Metatranscriptomic analysis of ectomycorrhizal roots reveals genes associated with <scp><i>P</i></scp><i>iloderma–</i><scp><i>P</i></scp><i>inus</i> symbiosis: improved methodologies for assessing gene expression <i>in situ</i>

<jats:title>Summary</jats:title><jats:p>Ectomycorrhizal (<jats:styled-content style="fixed-case">EM</jats:styled-content>) fungi form symbiotic associations with plant roots that regulate nutrient exchange between forest plants and soil. Environmental metagenomics approaches that employ next‐generation sequencing show great promise for studying <jats:styled-content style="fixed-case">EM</jats:styled-content> symbioses; however, metatranscriptomic studies have been constrained by the inherent difficulties associated with isolation and sequencing of <jats:styled-content style="fixed-case">RNA</jats:styled-content> from mycorrhizae. Here we apply an optimized method for combined <jats:styled-content style="fixed-case">DNA</jats:styled-content>/<jats:styled-content style="fixed-case">RNA</jats:styled-content> extraction using field‐collected <jats:styled-content style="fixed-case">EM</jats:styled-content> fungal–pine root clusters, together with protocols for taxonomic identification of expressed ribosomal <jats:styled-content style="fixed-case">RNA</jats:styled-content>, and inference of <jats:styled-content style="fixed-case">EM</jats:styled-content> function based on plant and fungal metatranscriptomics. We used transcribed portions of ribosomal <jats:styled-content style="fixed-case">RNA</jats:styled-content> genes to identify several transcriptionally dominant fungal taxa associated with loblolly pine including <jats:styled-content style="fixed-case"><jats:italic>A</jats:italic></jats:styled-content><jats:italic>mphinema</jats:italic>, <jats:styled-content style="fixed-case"><jats:italic>R</jats:italic></jats:styled-content><jats:italic>ussula</jats:italic> and <jats:styled-content style="fixed-case"><jats:italic>P</jats:italic></jats:styled-content><jats:italic>iloderma</jats:italic> spp. One taxon, <jats:styled-content style="fixed-case"><jats:italic>P</jats:italic></jats:styled-content><jats:italic>iloderma croceum</jats:italic>, has a publically available genome that allowed us to identify patterns of gene content and transcript abundance. Over 1500 abundantly expressed <jats:styled-content style="fixed-case"><jats:italic>P</jats:italic></jats:styled-content><jats:italic>iloderma</jats:italic> genes were detected from mycorrhizal roots, including genes for protein metabolism, cell signalling, electron transport, terpene synthesis and other extracellular activities. In contrast, <jats:styled-content style="fixed-case"><jats:italic>P</jats:italic></jats:styled-content><jats:italic>iloderma</jats:italic> gene encoding an ammonia transporter showed highest transcript abundance in soil samples. Our methodology highlights the potential of metatranscriptomics to identify genes associated with symbiosis and ecosystem function using field‐collected samples.</jats:p>