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.
Bibliographical noteFunding Information:
This work was made possible by User Project 49816 at the Environmental & Molecular Sciences Laboratory (EMSL), a national scientific user facility sponsored by the Department of Energy’s Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory (PNNL) , and by award DE-SC0012742 from the Department of Energy, Biological & Ecological Research program .
- Brown rot fungi
- Gene expression