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, 209 (4), 1705-19

Ectomycorrhizal Fungi Decompose Soil Organic Matter Using Oxidative Mechanisms Adapted From Saprotrophic Ancestors

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Ectomycorrhizal Fungi Decompose Soil Organic Matter Using Oxidative Mechanisms Adapted From Saprotrophic Ancestors

Firoz Shah et al. New Phytol.

Abstract

Ectomycorrhizal fungi are thought to have a key role in mobilizing organic nitrogen that is trapped in soil organic matter (SOM). However, the extent to which ectomycorrhizal fungi decompose SOM and the mechanism by which they do so remain unclear, considering that they have lost many genes encoding lignocellulose-degrading enzymes that are present in their saprotrophic ancestors. Spectroscopic analyses and transcriptome profiling were used to examine the mechanisms by which five species of ectomycorrhizal fungi, representing at least four origins of symbiosis, decompose SOM extracted from forest soils. In the presence of glucose and when acquiring nitrogen, all species converted the organic matter in the SOM extract using oxidative mechanisms. The transcriptome expressed during oxidative decomposition has diverged over evolutionary time. Each species expressed a different set of transcripts encoding proteins associated with oxidation of lignocellulose by saprotrophic fungi. The decomposition 'toolbox' has diverged through differences in the regulation of orthologous genes, the formation of new genes by gene duplications, and the recruitment of genes from diverse but functionally similar enzyme families. The capacity to oxidize SOM appears to be common among ectomycorrhizal fungi. We propose that the ancestral decay mechanisms used primarily to obtain carbon have been adapted in symbiosis to scavenge nutrients instead.

Keywords: decomposition; ectomycorrhizal fungi; evolution; soil organic matter; spectroscopy; transcriptome.

Figures

Figure 1
Figure 1
Species and gene expression phylogenies of analysed fungi. A maximum likelihood species tree (left) was reconstructed with sequences from 3148 putative 1: 1 orthologues from each of the nine analysed fungal species, with 1000 bootstrap replicates. The neighbour‐joining gene expression tree (right) was constructed with the expression levels of the orthologues upon fungal growth on soil organic matter extracts (asterisk) and mineral nutrient medium (MMN) (no asterisk), with 1000 bootstrap replicates. Both trees were rooted with Jaapia argillacea as an outgroup. The closed red triangles indicate the estimated origins of the ectomycorrhizal (ECM) fungi; the open red triangle indicates an alternative reconstruction with a single origin in the Boletales clade and at least one reversal to saprotrophy (Kohler et al., 2015). The last common ancestor of the Agaricomycetidae clade (indicated with an arrow) probably lived between 125 and 150 million yr ago (Floudas et al., 2012; Kohler et al., 2015). The designations in boldface letters indicate ECM fungi. COP, Coniophora puteana; HEC, Hebeloma cylindrosporum; HYP, Hydnomerulius pinastri; JAA, Jaapia argillacea; LAB, Laccaria bicolor; PAI, Paxillus involutus; PIC, Piloderma croceum; SEL, Serpula lacrymans; SUL, Suillus luteus.
Figure 2
Figure 2
Decomposition of soil organic matter (SOM) extract. (a) Fourier transform infrared (FTIR) spectra of the SOM extract before (FH0, initial material) and after 7 d of incubation with various ectomycorrhizal (ECM) and saprophytic fungi (au, arbitrary units). All spectra have been normalized to the same total area over the wave number region displayed (n = 3). Spectral changes were observed in six regions ascribed to different vibrational modes: C–O and C–O–C stretching of carbohydrates (970–1150 cm−1); C–O stretching of phenols (1150–1250 cm−1); C–O stretching of esters (1300 cm−1); O–H bending, aliphatic C–H deformation or ammonium N–H bending (1350–1450 cm−1); C–C stretching of aromatic rings (1510 cm−1); and C=O stretching of carbonyl groups (1620–1800 cm−1). (b) Principal component analysis (PCA) scores plot of the FTIR spectra of the SOM extract before (FH0) and after 7 d incubation with the ECM fungi and saprotrophic fungi (n = 3). (c) Pyrolysis‐GC/MS results (shown as sums of the major groups of organic compounds). The data are corrected for the total organic C concentration and normalized to the nonincubated SOM extract (FH0) (mean ± SE; n = 3). The identified pyrolytic compounds are listed in Supporting Information Table S2. The inset shows the ratio of guaiacylacetone to trans‐propenylguaiacol (Ox/C3‐G) (grey bars), which is a marker of the degree of oxidation of guaiacyl lignin (Buurman et al., 2008). Bars with different lowercase letters are significantly different according to Tukey's test (P < 0.05). The values are normalized to the nonincubated samples (mean ± SE; n = 3). The species abbreviations are listed in the legend of Fig. 1.
Figure 3
Figure 3
Phylogenetic distribution of soil organic matter (SOM)‐up‐regulated genes. The panel shows the expression and presence of SOM‐up‐regulated genes in orthologous groups (rows, fold change > 5 of pairwise comparisons in SOM extract vs modified Melin–Norkrans (MMN) medium; < 0.01; n = 3) shared by at least two species (columns). The species abbreviations and clade affiliations (colour coded) are shown in the legend of Fig. 1. The size of the circles indicates the number of genes in the orthologous groups within a given species (if any) and the black slice is proportional to the number of up‐regulated genes. The arrows indicate 14 1 : 1 orthologues. The left panel shows the orthologous groups that were up‐regulated in two to four species (sp.), and the middle and right panels show orthologous groups that were up‐regulated uniquely in Paxillus involutus (PAI), Hydnomerulius pinastri (HYP) and Suillus luteus (SUL). Orthologous groups that were uniquely up‐regulated in the other species are shown in Supporting Information Fig. S7. Annotations of orthologue clusters are shown in Table S6.
Figure 4
Figure 4
Functional categories of genes encoding secreted proteins and which are up‐regulated during growth on soil organic matter (SOM) extract. Shown is the proportion of proteins annotated as oxidases, hydrolases, peptidases, other enzymes, and cell wall proteins that were significantly up‐regulated as revealed by RNA‐Seq analysis (pairwise comparisons in SOM extract vs modified Melin–Norkrans (MMN) medium; < 0.01; n = 3). The category ‘Miscellaneous’ includes proteins with a diverse set of conserved protein family (Pfam) domains that could not be annotated as enzymes or cell wall proteins. Orphans are putative proteins that lack both Pfam domains and homologues. The species abbreviations are listed in the legend of Fig. 1. The total numbers of SOM‐up‐regulated genes are shown in Supporting Information Table S1.
Figure 5
Figure 5
Soil organic matter (SOM) regulation of selected genes encoding auxiliary redox activities/enzymes (AAs), peroxidases and carbohydrate‐modifying enzymes acting on cellulose. Shown is the average ratio of gene expression (n = 3) of pairwise comparisons in SOM extract vs modified Melin–Norkrans (MMN) medium. Within each subpanel, one for each species, the boxes represent individual gene models found within the family, and the colours show the fold change in expression. The complete sets of enzymes are shown in Supporting Information Figs S8–S10 and Tables S8–S10. The species abbreviations and clade affiliations (colour coded) are shown in the legend of Fig. 1.
Figure 6
Figure 6
Phylogeny and expression patterns of laccases. (a) An unrooted maximum likelihood tree of protein sequences of 71 laccases retrieved from sequences of genome models (cf. Supporting Information Table S8). Bootstrap values are shown for branches having > 50% support. The gene models were assigned trivial names according to the fold change (from highest to lowest) in pairwise comparisons in soil organic matter (SOM) extract vs modified Melin–Norkrans (MMN) medium. Vertical bars labelled with a ‘P’ indicate paralogue clades with > 50% bootstrap support grouping at least three sequences coming from only one species. (b) SOM regulation of the 71 laccase genes. The bars show the average fold change (= 3) in pairwise comparisons in media containing SOM extract vs MMN medium. Along the x‐axis are gene models from the different fungi. For each species, the models are arranged from highest to lowest fold changes. The arrows indicate that the gene is found in a clade of paralogues. A fold change value of 1 indicates equal transcription level in the two media. The species abbreviations and clade affiliations (colour coded) are shown in the legend of Fig. 1.

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