| 1. |
- Liu, Chen, et al.
(författare)
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Establishment of Proximity-Dependent Biotinylation Approaches in Different Plant Model Systems([OPEN])
- 2020
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Ingår i: Plant Cell. - : Oxford University Press (OUP). - 1040-4651 .- 1532-298X. ; 32, s. 3388-3407
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Tidskriftsartikel (refereegranskat)abstract
- Proximity labeling is a powerful approach for detecting protein-protein interactions. Most proximity labeling techniques use a promiscuous biotin ligase or a peroxidase fused to a protein of interest, enabling the covalent biotin labeling of proteins and subsequent capture and identification of interacting and neighboring proteins without the need for the protein complex to remain intact. To date, only a few studies have reported on the use of proximity labeling in plants. Here, we present the results of a systematic study applying a variety of biotin-based proximity labeling approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols that combine mass spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as a test case. We further benchmark the efficiency of various promiscuous biotin ligases in comparison with one-step affinity purification approaches. We identified both known and novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both nonbiotinylated and biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to suggest structural information of protein complexes.A systematic study applying a variety of biotin-based proximity labeling approaches in several plant systems using various conditions and bait proteins.
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| 2. |
- Rafiei, Vahideh, et al.
(författare)
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A Verticillium longisporum pleiotropic drug transporter determines tolerance to the plant host β-pinene monoterpene
- 2022
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Ingår i: Molecular plant pathology. - : Wiley. - 1464-6722 .- 1364-3703. ; 23:2, s. 291-303
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Tidskriftsartikel (refereegranskat)abstract
- Terpenes constitute a major part of secondary metabolites secreted by plants in the rhizosphere. However, their specific functions in fungal–plant interactions have not been investigated thoroughly. In this study we investigated the role of monoterpenes in interactions between oilseed rape (Brassica napus) and the soilborne pathogen Verticillium longisporum. We identified seven monoterpenes produced by B. napus, and production of α-pinene, β-pinene, 3-carene, and camphene was significantly increased upon fungal infection. Among them, β-pinene was chosen for further analysis. Transcriptome analysis of V. longisporum on exposure to β-pinene resulted in identification of two highly expressed pleotropic drug transporters paralog genes named VlAbcG1a and VlAbcG1b. Overexpression of VlAbcG1a in Saccharomyces cerevisiae increased tolerance to β-pinene, while deletion of the VlAbcG1a homologous gene in Verticillium dahliae resulted in mutants with increased sensitivity to certain monoterpenes. Furthermore, the VlAbcG1a overexpression strain displayed an increased tolerance to β-pinene and increased virulence in tomato plants. Data from this study give new insights into the roles of terpenes in plant–fungal pathogen interactions and the mechanisms fungi deploy to cope with the toxicity of these secondary metabolites.
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