Liao Et Al 2014.Pdf

bs_bs_banner Environmental Microbiology (2014) 16(12), 3730–3742 doi:10.1111/1462-2920.12619 Metatranscriptomic analysis of ectomycorrhizal roots reveals genes associated with Piloderma–Pinus symbiosis: improved methodologies for assessing gene expression in situ H.-L. Liao,1* Y. Chen,2 T. D. Bruns,3 K. G. Peay,4 activities. In contrast, Piloderma gene encoding an J. W. Taylor,3 S. Branco,3 J. M. Talbot4 and ammonia transporter showed highest transcript R. Vilgalys1 abundance in soil samples. Our methodology high- Departments of 1Biology and lights the potential of metatranscriptomics to identify 2Medicine, Duke University, PO box 90338, Biological genes associated with symbiosis and ecosystem Sciences Building, Durham, NC 27708, USA. function using field-collected samples. 3Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA. Introduction 4Department of Biology, Stanford University, Stanford, CA, USA. The symbiotic activities of ectomycorrhizal (EM) fungi are essential for pine forest establishment and sustainability (Hasselquist et al., 2005; Wang and Qiu, 2006; Smith and Summary Read, 2008). The composition of EM fungal communities Ectomycorrhizal (EM) fungi form symbiotic asso- can be affected by environmental factors (Cullings and ciations with plant roots that regulate nutrient Makhija, 2001; Cullings et al., 2003; Wolfe et al., 2008). exchange between forest plants and soil. Environ- The EM symbiosis is characterized by a hyphal mantle mental metagenomics approaches that employ that forms over root tips (Bonfante and Genre, 2010) next-generation sequencing show great promise for where both epidermal and cortical root cells are colonized studying EM symbioses; however, metatrans- by intercellular hyphae (the Hartig net). EM fungi, through criptomic studies have been constrained by the cell–cell interaction of their hyphal network with root cells, inherent difficulties associated with isolation and enjoy direct access to their plant host’s carbon and com- sequencing of RNA from mycorrhizae. Here we apply pensate the plant host by stimulating plant growth (Sung an optimized method for combined DNA/RNA ex- et al., 1995), mineral nutrient uptake (Van den Driessche, traction using field-collected EM fungal–pine root 1991), water absorption (Smith and Read, 2008) and clusters, together with protocols for taxonomic iden- disease resistance (Zhao et al., 2008). tification of expressed ribosomal RNA, and inference Genomic studies of EM fungi are providing novel of EM function based on plant and fungal insights into mechanisms of symbiosis. In Laccaria bicolor metatranscriptomics. We used transcribed portions (the first EM fungus to have its genome sequenced), of ribosomal RNA genes to identify several transcrip- approximately 20 000 protein-encoding genes were iden- tionally dominant fungal taxa associated with loblolly tified including ammonia transporters, GTPase and small- pine including Amphinema, Russula and Piloderma secreted proteins involved in symbiosis establishment spp. One taxon, Piloderma croceum, has a publically (Martin et al., 2008). Genome sequencing of the black available genome that allowed us to identify patterns truffle (Tuber melanosporum) has also revealed many of gene content and transcript abundance. Over genes that play a role in EM–fungal–plant interaction 1500 abundantly expressed Piloderma genes were including plant cell adhesion, plant defence evasion, and detected from mycorrhizal roots, including genes for plant cell wall modifications (Martin et al., 2010). protein metabolism, cell signalling, electron trans- Molecular dissection of mycorrhizal symbiosis is com- port, terpene synthesis and other extracellular plicated by many challenges, including the difficulty of culturing most EM fungi and the requirements for inocu- lation and successful EM colonization of living plant roots. Received 8 May, 2014; accepted 27 August, 2014. *For corre- spondence. E-mail [email protected]; Tel. 9196607362; Fax For most EM fungi, in vitro gene expression analyses are 919-660-7293. still not practical or possible. Fortunately, metagenomic © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd Metatranscriptomic analysis of ectomycorrhizae 3731 methods that follow gene expression in field-collected same soil core from which roots were collected. At least samples show much promise for understanding the three individual root and soil samples were collected from mechanisms by which different EM fungal lineages a single core as subreplicates. respond to their natural environment. Next-generation Application of metagenomic approaches presents chal- DNA sequencing studies using high-throughput amplicon lenges due to the difficulty of obtaining high-quality RNA sequencing especially show great promise for functional from a small amount of field tissues, especially from plant studies of EM fungal communities (Gottel et al., 2011; roots (Chang et al., 1993; Martin et al., 2004), and the Gugerli et al., 2013; Shakya et al., 2013; Talbot et al., challenge of assembly of whole-metatranscriptomic data. 2014). For example, a recent study of the North American Because initial attempts to isolate RNA from single pine soil ‘mycobiome’ showed strong geographic pattern- mycorrhizal root tip yielded insufficient RNA for HiSeq, ing of EM fungal communities but high degrees of func- we chose to focus on EM root clusters, which are larger tional convergence across these communities (Talbot and more suitable for obtaining high-quality RNA, et al., 2014). However, only few methods have been next-generation sequencing and comparative metatran- applied to study EM function and they are based on rela- scriptomics at the single-species level. Because high- tively coarse measures of community enzyme production quality mRNA is critical for transcriptomics, choosing the or gross hyphal morphology (Courty et al., 2005; Moeller right extraction method is critical for field-collected root et al., 2014). Consequently, application of finer resolution cores with small amounts of usable tissue. Of the metagenomic approaches shows great potential to methods we compared (Table S1), including kit-based advance the study of EM functional ecology. While and conventional methods, the best results were obtained transcriptomic tools are well developed for some model using a cetyltrimethyl ammonium bromide (CTAB)/ systems in vitro (Laccaria, Tuber) (Martin et al., 2008; chloroform extraction with LiCl precipitation of RNA. The 2010; Plett et al., 2011), metatranscriptomics has CTAB/LiCl method described by Chang and colleagues not been widely applied to study the function of (1993) was able to recover good quality RNA from root ectomycorrhizae in the natural environment. This is due to samples of P. taeda; however, the quantity of recovered the difficulty of obtaining high-quality RNA from a small RNA was relatively low (∼ 6 ηg RNA mg−1 root cluster). amount of field tissues and the challenge of assembling Several CTAB methods modified from Chang and and analysing metatranscriptomic data. To study the func- colleagues (1993) or Liao and colleagues (2004) were tional distribution of EM fungal hyphal network in both root applied to extract RNA from larger amount of pine tissues and soil systems, we collected root clusters and soils from (1–4 g) (Joosen et al., 2006; Lorenz et al., 2009; 2010; two Pinus taeda native forests. Here we propose a meth- Canales et al., 2011). In this study, we modified the RNA odology to study the metatranscriptome of EM fungi–pine extraction method from Chang and colleagues (1993) root symbiotic associations from small samples (pine root combined with genome grinding/beading strategy to clusters) using next-generation sequencing technology homogenize small amounts of root tissue. The improved and advanced assemblers that use data from the rapidly methods enabled us to obtain good quantity of DNA and growing number of transcriptomic sequence databases. RNA from the same extraction in a single root cluster To test our methodology, we used the Pinus–Piloderma (∼ 100 ηg RNA mg−1 tissues). RNA isolation from single system because of the large amount of publicly available root clusters yielded of 0.5 μg high-quality RNA, sufficient transcriptomic data from these two species (Grigoriev for the production of quality reads by Illumina RNAseq. et al., 2012; NCBI). Our methodology identifies a core set Except RNA PowerSoil kit (MoBio, Carlsbad, CA, USA), of Piloderma genes expressed under a variety of all of the RNA extraction methods we examined were not developmental/environmental conditions. adopted for soil samples collected in the field (Table S1). Results Integrative assembly of cDNA sequences Sampling and RNA extraction for field-collected samples We performed Illumina HiSeq sequencing to analyse the To study EM fungal–Pinus interactions in situ, we sampled transcriptomic activity for individual root clusters and soil at least 12 replicates of EM root clusters and soils from samples respectively. We recovered an average of 40 two P. taeda native forests 10 mile apart (Fig. S1). The million reads that passed quality control from a single EM organic soil horizons were collected using soil cores. In root cluster (Table S2). The computational workflow each plot, two soil cores were collected from two points sorted out the reads representing fungal ribosomal RNA 50 m apart from each other (i.e. four soil cores were (rRNA) using fungal rRNA databases (Fig. 1). De novo collected in total). Intact root clusters

Load more