A molecular method to evaluate basidiomycete laccase gene expression in forest soils

Abstract

Fungal laccases, exo-enzymes lacking high substrate specificity, play a central role in cycling of soil organic matter. In a precedent work based on a PCR technique on soil DNA extracts, we showed that the highest diversity of laccase genes occurred in the most organic horizon (i.e., O h) of a brown forest soil. In the present article, we develop a method of RNA extraction, RT-PCR, and semi-quantitative PCR to analyze the expression of Basidiomycete laccase genes. We performed a very first assessment of the methodological approach on five cores of the O h horizon of the same brown forest soil. The level of laccase transcripts was heterogeneous amongst the soil cores. Two samples gave strong expression levels, two showed very faint ones, and the last replicate presented no detectable laccase transcripts. A control with a transcript analysis of the actin gene, which is constitutively expressed in fungi, allowed to rule out that the differences in the transcript level of laccases were due to experimental failures or inhibiting substances in the RNA extracts. A cladistic analysis showed that most laccase transcript sequences detected were grouped in two clades closely related to the ectomycorrhizal fungus Xerocomus chrysenteron. Comparison between DNA laccase sequences found in our previous study and RNA laccase sequence profiles found here showed that less than 30% of the laccases genes detected in a soil core were expressed. This preliminary study demonstrates the potential of RT-PCR for gene expression profiling in forest soils. The number of analyzed samples cannot allow us to draw definitive ecological conclusions, but there are some indications that differences between rhizospheric and bulk soil samples (polyphenol abundances, microbial densities, etc.) might be a potential explanation for the variable laccase expression observed.