BioID is a well-established method for identifying protein–protein interactions and has been utilized within live cells and several animal models. However, the conventional labeling period requires 15–18 h for robust biotinylation which may not be ideal for some applications. Recently, two new ligases termed TurboID and miniTurbo were developed using directed evolution of the BioID ligase and were able to produce robust biotinylation following a 10 min incubation with excess biotin. However, there is reported concern about inducibility of biotinylation, cellular toxicity, and ligase stability. To further investigate the practical applications of TurboID and ascertain strengths and weaknesses compared to BioID, we developed several stable cell lines expressing BioID and TurboID fusion proteins and analyzed them via immunoblot, immunofluorescence, and biotin-affinity purification-based proteomics. For TurboID we observed signs of protein instability, persistent biotinylation in the absence of exogenous biotin, and an increase in the practical labeling radius. However, TurboID enabled robust biotinylation in the endoplasmic reticulum lumen compared to BioID. Induction of biotinylation could be achieved by combining doxycycline-inducible expression with growth in biotin depleted culture media. These studies should help inform investigators utilizing BioID-based methods as to the appropriate ligase and experimental protocol for their particular needs.

Comparison of quantitative disease resistance loci (QDRL) towards the diverse array of soilborne pathogens that affect soybean [Glycine max (L.) Merr.] is key to the incorporation of resistance in breeding programs. The water molds Phytophthora sojae (Kauffman & Gerdmann), Pythium irregulare (Buisman), Pythium ultimum var. ultimum (Trow), and Pythium ultimum var. sporangiiferum (Drechsler) contribute to soybean yield losses annually. Six Soybean Nested Association Mapping (SoyNAM) populations were evaluated for resistance to one or more of these pathogens. Four were screened with a tray test to measure lesion length after inoculation with Ph. sojae; cup assays were used to screen three, three, and two populations for resistance towards Py. irregulare, Py. ultimum var. ultimum, and Py. ultimum var. sporangiiferum, respectively. There were two to eight major or minor QDRL identified within each SoyNAM population towards one or more of these water molds for a total of 33 QDRL. The SoyNAM populations evaluated for resistance to two or more water molds had different QDRL towards each pathogen, indicating that within a source of resistance, mechanisms are potentially specific to the pathogen. Only 3 of the 33 QDRL were associated with resistance to more than one pathogen. There was a major QDRL on chromosome 3 associated with resistance to Py. ultimum var. ultimum and Py. ultimum var. sporangiiferum, and QDRL on chromosomes 13 and 17 shared a flanking marker for both Py. irregulare and Py. ultimum var. ultimum. The SoyNAM population can serve as a diverse resource to map QDRL and compare mechanisms across pathogens and isolates.

Although secondary metabolites are typically associated with competitive or pathogenic interactions, the high bioactivity of endophytic fungi in the Xylariales, coupled with their abundance and broad host ranges spanning all lineages of land plants and lichens, suggests that enhanced secondary metabolism might facilitate symbioses with phylogenetically diverse hosts.Here, we examined secondary metabolite gene clusters (SMGCs) across 96 Xylariales genomes in two clades (Xylariaceae s.l. and Hypoxylaceae), including 88 newly sequenced genomes of endophytes and closely related saprotrophs and pathogens. We paired genomic data with extensive metadata on endophyte hosts and substrates, enabling us to examine genomic factors related to the breadth of symbiotic interactions and ecological roles.All genomes contain hyperabundant SMGCs; however, Xylariaceae have increased numbers of gene duplications, horizontal gene transfers (HGTs) and SMGCs. Enhanced metabolic diversity of endophytes is associated with a greater diversity of hosts and increased capacity for lignocellulose decomposition.Our results suggest that, as host and substrate generalists, Xylariaceae endophytes experience greater selection to diversify SMGCs compared with more ecologically specialised Hypoxylaceae species. Overall, our results provide new evidence that SMGCs may facilitate symbiosis with phylogenetically diverse hosts, highlighting the importance of microbial symbioses to drive fungal metabolic diversity.

The novel coronavirus SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic and has caused a major health and economic burden worldwide. Understanding how SARS-CoV-2 viral proteins behave in host cells can reveal underlying mechanisms of pathogenesis and assist in development of antiviral therapies. Here, the cellular impact of expressing SARS-CoV-2 viral proteins was studied by global proteomic analysis, and proximity biotinylation (BioID) was used to map the SARS-CoV-2 virus–host interactome in human lung cancer-derived cells. Functional enrichment analyses revealed previously reported and unreported cellular pathways that are associated with SARS-CoV-2 proteins. We have established a website to host the proteomic data to allow for public access and continued analysis of host–viral protein associations and whole-cell proteomes of cells expressing the viral–BioID fusion proteins. Furthermore, we identified 66 high-confidence interactions by comparing this study with previous reports, providing a strong foundation for future follow-up studies. Finally, we cross-referenced candidate interactors with the CLUE drug library to identify potential therapeutics for drug-repurposing efforts. Collectively, these studies provide a valuable resource to uncover novel SARS-CoV-2 biology and inform development of antivirals.