The endohyphal microbiome: current progress and challenges for scaling down integrative multi-omic microbiome research

Abstract

As microbiome research has progressed, it has become clear that most, if not all, eukaryotic organisms are hosts to microbiomes composed of prokaryotes, other eukaryotes, and viruses. Fungi have only recently been considered holobionts with their own microbiomes, as filamentous fungi have been found to harbor bacteria (including cyanobacteria), mycoviruses, other fungi, and whole algal cells within their hyphae. Constituents of this complex endohyphal microbiome have been interrogated using multi-omic approaches. However, a lack of tools, techniques, and standardization for integrative multi-omics for small-scale microbiomes (e.g., intracellular microbiomes) has limited progress towards investigating and understanding the total diversity of the endohyphal microbiome and its functional impacts on fungal hosts. Understanding microbiome impacts on fungal hosts will advance explorations of how "microbiomes within microbiomes" affect broader microbial community dynamics and ecological functions. Progress to date as well as ongoing challenges of performing integrative multi-omics on the endohyphal microbiome is discussed herein. Addressing the challenges associated with the sample extraction, sample preparation, multi-omic data generation, and multi-omic data analysis and integration will help advance current knowledge of the endohyphal microbiome and provide a road map for shrinking microbiome investigations to smaller scales. Video Abstract.

Keywords: Endobacteria; Endohyphal microbiome; Integrative bioinformatics; Multi-omics; Mycovirus.

Fig. 1

The known inhabitants of the endohyphal microbiome (microalgae, mycoviruses, bacteria [including cyanobacteria], and fungi)

Fig. 2

Combinations of omics techniques utilized in endohyphal microbiome studies. The publications listed in each Venn diagram section are not exhaustive: one example was chosen for each omics combination that had multiple reference options. The Moebius et al. (2014) publication is highlighted in two sections, and no publications have yet used the three remaining combinations of omics types (genomics + proteomics + metabolomics, transcriptomics + proteomics + metabolomics, and genomics + transcriptomics + proteomics + metabolomics) to investigate the endohyphal microbiome

Fig. 3

Challenges, considerations, and limitations for conducting integrative multi-omics experiments on the endohyphal microbiome. The overall multi-omics workflow consists of sample harvesting, sample extraction, and sample preparation, as well as data generation, data analysis, and data integration steps. The blue arrow denotes progression through the experimental process (from sample isolation to integration of multi-omics data), and the orange triangle denotes the decrease in sample yield as the workflow progresses from sample harvesting and extraction to sample preparation. Created with BioRender.com; adapted from “Multi-Panel Horizontal Timeline (Layout 2 × 2),” by BioRender.com (2023). Retrieved from https://app.biorender.com/biorender-templates