphosphates were not converted.When transiently expressed in tobacco (Nicotiana tabacum) pollen tubes, a StIPP-YFP fusion localized to the subapical plasma membrane and antagonized PtdIns(4,5)P2-dependent effects on cell morphology, indicating in vivo functionality. Phytophthora infestans-infection of N. benthamiana leaf epidermis cells resulted in relocalization of StIPP-GFP from the plasma membrane to the extra-haustorial membrane (EHM). Colocalizion with the effector protein RFP-AvrBlb2 at infection sites is consistent with a role of StIPP in the plant–oomycete interaction. Correlation analysis of fluorescence distributions of StIPP-GFP and biosensors for PtdIns(4,5)P2 or phosphatidylinositol 4-phosphate (PtdIns4P) indicate StIPP activity predominantly at the EHM.In Arabidopsis protoplasts, expression of StIPP resulted in the stabilization of the PAMP receptor, FLAGELLIN-SENSITIVE 2, indicating that StIPP may act as a PAMP-induced and localized antagonist of PtdIns(4,5)P2-dependent processes during plant immunity.
Publikation

Brillada, C.; Teh, O.-K.; Ditengou, F. A.; Lee, C.-W.; Klecker, T.; Saeed, B.; Furlan, G.; Zietz, M.; Hause, G.; Eschen-Lippold, L.; Hoehenwarter, W.; Lee, J.; Ott, T.; Trujillo, M.; Exocyst subunit Exo70B2 is linked to immune signaling and autophagy Plant Cell 33, 404-419, (2021) DOI: 10.1093/plcell/koaa022

During the immune response, activation of the secretory pathway is key to mounting an effective response, while gauging its output is important to maintain cellular homeostasis. The Exo70 subunit of the exocyst functions as a spatiotemporal regulator by mediating numerous interactions with proteins and lipids. However, a molecular understanding of the exocyst regulation remains challenging. We show that, in Arabidopsis thaliana, Exo70B2 behaves as a bona fide exocyst subunit. Conversely, treatment with the salicylic acid (SA) defence hormone analog benzothiadiazole (BTH), or the immunogenic peptide flg22, induced Exo70B2 transport into the vacuole. We reveal that Exo70B2 interacts with AUTOPHAGY-RELATED PROTEIN 8 (ATG8) via two ATG8-interacting motives (AIMs) and its transport into the vacuole is dependent on autophagy. In line with its role in immunity, we discovered that Exo70B2 interacted with and was phosphorylated by the kinase MPK3. Mimicking phosphorylation had a dual impact on Exo70B2: first, by inhibiting localization at sites of active secretion, and second, it increased the interaction with ATG8. Phosphonull variants displayed higher effector-triggered immunity (ETI) and were hypersensitive to BTH, which induce secretion and autophagy. Our results suggest a molecular mechanism by which phosphorylation diverts Exo70B2 from the secretory into the autophagy pathway for its degradation, to dampen secretory activity.
Publikation

Orosa, B.; Üstün, S.; Calderón Villalobos, L. I. A.; Genschik, P.; Gibbs, D.; Holdsworth, M. J.; Isono, E.; Lois, M.; Trujillo, M.; Sadanandom, A.; Plant proteostasis – shaping the proteome: a research community aiming to understand molecular mechanisms that control protein abundance New Phytol. 227, 1028-1033, (2020) DOI: 10.1111/nph.16664

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Preprints

Teh, O.-K.; Lee, C.-W.; Ditengou, F. A.; Klecker, T.; Furlan, G.; Zietz, M.; Hause, G.; Eschen-Lippold, L.; Hoehenwarter, W.; Lee, J.; Ott, T.; Trujillo, M.; Phosphorylation of the exocyst subunit Exo70B2 contributes to the regulation of its function bioRxiv (2018) DOI: 10.1101/266171

The exocyst is a conserved hetero-octameric complex that mediates early tethering of post-Golgi vesicles during exocytosis. Its Exo70 subunit functions as a spatiotemporal regulator by mediating numerous interactions with proteins and lipids. However, a molecular understanding of the exocyst functions remains challenging. Exo70B2 localized to dynamic foci at the plasma membrane and transited through Brefeldin A (BFA)-sensitive compartments, indicating that it participates in conventional secretion. Conversely, treatment with the immunogenic peptide flg22 or the salicylic acid (SA) defence hormone analogue Benzothiadiazole (BTH), induced Exo70B2 transport into the vacuole where it colocalized with autophagic markers AUTOPHAGY-RELATED PROTEIN 8 (ATG8) and NEIGHBOR OF BRCA1 GENE 1 (NBR1). According with its role in immunity, we discovered that Exo70B2 interacts with and is phosphorylated by the MITOGEN-ACTIVATED PROTEIN KINASE 3 (MPK3). Mimicking phosphorylation inhibited Exo70B2 localization at sites of active secretion. By contrast, lines expressing phosphonull variants displayed higher Effector-Triggered Immunity and were hypersensitive to BTH, conditions known to induce the secretory pathway. Our results suggest a molecular mechanism by which phosphorylation of Exo70B2 regulates interaction with the plasma membrane, and couples the secretory pathway with cellular signalling.
Publikation

Turek, I.; Tischer, N.; Lassig, R.; Trujillo, M.; Multi-tiered pairing selectivity between E2 ubiquitin-conjugating enzymes and E3 ligases J. Biol. Chem. 293, 16324-16336, (2018) DOI: 10.1074/jbc.RA118.004226

Ubiquitination is a prevalent post-translational modification involved in all aspects of cell physiology. It is mediated by an enzymatic cascade and the E2 ubiquitin-conjugating enzymes (UBCs) lie at its heart. Even though E3 ubiquitin ligases determine the specificity of the reaction, E2s catalyse the attachment of ubiquitin and have emerged as key mediators of chain assembly. They are largely responsible for the type of linkage between ubiquitin moieties and thus, the fate endowed onto the modified substrate. However, in vivo E2-E3 pairing remains largely unexplored. We therefore interrogated the interaction selectivity between 37 Arabidopsis E2s and PUB22, a U-box type E3 ubiquitin ligase that is involved in the dampening of immune signalling. We show that while the U-box domain, which mediates E2 docking, is able to interact with 18 out of 37 tested E2s, the substrate interacting armadillo (ARM) repeats impose a second layer of specificity, allowing the interaction with eleven E2s. In vitro activity assayed by autoubiquitination only partially recapitulated the in vivo selectivity. Moreover, in vivo pairing was modulated during the immune response; pairing with group VI UBC30 was inhibited, while interaction with the K63 chain-building UBC35 was increased. Functional analysis of ubc35 ubc36 mutants shows that they partially mimic pub22 pub23 pub24 enhanced activation of immune responses. Together, our work provides a framework to interrogate in vivo E2-E3 pairing and reveals a multi-tiered and dynamic E2-E3 network.
Publikation

Trujillo, M.; News from the PUB: plant U-box type E3 ubiquitin ligases J. Exp. Bot. 69, 371-384, (2018) DOI: 10.1093/jxb/erx411

Plant U-box type E3 ubiquitin ligases (PUBs) are well known for their functions in a variety of stress responses, including immune responses and the adaptation to abiotic stresses. First linked to pollen self-incompatibility, their repertoire of roles has grown to encompass also the regulation of developmental processes. Notably, new studies provide clues to their mode of action, underline the existence of conserved PUB–kinase modules, and suggest new links to G-protein signalling, placing PUBs at the crossroads of major signalling hubs. The frequent association with membranes, by interacting and/or targeting membrane proteins, as well as through a recently reported direct interaction with phospholipids, indicates a general function in the control of vesicle transport and their cargoes. This review aims to give an overview of the most significant advances in the field, while also trying to identify common themes of PUB function.
Publikation

Rajaraman, J.; Douchkov, D.; Lück, S.; Hensel, G.; Nowara, D.; Pogoda, M.; Rutten, T.; Meitzel, T.; Brassac, J.; Höfle, C.; Hückelhoven, R.; Klinkenberg, J.; Trujillo, M.; Bauer, E.; Schmutzer, T.; Himmelbach, A.; Mascher, M.; Lazzari, B.; Stein, N.; Kumlehn, J.; Schweizer, P.; Evolutionarily conserved partial gene duplication in the Triticeae tribe of grasses confers pathogen resistance Genome Biol. 19, 116, (2018) DOI: 10.1186/s13059-018-1472-7

BackgroundThe large and highly repetitive genomes of the cultivated species Hordeum vulgare (barley), Triticum aestivum (wheat), and Secale cereale (rye) belonging to the Triticeae tribe of grasses appear to be particularly rich in gene-like sequences including partial duplicates. Most of them have been classified as putative pseudogenes. In this study we employ transient and stable gene silencing- and over-expression systems in barley to study the function of HvARM1 (for H. vulgare Armadillo 1), a partial gene duplicate of the U-box/armadillo-repeat E3 ligase HvPUB15 (for H. vulgare Plant U-Box 15).ResultsThe partial ARM1 gene is derived from a gene-duplication event in a common ancestor of the Triticeae and contributes to quantitative host as well as nonhost resistance to the biotrophic powdery mildew fungus Blumeria graminis. In barley, allelic variants of HvARM1 but not of HvPUB15 are significantly associated with levels of powdery mildew infection. Both HvPUB15 and HvARM1 proteins interact in yeast and plant cells with the susceptibility-related, plastid-localized barley homologs of THF1 (for Thylakoid formation 1) and of ClpS1 (for Clp-protease adaptor S1) of Arabidopsis thaliana. A genome-wide scan for partial gene duplicates reveals further events in barley resulting in stress-regulated, potentially neo-functionalized, genes.ConclusionThe results suggest neo-functionalization of the partial gene copy HvARM1 increases resistance against powdery mildew infection. It further links plastid function with susceptibility to biotrophic pathogen attack. These findings shed new light on a novel mechanism to employ partial duplication of protein-protein interaction domains to facilitate the expansion of immune signaling networks.
Publikation

Kowarschik, K.; Hoehenwarter, W.; Marillonnet, S.; Trujillo, M.; UbiGate: a synthetic biology toolbox to analyse ubiquitination New Phytol. 217, 1749-1763, (2018) DOI: 10.1111/nph.14900

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.
Preprints

Rajaraman, J.; Douchkov, D.; Lück, S.; Hensel, G.; Nowara, D.; Pogoda, M.; Rutten, T.; Meitzel, T.; Höfle, C.; Hückelhoven, R.; Klinkenberg, J.; Trujillo, M.; Bauer, E.; Schmutzer, T.; Himmelbach, A.; Mascher, M.; Lazzari, B.; Stein, N.; Kumlehn, J.; Schweizer, P.; The partial duplication of an E3-ligase gene in Triticeae species mediates resistance to powdery mildew fungi bioRxiv (2017) DOI: 10.1101/190728

In plant-pathogen interactions, components of the plant ubiquitination machinery are preferred targets of pathogen-encoded effectors suppressing defense responses or co-opting host cellular functions for accommodation. Here, we employed transient and stable gene silencing-and over-expression systems in Hordeum vulgare (barley) to study the function of HvARM1 (for H. vulgare Armadillo 1), a partial gene duplicate of the U-box/armadillo-repeat E3 ligase HvPUB15 (for H. vulgare Plant U-Box 15). The partial ARM1 gene was derived from an ancient gene-duplication event in a common ancestor of the Triticeae tribe of grasses comprising the major crop species H. vulgare, Triticum aestivum and Secale cereale. The barley gene HvARM1 contributed to quantitative host as well as nonhost resistance to the biotrophic powdery mildew fungus Blumeria graminis, and allelic variants were found to be associated with powdery mildew-disease severity. Both HvPUB15 and HvARM1 proteins interacted in yeast and plant cells with the susceptibility-related, plastid-localized barley homologs of THF1 (for Thylakoid formation 1) and of ClpS1 (for Clp-protease adaptor S1) of Arabidopsis thaliana. The results suggest a neo-functionalization HvARM1 to increase resistance against powdery mildew and provide a link to plastid function in susceptibility to biotrophic pathogen attack.
Publikation

Matsui, H.; Nomura, Y.; Egusa, M.; Hamada, T.; Hyon, G.-S.; Kaminaka, H.; Watanabe, Y.; Ueda, T.; Trujillo, M.; Shirasu, K.; Nakagami, H.; The GYF domain protein PSIG1 dampens the induction of cell death during plant-pathogen interactions PLOS Genet. 13, e1007037, (2017) DOI: 10.1371/journal.pgen.1007037

The induction of rapid cell death is an effective strategy for plants to restrict biotrophic and hemi-biotrophic pathogens at the infection site. However, activation of cell death comes at a high cost, as dead cells will no longer be available for defense responses nor general metabolic processes. In addition, necrotrophic pathogens that thrive on dead tissue, take advantage of cell death-triggering mechanisms. Mechanisms by which plants solve this conundrum remain described. Here, we identify PLANT SMY2-TYPE ILE-GYF DOMAIN-CONTAINING PROTEIN 1 (PSIG1) and show that PSIG1 helps to restrict cell death induction during pathogen infection. Inactivation of PSIG1 does not result in spontaneous lesions, and enhanced cell death in psig1 mutants is independent of salicylic acid (SA) biosynthesis or reactive oxygen species (ROS) production. Moreover, PSIG1 interacts with SMG7, which plays a role in nonsense-mediated RNA decay (NMD), and the smg7-4 mutant allele mimics the cell death phenotype of the psig1 mutants. Intriguingly, the psig1 mutants display enhanced susceptibility to the hemi-biotrophic bacterial pathogen. These findings point to the existence and importance of the SA- and ROS-independent cell death constraining mechanism as a part of the plant immune system.
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