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Large-scale activity profiling of the apoplast upon bacterial infection

ABSTRACT: Pathogens suppress hydrolases in the plant apoplast to cause disease. Despite the importance of these enzyme-inhibitor interaction to plant diseases, to date only a handful examples have been described. Unravelling new plant targets and their inhibitory mechanisms will identify novel components of the plant immune system and point to molecular coevolution operating in the apoplast. Here, we simultaneously monitor the changes in activity of over 170 extracellular enzymes during bacterial infection.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Nicotiana Benthamiana Pseudomonas Syringae Pv. Tomato Str. Dc3000

SUBMITTER: Farnusch Kaschani

LAB HEAD: Farnusch Kaschani

PROVIDER: PXD034869 | Pride | 2023-02-23

REPOSITORIES: Pride

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Large-scale activity profiling of the apoplast upon bacterial infection

Project description:Pathogens suppress hydrolases in the plant apoplast to cause disease. Despite the importance of these enzyme-inhibitor interaction to plant diseases, to date only a handful examples have been described. Unravelling new plant targets and their inhibitory mechanisms will identify novel components of the plant immune system and point to molecular coevolution operating in the apoplast. Here, we simultaneously monitor the changes in activity of over 170 extracellular enzymes during bacterial infection.

2023-02-23 | PXD039897 | Pride

Identification of root specific papain-like cysteine proteases in mays

Project description:Plant proteases are key regulators of plant cell processes such as seed development, immune responses, senescence and programmed cell death (PCD). Apoplastic papain-like cysteine proteases (PLCPs) are hubs in plant-microbe interactions and play an important role during abiotic stresses. The apoplast is a crucial interface for the interaction between plant and microbes. So far, apoplastic maize PLCPs and their function have been mostly described for aerial parts. In this study, we focused on apoplastic PLCPs in the roots of maize plants. We have analyzed the phylogeny of maize PLCPs and investigated their protein abundance after salicylic acid treatment. Using activity-based protein profiling (ABPP) we have identified a novel root-specific PLCP belonging to the RD21-like subfamily, as well as three salicylic acid activated PLCPs. The root specific PLCP CP1C shares sequence and structural similarities to known CP1-like proteases. Biochemical analysis of recombinant CP1C revealed different substrate specificities and inhibitor affinities compared to the related proteases. This study characterized a root-specific PLCP and identifies differences between the SA-dependent activation of PLCPs in roots and leaves.

2019-05-27 | PXD013124 | Pride

Broad-range metalloprotease profiling in plants uncovers immunity provided by defence-related metalloenzyme

Project description:Plants encode for >100 metalloproteases representing >19 different protein families. Tools to study this large and diverse class of proteases have not yet been introduced into plant science. Here, we used hydroxamate-based photoaffinity probes to explore plant proteomes for metalloproteases. We detected labeling of 23 metalloproteases in leaf extracts of the model plant Arabidopsis thaliana that belong to eight unrelated metalloprotease families and localize to different subcellular compartments. We identified several chloroplast localized FtsH proteases; vacuolar aspartyl aminopeptidase DAP1; peroxisomal metalloprotease PMX16; and many cytosolic metalloproteases. We also identified non-proteolytic metallohydrolases involved the release of auxin and in the urea cycle. Studies on tobacco plant Nicotiana benthamiana infected with the bacterial plant pathogen Pseudomonas syringae uncovered PRp27, a secreted protein with anticipated metalloprotease activity. PRp27 depletion increases susceptibility, whilst its overexpression increases resistance, dependent on the presence of active site residues. Collectively, these studies uncover the strength of hydroxamate-based probes to study metalloenzymes in plants

2022-08-12 | PXD032265 | 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

Protease activities induced in the apoplast of the resistant tomato cultivar Hawaii 7996 in response to Ralstonia solanacearum infection

Project description:In the work presented here we aimed to identify the active proteases secreted in the apoplast of the resistant tomato Hawaii 7996 when challenged with R. solanacearum and compared it with that of the susceptible cultivar Marmande. Using ABPP we characterized a variety-specific induction of papain-like cysteine proteases and serine hydrolases.. Altogether this work denotes the importance of novel proteomic approaches –such as ABPP– to provide new insights on old yet elusive questions regarding the molecular basis of resistance to R. solanacearum.

2018-05-18 | PXD005603 | Pride

Transcriptome and proteome profiles of the diazotroph Nitrospirillum amazonense strain CBAmC in response to the sugarcane apoplast fluid

Project description:Background and aims The endophytic diazotrophic strain CBAmC of Nitrospirillum amazonense has been reported as a plant growth promoter of sugarcane variety RB867515 when grown under field conditions. The present work aimed to assess the influence of apoplast fluid from RB867515 on the transcriptomic and proteomic profiles of CBAmC cultured in vitro. Methods RNA-Seq in Ion Proton™ and ESI-LC-MS/MS peptide analysis were used to evaluate the transcriptomic and proteomic profiles, respectively, of CBAmC exposed for 2 h to the sugarcane apoplast fluid. Results The bacterial transcriptomic and proteomic profiles were well correlated. The overall response of CBAmC to the apoplast fluid included overexpression of defense systems against reactive oxygen species (ROS) and osmotic stress, RND efflux pumps for toxic compounds, Sec and Tat secretory systems, and assimilative metabolism of iron. In contrast, active transporters of organic compounds, chemotaxis system and flagellum structure were underexpressed. Conclusions The bacterial metabolic pathways / functions activated in response to the sugarcane apoplast fluid are most likely related to its adaptation to the peculiar characteristics of the fluid. The activation of some of those functions could be determinant for its adaptation to the sugarcane apoplastic niche, and perhaps be involved in the previously observed effect of promoting plant growth. SUBMITTER_CITATION: Terra, L.A., de Soares, C.P., Meneses, C.H.S.G. et al. Plant Soil (2019). Transcriptome and proteome profiles of the diazotroph Nitrospirillum amazonense strain CBAmC in response to the sugarcane apoplast fluid.

2019-04-26 | GSE130321 | GEO

Trichoderma-plant root colonization: escaping early plant defense responses and activation of the antioxidant machinery

Project description:Trichoderma spp. are versatile opportunistic plant symbionts which can colonize the apoplast of plant roots. Microarrays analysis of Arabidopsis thaliana roots inoculated with Trichoderma asperelloides T203, coupled with qPCR analysis of 137 stress-responsive genes and transcription factors, revealed wide gene transcript reprogramming, preceded by a transient repression of the plant immune responses supposedly to allow root colonization. Enhancement in the expression of WRKY18 and 40, which stimulate JA-signaling via suppression of JAZ repressors and negative-regulate the expression of the defense genes FMO1, PAD3 and CYP71A13, was detected in Arabidopsis roots upon Trichoderma colonization. Reduced root colonization was observed in the wrky18/wrky40 double mutant line, while partial phenotypic complementation was achieved by over-expressing WRKY40 in the wrky18 wrky40 background. On the other hand, an increased colonization rate was found in roots of the FMO1 knockout mutant. Two-condition experiment: Roots treated with Trichoderma vs. Control untreated roots. Biological replicates: 2 control replicates, 2 treated replicates. 1 dye-swap.

2012-11-09 | E-GEOD-42113 | biostudies-arrayexpress

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

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

Identification of novel interaction partners of threonine aspartase1

Project description:The goal of this project was to identify interaction partners of human Taspase 1 using a combination of affinity enrichment and mass spectrometry.

2023-03-11 | PXD035546 | Pride

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