Comparative Fungal Community Analyses Using Metatranscriptomics and Internal Transcribed Spacer Amplicon Sequencing from Norway Spruce

Abstract

The 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.

New insight to the role of microbes in the methane exchange in trees: evidence from metagenomic sequencing

“…Still, based on the project descriptions behind the retrieved SRA‐database entries, metagenomic sequencing is still mostly targeting the host tree genomes more than the associated microbiomes. In addition, owing to the large genome size of the host tree compared with the epi‐ and endophytes, microbiome sequencing through the regular shotgun approach is hindered by a low signal‐to‐noise ratio (Schneider et al ., 2021). In that sense, targeted capture metagenomics shows greater potential to uncover even rare microbial genes among the plant‐cell DNA, as we showed here for the spruce shoots.…”

“…Though the metagenomic tools can generate a vast amount of genomic data, linking unknown DNA fragments to given functions and species is limited by the low amount of annotated reference sequences/genomes in the databases (Kaul et al ., 2016; Schneider et al ., 2021). To solve this, traditional cultivation approaches are still needed to complement the sequencing methods.…”

“…To discern the true contribution of microbes on the tree CH 4 exchange, we need to analyze active species/genes through methods such as metatranscriptomics. Like metagenomics, metatranscriptomics is challenging to apply to endophytes owing to the vast amount of plant‐derived background noise and the limited amount of reference data, but it has been successfully used to examine fungal endophytes in Norway spruce roots and needles (Schneider et al ., 2021). Considering the tree CH 4 cycle, expression analyses of the whole microbiome would enable detailed studies on the interactions between CH 4 cycling and other community members – potentially unfolding the role of other microbes; for example, as providers of methanogenic substrates or in controlling the O 2 level.…”

The search for Schizosaccharomyces fission yeasts in environmental metatranscriptomes

“…This was later extended to functional profiling, which required knowledge of the physiological activity of species (Franzosa et al, 2018). Transcriptomic activity can be used as a proxy for physiological activity and might better reflect the functional niche of a species than its mere occurrence based on genetic markers (Schneider et al, 2021; Shakya et al, 2019). In the last decade, transcriptomic meta‐community research has given important insights into the occurrence, abundance and interactions of microorganisms in their local communities (Westermann & Vogel, 2021).…”

Genomes of keystone Mortierella species lead to better in silico prediction of soil mycobiome functions from Taiwan’s offshore islands

“…Metatranscriptomics is a direct approach to elucidating the relationship between the fungal community and its surroundings. For example, the ectomycorrhizae community metatranscriptome and its metabolic pathways have been shown to respond to perturbations caused by different fertilisation strageties in Norwegian spruce trees [17]. Unfortunately, a major technical hurdle in metatranscriptomic approach is the strong bias in the host-fungi biomass ratio [18].…”