Abstract
ABSTRACTThe health, growth, and fitness of boreal forest trees are impacted and improved by their associated microbiomes. Microbial gene expression and functional activity can be assayed with RNA sequencing (RNA-Seq) data from host samples. In contrast, phylogenetic marker gene amplicon sequencing data are used to assess taxonomic composition and community structure of the microbiome. Few studies have considered how much of this structural and taxonomic information is included in transcriptomic data from matched samples. Here, we described fungal communities using both host-derived RNA-Seq and fungal ITS1 DNA amplicon sequencing to compare the outcomes between the methods. We used a panel of root and needle samples from the coniferous tree species Picea abies (Norway spruce) growing in untreated (nutrient-deficient) and nutrient-enriched plots at the Flakaliden forest research site in boreal northern Sweden. We show that the relationship between samples and alpha and beta diversity indicated by the fungal transcriptome is in agreement with that generated by the ITS data, while also identifying a lack of taxonomic overlap due to limitations imposed by current database coverage. Furthermore, we demonstrate how metatranscriptomics data additionally provide biologically informative functional insights. At the community level, there were changes in starch and sucrose metabolism, biosynthesis of amino acids, and pentose and glucuronate interconversions, while processing of organic macromolecules, including aromatic and heterocyclic compounds, was enriched in transcripts assigned to the genus Cortinarius.IMPORTANCE A deeper understanding of microbial communities associated with plants is revealing their importance for plant health and productivity. RNA extracted from plant field samples represents the host and other organisms present. Typically, gene expression studies focus on the plant component or, in a limited number of studies, expression in one or more associated organisms. However, metatranscriptomic data are rarely used for taxonomic profiling, which is currently performed using amplicon approaches. We created an assembly-based, reproducible, and hardware-agnostic workflow to taxonomically and functionally annotate fungal RNA-Seq data obtained from Norway spruce roots, which we compared to matching ITS amplicon sequencing data. While we identified some limitations and caveats, we show that functional, taxonomic, and compositional insights can all be obtained from RNA-Seq data. These findings highlight the potential of metatranscriptomics to advance our understanding of interaction, response, and effect between host plants and their associated microbial communities.
Highlights
The health, growth, and fitness of boreal forest trees are impacted and improved by their associated microbiomes
The aim of this study was 3-fold: (i) to implement a bioinformatic workflow for metatranscriptomic RNA sequencing (RNA-Seq) data that filters host-derived reads and assigns taxonomic and functional annotations to assembled fungal transcripts; (ii) to compare results derived using this pipeline to rRNA gene amplicon-based data of the fungal internal transcribed spacer (ITS) region [28]; and (iii) to demonstrate the potential of our metatranscriptomic data for providing multifaceted, functional insight into actively expressed genes, for example, identifying biological processes that are enriched in response to longterm nutrient enrichment (NE) both at community level and for the selected genus Cortinarius
Functional annotation of open reading frames (ORFs) was performed, with 92.7% of ORFs having a hit in the eggnog database, of which 64.5% were assigned to a Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog and 59.8% were assigned Gene Ontology (GO) terms
Summary
The health, growth, and fitness of boreal forest trees are impacted and improved by their associated microbiomes. More such studies are needed to understand whether both methods provide similar insight into community diversity, species composition, and biological function This question was addressed by performing a comparison of taxonomic information and community structure obtained from mRNA-based metatranscriptomics and amplicon sequencing of the fungal ITS1 region. The aim of this study was 3-fold: (i) to implement a bioinformatic workflow for metatranscriptomic RNA-Seq data that filters host-derived reads and assigns taxonomic and functional annotations to assembled fungal transcripts; (ii) to compare results derived using this pipeline to rRNA gene amplicon-based data of the fungal ITS region [28]; and (iii) to demonstrate the potential of our metatranscriptomic data for providing multifaceted, functional insight into actively expressed genes, for example, identifying biological processes that are enriched in response to longterm NE both at community level and for the selected genus Cortinarius
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