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Identification of flagellin peptides in N. benthamiana apoplastic fluid

ABSTRACT: Purified flagellin from Pseudomonas Pta6605 WT was supplemented with N. benthamiana apoplastic fluids from plants transiently expressing only p19 or p19 and bGAL1 (a galactosidase). By performing subsequent LC/MSMS we wanted to identify and quantify the levels of flagellin peptides released by endogenous peptidases in the AF.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Nicotiana Benthamiana Pseudomonas Amygdali Pv. Tabaci

SUBMITTER: Farnusch Kaschani

LAB HEAD: Farnusch Kaschani

PROVIDER: PXD011823 | Pride | 2019-04-15

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	ACE_0383_SOI_v02.fasta	Fasta
	ACE_0387_PB28.raw	Raw
	ACE_0387_PB29.raw	Raw
	ACE_0387_PB30.raw	Raw
	ACE_0387_PB31.raw	Raw

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1 - 5 of 16

Publications

Glycosidase and glycan polymorphism control hydrolytic release of immunogenic flagellin peptides.

Buscaill Pierre P Chandrasekar Balakumaran B Sanguankiattichai Nattapong N Kourelis Jiorgos J Kaschani Farnusch F Thomas Emma L EL Morimoto Kyoko K Kaiser Markus M Preston Gail M GM Ichinose Yuki Y van der Hoorn Renier A L RAL

Science (New York, N.Y.) 20190401 6436

Plants and animals recognize conserved flagellin fragments as a signature of bacterial invasion. These immunogenic elicitor peptides are embedded in the flagellin polymer and require hydrolytic release before they can activate cell surface receptors. Although much of flagellin signaling is understood, little is known about the release of immunogenic fragments. We discovered that plant-secreted β-galactosidase 1 (BGAL1) of <i>Nicotiana benthamiana</i> promotes hydrolytic elicitor release and acts ...[more]

PMID: 30975858

Similar Datasets

Re-annotated role of beta-Galactosidase in Flagellin deglycosylation

Project description:Most Eukaryotes recognise flagellin as a signature of bacterial invasion. In contrast to animals, plants do not recognise flagellin proteins, but conserved peptides released from flagellin (Felix et al., 1999). However, these peptides (e.g. flg22) are folded and buried deeply inside the flagellin polymer and would need to be released before they can interact with cell surface receptors, such as FLS2 (Fliegman & Felix, 2016). Here we discovered that the hydrolytic pathway releasing the flagellin elicitor in plants is initiated by a host-secreted beta-galactosidase (BGAL), which removes the terminal modified viosamine (mVio) from the O-glycan that cloaks the flagellin polymer. BGAL contributes to flagellin-dependent immunity but only against bacterial Pseudomonas syringae strains that carry mVio. Signatures of arms races at this new level of antagonistic interactions are that BGAL is suppressed during infection by a heat stable metabolite secreted by bacteria, and that other P. syringae strains carry BGAL-insensitive O-glycans.

2019-04-15 | PXD010461 | Pride

The transcriptome, extracellular proteome and active secretome of agroinfiltrated N. benthamiana uncover an expanded protease repertoire

Project description:Background: The infiltration of disarmed Agrobacterium tumefaciens into leaves of Nicotiana benthamiana (agroinfiltration) is a widely-adopted procedure to produce and study proteins in plant science, for instance to determine subcellular localization and protein-protein interactions. Agroinfiltration also facilitates quick and safe production of biopharmaceuticals. However, the agroinfiltration expression platform is hampered by unintended proteolysis, some of which might be associated with an elevated immune response. Results: Here, we studied the transcriptome, extracellular proteome and active secretome of agroinfiltrated leaves over a time course, with and without the p19 silencing inhibitor. Remarkably, p19 expression had little effect on the leaf transcriptome and no effect on the extracellular proteome. 25% of the detected transcripts changed in abundance upon agroinfiltration, associated with a gradual upregulation of immunity and a downregulation of photosynthesis. By contrast, 70% of the extracellular proteins increase in abundance and for some proteins this coincides with increased efficiency of extracellular delivery. The protease repertoire of N. benthamiana is expanded compared to other model plants and consists of 1245 proteases and protease homologs. We detect transcripts encoding for 975 proteases, identified extracellular peptides from 196 proteases and monitored 17 active extracellular Ser and Cys proteases in agroinfiltrated leaves. Conclusions: Analysis of agroinfiltrated leaves at transcript, protein and activity levels uncover transcriptional and post-transcriptional/translational reprogramming upon agroinfiltration, associated with substantial increase of the extracellular proteome, and a complex, expanded protease repertoire. This dataset increases our understanding of the plant response to agroinfiltration and indicates ways to improve a key expression platform for both plant science and molecular farming.

2017-10-24 | PXD006708 | Pride

Ubiquitin-independent disassembly by a p97 AAA-ATPase complex drives PP1 holoenzyme formation

Project description:Protein phosphatase-1 (PP1) dephosphorylates a broad spectrum of substrates to regulate a myriad of cellular processes. This functional diversity relies on the ordered assembly of alternative PP1 holoenzymes. Here, we show that newly synthesized PP1 is first held in an inactive complex by its partners SDS22 and inhibitor-3 (I3) that needs to be disassembled by the p97 AAA-ATPase to promote exchange to substrate specifiers. Unlike p97-mediated degradative processes that require the Ufd1-Npl4 ubiquitin adapters, p97 is targeted to PP1 by p37 and related adapter proteins. Reconstitution with purified components revealed direct interaction of the p37 SEP domain with I3 without the need of ubiquitination, and ATP-driven pulling of I3 into the central channel of the p97 hexamer, which triggers dissociation of I3 and SDS22. Thus, we establish regulatory ubiquitin-independent protein complex disassembly as part of the functional arsenal of p97 and define an unanticipated essential step in PP1 biogenesis that illustrates the molecular challenges of ordered subunit exchange.

2018-10-23 | PXD010750 | Pride

Cathepsin L Regulates Metabolic Networks Controlling Rapid Cell Growth and Proliferation

Project description:Rapidly proliferating cells reshape their metabolism to satisfy their ever-lasting need for building blocks. This phenomenon is exemplified in certain malignant conditions but also occurs during embryonic development, when cells rely heavily on glycolytic metabolism to exploit their metabolic intermediates for biosynthetic routes. How cells regulate this metabolic makeover is currently unknown. Here we report that loss of cathepsin L (CtsL) is associated with fast proliferation rate and enhanced glycolytic metabolism that depend on lactate dehydrogenase A (LDHA) activity. Furthermore, we show that CtsL inhibition led to increased LDHA expression, suggesting of a causal relationship between LDHA expression and function. In conclusion, we propose that CtsL regulate this metabolic circuit to keep cell division under check.

2019-05-06 | PXD011588 | Pride

Isolation and characterisation of a 14kDa adhesin protein from Rhizobium leguminosarum biovar viciae 3841

Project description:In this project we aimed to identify a 14 kDa adhesin protein from Rhizobium leguminosarum biovar viciae 3841 unsing in gel digestion coupled to LC-MS/MS.

2024-08-19 | PXD029089 | Pride

Proteasome activity profiling uncovers alteration of catalytic β2 and β5 subunits of the stress-induced proteasome during salinity stress in tomato roots

Project description:The stress proteasome in the animal kingdom facilitates faster conversion of oxidized proteins during stress conditions by incorporating different catalytic β subunits. Plants deal with similar kind of stresses and also carry multiple paralogous genes encoding for each of the three catalytic β subunits. Here we investigated the existence of stress proteasomes upon salt stress in tomato roots. In contrast to Arabidopsis thaliana, tomato has a simplified proteasome gene set with single genes encoding each β subunit except for two genes encoding β2. Using proteasome activity profiling on tomato roots during salt stress we discovered a transient modification of the catalytic subunits of the proteasome coinciding with a loss of viability. This stress-induced proteasome disappears at later time points and coincides with the need to degrade oxidized proteins during salt stress. Subunit-selective proteasome probes and MS analysis of fluorescent 2D gels demonstrated that the stress-induced proteasome is not caused by altered subunit assembly, but involves an increased molecular weight (MW) of both β2 and β5 subunits, and an additional acidic pI shift for β5, whilst β1 remains mostly unchanged. Although the underlying subunit modification is unknown, we can rule out ubiquitination, phosphorylation and glycosylation as underlying molecular mechanisms. This stress-induced proteasome may play an important role in PCD during abiotic stress.

2017-02-22 | PXD005266 | Pride

Project description:Agroinfiltrated Nicotiana benthamiana is a flexible and scalable recombinant protein (RP) production platform, but its great potential is hampered by plant proteases that degrade RPs. Here, we tested 29 candidate protease inhibitors (PIs) in agroinfiltrated N. benthamiana leaves for enhancing accumulation of three unrelated RPs: glycoenzyme α-Galactosidase, glycohormone erythropoietin (EPO) and IgG antibody VRC01. Of the previously described RP accumulation enhancing PIs, we found the cystatin SlCYS8 to be effective. We identified three additional new, unrelated PIs that enhanced RP accumulation: N. benthamiana NbPR4, NbPot1 and human HsTIMP, which are described to inhibit cysteine, serine and metalloproteases, respectively. Remarkably, accumulation of three unrelated RPs is enhanced by each PI, suggesting the mechanism of RP degradation may contain universal elements. Inhibitory motifs of HsTIMP and SlCYS8 are required to enhance RP accumulation, suggesting their target proteases may degrade RPs. Different PIs additively enhance RP accumulation, but the effect of each PI is dose-dependent. Activity-based Protein Profiling (ABPP) revealed that the activity profiles of Papain-like Cys proteases (PLCPs), Ser hydrolases (SHs) or Vacuolar Processing Enzymes (VPEs) in leaves are unaffected by the new PIs, whereas SlCYS8 specifically only suppresses PLCP activity. Quantitative leaf proteome data indicate that the three new PIs affect agroinfiltrated tissues similarly and that they all increase plant immunity. Our data indicate that RPs are degraded stepwise by a redundant, dynamic network of plant proteases. NbPR4, NbPot1 and HsTIMP can be used to identify and control new plant proteases and to enhance RP accumulation in industrial molecular farming.

2018-05-17 | PXD008538 | Pride

Effect of Cpd1 on melanoma proteome

Project description:Investigation of the time dependent effect of Cpd1 (small molecule interactor) on the proteome composition of melanoma WM3734 line by label-free quantification using mass spectrometry. The goal was to identify which protein abundances are increased upon treatment with Cpd1. Melanoma cell line WM3734 was treated with Cpd1 and with the control DMSO. Samples were taken at two time points, 24h and 72h.

2022-03-14 | PXD008757 | Pride

Activity profiling of αlpha-glycosidases in plants

Project description:With nearly 140 α-glycosidases in 14 different families, plants are well equipped with enzymes that can break the α-glucosidic bonds in a large diversity of molecules. Here, we introduce activity profiling of α-glycosidases in plants using α-configured cyclophellitol aziridine probes carrying various fluorophores or biotin. In Arabidopsis, these probes label members of the GH31 family of Glycosyl Hydrolases, including ER-resident α-glucosidase-II RSW3/PSL5 and Golgi-resident α-mannosidase-II HGL1, which both trim N-glycans on glycoproteins. We also detect the active state of extracellular α-glycosidases such as α-xylosidase XYL1, which acts on xyloglucan in the cell wall to promote cell expansion, and α-glucosidase AGLU1, which acts in starch hydrolysis and can suppress fungal invasion. α-glycosidase labelling causes specific signals at 100-130 kDa that are pH-dependent and can be supressed by α-glycosidase inhibitors miglitol and acarbose. The α-glycosidase probes display similar miglitol-sensitive signals in leaf extracts of in a broad range of plant species. To show its use on a non-model plant, we applied glycosidase activity profiling on Crocus sativa, a cash crop for the production of saffron spice. Using a combination of biotinylated glycosidase probes, we identified and quantified 70 active glycosidases in stigma stages 1 and 4 of saffron (Crocus sativa L.), ten of which are differentially active. We also uncover massive changes in hydrolase activities in corms upon infection with Fusarium oxysporum using multiplex fluorescent labelling in combination with probes for serine hydrolases and cysteine proteases. These experiments demonstrate the ease by which active α-glycosidases and other hydrolases can be displayed in non-model plants.

2018-05-17 | PXD009014 | Pride

Using Proteomics for an Insight into the Performance of Activated Sludge

Project description:The performance of a lab-scale wastewater treatment plant during the start-up phase was investigated. A period of varying pH resulted in the loss of ammonium removal efficiency together with a decrease in the specific autotrophic oxygen uptake rate (OUR). From the OUR, it was inferred that the ammonium oxidizing bacteria (AOB) were inhibited by the fluctuation in the pH values. However, OUR alone could not provide the information as to how the AOB were affected at the molecular level. To gain a better insight, shotgun proteomic method was used in this work to quantify the total proteins in the system. Label-free quantification (LFQ) showed that during the time of poor ammonium removal, the marker enzyme hydroxylamine oxidase found in Nitrosomonas sp. was at the lowest LFQ intensity. Based on these results, proteomics has the potential to be used as a monitoring tool. Nevertheless, there are still some restrictions when measuring activated sludge using proteomic method such as the availability of a suitable proteomic database. In this paper, we describe our experience of using publicly available database for identification of activated sludge proteins.

2020-05-18 | PXD014156 | Pride

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