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Profiling FERONIA interaction proteins by Pupylation-based interacting tagging in Arabidopsis

ABSTRACT: FERONIA (FER) is a ubiquitious expressed and a plasma memberane-localized receptor-like protein kinase which modulate many biological functions. Elucidating the interacting proteins is the crucial step in understanding the molecular function of FER in regulating plant development. However, weak and transient interactions make identifying FER interacting proteins a challenge. Here, we adapted a novel proximity-based labelling technique, pupylation-based interaction tagging (PUP-IT), to label interacting proteins, subsequent enriched by affinity capture, and analyzed by liquid chromatography-coupled tandem mass spectrometery (LC-MS/MS) to profile FER interacting proteins. To perform a quick screen, a transient protoplasts expression system was conducted to identify interacting proteins in mesophyll cells. In addition, we generated a transgenic line that harboring a tagging substrate-inducible system and FER-ligase recombinant protein driven by a native promoter for surveying the interacting proteins in seedlings and flower organ. We introduce and prove the concept of PUP-IT tool designed to detect protein interactions of significance in plants.

INSTRUMENT(S): Orbitrap Fusion Lumos

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

TISSUE(S): Flower, Root, Leaf, Shoot

SUBMITTER: Zhuoran Lin

LAB HEAD: Kun-Hsiang Liu

PROVIDER: PXD052510 | Pride | 2024-08-13

REPOSITORIES: Pride

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Project description:Pupylation is a posttranslational protein modification in bacteria resembling ubiquitination in eukaryotes. The prokaryotic ubiquitin-like protein Pup is covalently attached to other proteins via an isopeptide bond between its carboxyterminal glutamate residue and a lysine residue in the target. In mycobacteria, pupylation was shown to mark proteins for unfolding by the ATPase Mpa and subsequent degradation by the proteasome. However, the occurrence of pupylation in species without a proteasome like Corynebacterium glutamicum suggests that degradation may not be the only fate of pupylated proteins. The Îpup mutant senses a stronger iron limitation than the wild type. Among the 125 genes showing at least 2-fold changes in transcript levels in the Îpup mutant (p-value â¤ 0.05) were 54% of all genes known to be regulated by the master regulator of iron homeostasis DtxR (Brune et al., 2006; Wennerhold and Bott, 2006). Except for ftn, which is activated by DtxR and showed a 2-fold decreased mRNA level, the other DtxR target genes showed increased mRNA levels in the Îpup strain. These included ripA, encoding a transcriptional regulator of iron proteins, which represses a number of prominent iron-containing proteins under iron limitation, such as aconitase or succinate dehydrogenase (Wennerhold et al., 2005). 79% of the known RipA target genes showed decreased mRNA levels in the Îpup strain. DNA microarray analyses were performed to compare the mRNA levels of the C. glutamicum Îpup mutant and its parent wild type under iron-limited conditions. The two strains precultivated in CGXII medium with 4% (w/v) glucose and 1 ÂµM FeSO4 were inoculated into fresh medium to an OD600 of 1, cultured for 2 h, and harvested on ice by centrifugation (5 min at 4,000 g and 4Â°C). Please note that the GPL16989 array design comprises oligos for 4 different bacterial genomes. In the GPR-files in this study, all IDs/Names (oligonucleotides) which are not from the host C. glutamicum were replaced by the text EMPTY since only C. glutamicum expression was analyzed.

			Action	DRS
	16.pdResult	Other
	16.raw	Raw
	17.pdResult	Other
	17.raw	Raw
	18.pdResult	Other

Dataset Information

Profiling FERONIA interaction proteins by Pupylation-based interacting tagging in Arabidopsis

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