Fluorescence in situ mRNA hybridization for gene expression detection in a wood decay fungus

J. Zhang, Dehong Hu, G. Orr, Jonathan Schilling

Research output: Contribution to journalArticle

Abstract

Wood-degrading fungi efficiently decompose wood using a series of steps that are regulated across time and space. RNA sequencing with “bulk” sampling method has indicated processes controlled at the transcription level at different decay stages and locations along hyphal networks. To further understand these regulatory mechanisms, a high-resolution technique for gene expression determination is required. In this work, Fluorescence In Situ Hybridization (FISH) of mRNA was tested and optimized in the ‘brown rot’-type (carbohydrate-selective; lignin-rich residues) model fungus Postia placenta to image gene expression at the hyphal scale, <10 μm. The mRNA of one housekeeping gene Tef1α and three wood-decaying genes Cel28a, Cel5b and Qrd1 were tested in hyphae grown in liquid nutrients and wood substrate. The 20 bp-long DNA probes were successfully delivered intracellularly to give sufficient hybridization signal for imaging. Ahead of hybridization, permeabilization by lyticase and proteinase K treatments proved to be the critical step to increase probe and mRNA accessibility. Tyramide signal amplification was used after hybridization to enhance signal. This gave qualitative measures of mRNA of the four target genes, which were generally in line with previous expression patterns generated from coarser-scale RNA-seq and qRT-PCR studies. These techniques developed here were not without challenges, and future optimization using multiple probe fragments for each mRNA molecule is proposed.

Original languageEnglish (US)
Article number104731
JournalInternational Biodeterioration and Biodegradation
Volume143
DOIs
StatePublished - Sep 2019

Fingerprint

Decay (organic)
Fungi
Fluorescence In Situ Hybridization
Gene expression
gene expression
fluorescence
Fluorescence
fungus
Wood
Gene Expression
Messenger RNA
probe
Genes
RNA
gene
RNA Sequence Analysis
Endopeptidase K
accessibility
Hyphae
Lignin

Keywords

  • Brown rot fungi
  • Decomposition
  • FISH
  • Gene expression
  • Wood

Cite this

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abstract = "Wood-degrading fungi efficiently decompose wood using a series of steps that are regulated across time and space. RNA sequencing with “bulk” sampling method has indicated processes controlled at the transcription level at different decay stages and locations along hyphal networks. To further understand these regulatory mechanisms, a high-resolution technique for gene expression determination is required. In this work, Fluorescence In Situ Hybridization (FISH) of mRNA was tested and optimized in the ‘brown rot’-type (carbohydrate-selective; lignin-rich residues) model fungus Postia placenta to image gene expression at the hyphal scale, <10 μm. The mRNA of one housekeeping gene Tef1α and three wood-decaying genes Cel28a, Cel5b and Qrd1 were tested in hyphae grown in liquid nutrients and wood substrate. The 20 bp-long DNA probes were successfully delivered intracellularly to give sufficient hybridization signal for imaging. Ahead of hybridization, permeabilization by lyticase and proteinase K treatments proved to be the critical step to increase probe and mRNA accessibility. Tyramide signal amplification was used after hybridization to enhance signal. This gave qualitative measures of mRNA of the four target genes, which were generally in line with previous expression patterns generated from coarser-scale RNA-seq and qRT-PCR studies. These techniques developed here were not without challenges, and future optimization using multiple probe fragments for each mRNA molecule is proposed.",
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