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.

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.

The Plant Cell, Volume 32, Issue 11, November 2020, Pages 3388–3407; https://doi.org/10.1105/tpc.20.00235 The authors note that they mistakenly omitted an acknowledgment for Ghent University (BOF18-GOA-013 to P.V.D and A.G.). from the final version of this article. The revised acknowledgment section is as follows: This work was supported by the European Research Council (T-Rex project 682436 to D.V.D. and PROPHECY project 803972 to P.V.D.), the National Science Foundation Flanders (grant G009415N to D.V.D. and G.D.J.), the Deutsche Forschungsgemeinschaft in frame of the Collaborative Research Center 924 (Sonderforschungsbereich 924, grant INST 95/1126-2, Project B4 to T.O. and N.B.A.), the Vetenskapsrådet (VR; grant 21679000 to P.N.M.), the Swedish Research Council Formas (grant 22924-000 to P.N.M.), the Carl Trygger Foundation for Scientific Research (grants 15:336 and 17:317 to P.M.), the Foundation of Research and Technology Hellas (IMBB-FORTH; to P.N.M.), and Ghent University (BOF18-GOA-013 to P.V.D and A.G.). These details have been corrected only...