Advances and Challenges in Fluorescence in situ Hybridization for Visualizing Fungal Endobacteria

Frontiers in Microbiology

Julia M. Kelliher¹  Aaron J. Robinson¹  Patrick S. G. Chain¹  James H. Werner²  Demosthenes P. Morales²  Pilar Junier⁴  Caitlyn Ark⁵  Andrew C. Pawlowski⁵ 

[1] Biosecurity and Public Health, Los Alamos National Laboratory, Los Alamos, NM, United States;Center of Integrated Nanotechnologies, Los Alamos National Laboratory, Los Alamos, NM, United States;Department of Genetics, Harvard Medical School, Boston, MA, United States;Institute of Biology, University of Neuchâtel, Neuchâtel, Switzerland;Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, United States;

关键词: fungi;    fluorescence in situ hybridization;    endobacteria;    symbiosis;    bioimaging;   

DOI  :  10.3389/fmicb.2022.892227

来源: DOAJ

【 摘 要 】

Several bacteria have long been known to interact intimately with fungi, but molecular approaches have only recently uncovered how cosmopolitan these interactions are in nature. Currently, bacterial–fungal interactions (BFI) are inferred based on patterns of co-occurrence in amplicon sequencing investigations. However, determining the nature of these interactions, whether the bacteria are internally or externally associated, remains a grand challenge in BFI research. Fluorescence in situ hybridization (FISH) is a robust method that targets unique sequences of interest which can be employed for visualizing intra-hyphal targets, such as mitochondrial organelles or, as in this study, bacteria. We evaluate the challenges and employable strategies to resolve intra-hyphal BFI to address pertinent criteria in BFI research, such as culturing media, spatial distribution of bacteria, and abundance of bacterial 16S rRNA copies for fluorescent labeling. While these experimental factors influence labeling and detection of endobacteria, we demonstrate how to overcome these challenges thorough permeabilization, appropriate media choice, and targeted amplification using hybridization chain reaction FISH. Such microscopy imaging approaches can now be utilized by the broader research community to complement sequence-based investigations and provide more conclusive evidence on the nature of specific bacterial–fungal relationships.

【 授权许可】

Unknown