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

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 inve...

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Autores principales: Morales, Demosthenes P., Robinson, Aaron J., Pawlowski, Andrew C., Ark, Caitlyn, Kelliher, Julia M., Junier, Pilar, Werner, James H., Chain, Patrick S. G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9199388/
https://www.ncbi.nlm.nih.gov/pubmed/35722318
http://dx.doi.org/10.3389/fmicb.2022.892227
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author Morales, Demosthenes P.
Robinson, Aaron J.
Pawlowski, Andrew C.
Ark, Caitlyn
Kelliher, Julia M.
Junier, Pilar
Werner, James H.
Chain, Patrick S. G.
author_facet Morales, Demosthenes P.
Robinson, Aaron J.
Pawlowski, Andrew C.
Ark, Caitlyn
Kelliher, Julia M.
Junier, Pilar
Werner, James H.
Chain, Patrick S. G.
author_sort Morales, Demosthenes P.
collection PubMed
description 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.
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spelling pubmed-91993882022-06-16 Advances and Challenges in Fluorescence in situ Hybridization for Visualizing Fungal Endobacteria Morales, Demosthenes P. Robinson, Aaron J. Pawlowski, Andrew C. Ark, Caitlyn Kelliher, Julia M. Junier, Pilar Werner, James H. Chain, Patrick S. G. Front Microbiol Microbiology 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. Frontiers Media S.A. 2022-05-26 /pmc/articles/PMC9199388/ /pubmed/35722318 http://dx.doi.org/10.3389/fmicb.2022.892227 Text en Copyright © 2022 Morales, Robinson, Pawlowski, Ark, Kelliher, Junier, Werner and Chain. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Morales, Demosthenes P.
Robinson, Aaron J.
Pawlowski, Andrew C.
Ark, Caitlyn
Kelliher, Julia M.
Junier, Pilar
Werner, James H.
Chain, Patrick S. G.
Advances and Challenges in Fluorescence in situ Hybridization for Visualizing Fungal Endobacteria
title Advances and Challenges in Fluorescence in situ Hybridization for Visualizing Fungal Endobacteria
title_full Advances and Challenges in Fluorescence in situ Hybridization for Visualizing Fungal Endobacteria
title_fullStr Advances and Challenges in Fluorescence in situ Hybridization for Visualizing Fungal Endobacteria
title_full_unstemmed Advances and Challenges in Fluorescence in situ Hybridization for Visualizing Fungal Endobacteria
title_short Advances and Challenges in Fluorescence in situ Hybridization for Visualizing Fungal Endobacteria
title_sort advances and challenges in fluorescence in situ hybridization for visualizing fungal endobacteria
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9199388/
https://www.ncbi.nlm.nih.gov/pubmed/35722318
http://dx.doi.org/10.3389/fmicb.2022.892227
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