Dataset Information

Arabidopsis thaliana ASK1 proximity labeling upon Pst DC3000 infection

ABSTRACT: we used proximity labeling of ASK1, a core SCF adaptor, to profile dynamic changes in SCF-associated networks during Pseudomonas syringae infection. This analysis revealed widespread remodeling of F-box proteins and immune-related regulators, including several previously uncharacterized components.

INSTRUMENT(S):

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Leaf

SUBMITTER: Christian Montes Serey

LAB HEAD: Justin Walley

PROVIDER: PXD063820 | Pride | 2026-03-05

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	1137_CMS_230725_ASK1-Pst-24h-TID_TMTpro_4x1000ng-QE2-01.raw	Raw
	1137_CMS_230725_ASK1-Pst-24h-TID_TMTpro_4x1000ng-QE2-02.raw	Raw
	1137_CMS_230725_ASK1-Pst-24h-TID_TMTpro_4x1000ng-QE2-03.raw	Raw
	1137_CMS_230725_ASK1-Pst-24h-TID_TMTpro_4x1000ng-QE2-04.raw	Raw
	1138_CMS_230725_ASK1-Pst-1h-TID_TMTpro_4x1000ng-QE2-01.raw	Raw

Items per page:

1 - 5 of 14

Similar Datasets

Project description:Identifying specific protein interactors and spatially or temporally restricted local proteomes contributes significantly to our understanding of cellular processes in virtually all aspects of life. Obtaining such data is challenging, especially when the protein, cell type or event of interest is rare. In recent years, different proximity labeling techniques have been developed that have greatly improved our ability to tackle these questions. However, while effective in mammalian systems, use in plants has been extremely limited due to technical challenges. Recent technological improvements in the form of two highly active versions of the biotin ligase BirA* (TurboID and miniTurboID), prompted us to test this new system on two challenging but widely asked questions in plants: what are interaction partners of low-abundant proteins and what are organellar proteomes in rare and transient cell types. To address the first question, we used the transcription factor FAMA as a test case. FAMA is a master regulator of guard cell development and promotes terminal differentiation of the guard cell precursor by both activating and repressing hundreds of genes. FAMA-expressing young guard cells are rare and restricted to the epidermis of developing aerial tissues, which makes them a good model system to test TurboID applicability under material-limiting conditions. For this experiment, young Arabidopsis seedlings expressing FAMA-TurboID were treated with biotin to label FAMA complexes. Col-0 wild type and seedlings expressing nuclear TurboID under the FAMA promoter were used as controls for unspecific binding of proteins to the beads and stochastic labeling of nuclear proteins, respectively. Biotinylated proteins were isolated by affinity purification with streptavidin-coupled beads and identified by LC-MS/MS. Analysis of proteins labeled by FAMA-TurboID fusions revealed known interactors of this late stomatal lineage specific transcription factor, as well as novel proteins that could explain its dual function as an activator and repressor. Comparison with proteins obtained from classical co-immunoprecipitation approaches with FAMA showed that proximity labeling is superior for identification of meaningful interaction partners of low-abundant proteins.

Project description:Identifying specific protein interactors and spatially or temporally restricted local proteomes contributes significantly to our understanding of cellular processes in virtually all aspects of life. Obtaining such data is challenging, especially when the protein, cell type or event of interest is rare. In recent years, different proximity labeling techniques have been developed that have greatly improved our ability to tackle these questions. However, while effective in mammalian systems, use in plants has been extremely limited due to technical challenges. Recent technological improvements in the form of two highly active versions of the biotin ligase BirA* (TurboID and miniTurboID), prompted us to test this new system on two challenging but widely asked questions in plants: what are interaction partners of low-abundant proteins and what are organellar proteomes in rare and transient cell types. To address the second question, we used nuclei of developing stomatal guard cells, which express the transcription factor FAMA, as our test case. FAMA is a master regulator of guard cell development and promotes terminal differentiation of the guard cell precursor by both activating and repressing hundreds of genes. FAMA-expressing young guard cells are rare and restricted to the epidermis of developing aerial tissues, which makes them a good model system to test TurboID applicability under material-limiting conditions. For this experiment, young Arabidopsis seedlings expressing nuclear TurboID under the FAMA or UBIQUITIN10 (UBQ10) promoter were treated with biotin to label nuclear proteomes. Col-0 wild type was used as controls for unspecific binding of proteins to the beads. Biotinylated proteins were isolated by affinity purification with streptavidin-coupled beads and identified by LC-MS/MS. By targeting TurboID to the nucleus we were able to purify nuclear proteins with relatively high specificity. Enrichment of FAMA-stage specific proteins, when nuclear TurboID was driven by the FAMA compared to the UBQ10 promoter, supports general applicability of TurboID for identification of subcellular proteomes.

Project description:Endoplasmic reticulum (ER)-localized cytochrome P450 enzymes rely on membrane-bound redox partners to establish the P450 monooxygenase system, facilitating electron transfer from pyridine dinucleotide cofactors NADPH and/or NADH. The eukaryotic ER membrane houses two electron transfer pathways: one mediated by NADPH-dependent cytochrome P450 oxidoreductase (CPR) and the other by NADH-dependent cytochrome b5 reductase (CBR) and cytochrome b5 (CB5). In some cases, CB5 can also deliver electrons for P450 enzymes. These systems support P450s and other oxidases across various biological processes. The Arabidopsis thaliana genome encodes five canonical CB5 genes—AtCB5A (At1g26340), AtCB5B (At2g32720), AtCB5C (At2g46650), AtCB5D (At5g48810), and AtCB5E (At5g53560)—all of which encode tail-anchored proteins with an N-terminal heme-binding (cyt-b5) domain and a C-terminal transmembrane (TM) domain. Additionally, Arabidopsis harbors a non-canonical CB5-like protein (AtCB5LP, At1g60660) that shares 36–46% sequence identity with canonical CB5s but exhibits an inverted topology, with its TM domain at the N-terminus and the cyt-b5 domain at the C-terminus. Despite this distinct structural arrangement, the biochemical and physiological roles of AtCB5LP remain largely unknown. Loss of CB5LP in Arabidopsis leads to embryonic defects and post-embryonic lethality. To explore its biological function, we employed proximity labeling using TurboID-tagged CB5LP, which identified 34 potential cytochrome P450 interactors, including CYP51A2. Broad metabolite profiling revealed significant alterations in the phytosterol composition of CB5LP knockout mutants, with elevated 14α-methyl-sterols and reduced sitosterol and stigmasterol levels, underscoring CB5LP’s pivotal role in sterol 14α-demethylation. In vitro assays using yeast microsomal extracts confirmed that CB5LP is essential for CYP51A2 activity, while a mutant variant lacking electron transfer capability failed to support CYP51 function. Collectively, these findings establish CB5LP as a critical electron donor required for CYP51-mediated phytosterol biosynthesis.

Dataset Information

Arabidopsis thaliana ASK1 proximity labeling upon Pst DC3000 infection

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets