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Title: Fluorescence In Situ mRNA Hybridization for Gene Expression Detection in a Wood Decay Fungus

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 (Rhodonia) placenta to image gene expression at the hyphal scale, <10 micrometers. The mRNA of one housekeeping gene Tef1a 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 were not without challenges, and futuremore » optimization using multiple probe fragments for each mRNA molecule is proposed.« less

Authors:
 [1]; ORCiD logo [2]; ORCiD logo [2];  [1]
  1. UNIVERSITY OF MINNESOTA
  2. BATTELLE (PACIFIC NW LAB)
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1557153
Report Number(s):
PNNL-SA-140382
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
International Biodeterioration & Biodegradation
Additional Journal Information:
Journal Volume: 143
Country of Publication:
United States
Language:
English
Subject:
Brown rot fungi, decomposition, FISH, gene expression, wood

Citation Formats

Zhang, Jiwei, Hu, Dehong, Orr, Galya, and Schilling, Jonathan S. Fluorescence In Situ mRNA Hybridization for Gene Expression Detection in a Wood Decay Fungus. United States: N. p., 2019. Web. doi:10.1016/j.ibiod.2019.104731.
Zhang, Jiwei, Hu, Dehong, Orr, Galya, & Schilling, Jonathan S. Fluorescence In Situ mRNA Hybridization for Gene Expression Detection in a Wood Decay Fungus. United States. doi:10.1016/j.ibiod.2019.104731.
Zhang, Jiwei, Hu, Dehong, Orr, Galya, and Schilling, Jonathan S. Mon . "Fluorescence In Situ mRNA Hybridization for Gene Expression Detection in a Wood Decay Fungus". United States. doi:10.1016/j.ibiod.2019.104731.
@article{osti_1557153,
title = {Fluorescence In Situ mRNA Hybridization for Gene Expression Detection in a Wood Decay Fungus},
author = {Zhang, Jiwei and Hu, Dehong and Orr, Galya and Schilling, Jonathan S.},
abstractNote = {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 (Rhodonia) placenta to image gene expression at the hyphal scale, <10 micrometers. The mRNA of one housekeeping gene Tef1a 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 were not without challenges, and future optimization using multiple probe fragments for each mRNA molecule is proposed.},
doi = {10.1016/j.ibiod.2019.104731},
journal = {International Biodeterioration & Biodegradation},
number = ,
volume = 143,
place = {United States},
year = {2019},
month = {9}
}