Fig 1.
Ectomycorrhizal compatibility and incompatibility between Suillus—Pinus species pairings.
(A) During a compatible EMF interaction, Suillus-inoculated roots develop characteristic ectomycorrhizas with short swollen (bifurcated) root tips with well-developed hyphal sheath and Hartig-net (observed in cross-sections of root tips, S1 Fig). Incompatible EMF interactions fail to establish mycorrhizae, with little or no fungal colonization, and are morphologically indistinguishable from un-inoculated (non-mycorrhizal) control roots (also shown). (B) Ectomycorrhizal compatibility between different species of Suillus and Pinus measured as a proportion of EMF root tips versus total bare root tips (n≥3). Tukey test was used to test significance across Pinus species within a Suillus species (P<0.05). Means marked by the same letters were not significantly different. The complete list of fungal cultures and spore prints used is listed in S3 Table.
Table 1.
Compatible and incompatible Suillus-Pinus species pairings used for comparative transcriptomic analysis.
For each Suillus-Pinus species pair, spore prints from two to three different fruit bodies (biological replicates) were used to inoculate Pinus seedlings. Collection data with source information for Suillus cutures, spore prints (and voucher specimens) are listed in S1 Dataset & S3 Table.
Fig 2.
Proportion of metatranscriptomic RNASeq reads assigned to Suillus (black), Pinus (white), or other fungi (grey) during compatible (+) vs. incompatible (-) EMF interactions.
Nested circles within graph represent individual pine seedlings (biological replicates) inoculated with basidiospores of different Suillus species. Controls (C) are uninoculated Pinus roots. Details with read numbers and gene annotations are shown in S1 Dataset.
Fig 3.
Experimental design showing compatible (c) and incompatible (i) mycorrhizal pairings used for RNASeq analysis to identify common compatible and common incompatible gene sets.
After de novo assembly and annotation, common and unique compatible/incompatible gene sets are identified for each species pair of mycobiont (Suillus) and phycobiont (Pinus).
Fig 4.
Unique and common Suillus genes that are upregulated in response to different pine hosts.
(A) Venn diagrams illustrating number of shared and unique genes for 4 Suillus species interacting with 3 Pinus hosts. Color backgrounds indicate normalized counts of upregulated host-specific transcripts (“unique genes”) identified from the individual pair combinations. Color coding is same for panels A and B. False discovery rate of 5% was used to identify unique genes with at least twofold change in expression (n = 3 for the compatible pairs and n = 2 for incompatible pairs). (B) Color-bar graph showing normalized functional categories of upregulated genes expressed during compatible and incompatible interactions (from Fig 5A). Abbreviations used for Suillus and Pinus: S. americanus (Sa), S. granulatus (Sg), and S. spraguei (Ss).P. monticola (Pm); P. strobus (Ps); P. taeda (Pt).
Fig 5.
Comparative gene expression of Suillus genes expressed with different Pinus hosts during compatible (Fig 6A) and incompatible (Fig 6B) mycorrhizal interactions.
Suillus genes identified from Fig 4 were grouped according to function and relative expression rate (SI text A4), and plotted as a heatmap (color coding same as in Fig 4). Suillus/Pinus species pairs with over 500 unique genes are shown in Fig 4A. Only 10 unique genes were identified from S. decipiens/Pinus pairs. These include 2 SSPs, 1 P-loop, 1 WD40 and 6 unknown; their annotations are provided (with other 3 Suillus spp. in S1 Dataset. Significance was determined by normalization of reads across pairings (using DESeq) with false discovery rate (FDR) of 5% using Benjamini-Hochberg test to identify highly expressed transcripts with at least 2-fold change. The color key shows log2 fold changes of the normalized read number. Gene expression in uninoculated P. taeda roots (“C”) is also shown. Complete read count data for all genes and treatments are shown in S1 Dataset.
Fig 6.
Expression of unique Suillus small-secreted proteins (SSPs) during compatible EMF interactions with Pinus.
Heatmap shows normalized gene expression of SSPs for individual Suillus spp. (Sa, Sg, Ss, or Sd) paired with different Pinus spp. Each gene was significantly overexpressed in one of the pair combinations as determined by comparisons with FDR<0.05 using Benjamini-Hochberg test. Gene expression in uninoculated P. taeda roots (“C”) is also shown. See S1 Dataset for complete gene annotations and read counts.
Fig 7.
Relative expression of Pinus monticola (A) and Pinus taeda (B) functional gene groups in response to individual Suillus species (Sa, Sg, Ss and Sd).
Gene expression in uninoculated P. taeda roots (“C” in Fig 7B) is also shown. Significance was determined using normalized read counts with FDR<0.05 using Benjamini-Hochberg test. Unique functional genes shared across all pairings are marked by an asterisk. Unique Pinus genes were further characterized by functional annotation (SI text A4).