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The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis

Mycorrhizal symbioses—the union of roots and soil fungi—are universal in terrestrial ecosystems and may have been fundamental to land colonization by plants. Boreal, temperate and montane forests all depend on ectomycorrhizae. Identification of the primary factors that regulate symbiotic development and metabolic activity will therefore open the door to understanding the role of ectomycorrhizae in plant development and physiology, allowing the full ecological significance of this symbiosis to be explored. Here we report the genome sequence of the ectomycorrhizal basidiomycete Laccaria bicolor (Fig. 1) and highlight gene sets involved in rhizosphere colonization and symbiosis. This 65-megabase genome assembly contains ∼20,000 predicted protein-encoding genes and a very large number of transposons and repeated sequences. We detected unexpected genomic features, most notably a battery of effector-type small secreted proteins (SSPs) with unknown function, several of which are only expressed in symbiotic tissues. The most highly expressed SSP accumulates in the proliferating hyphae colonizing the host root. The ectomycorrhizae-specific SSPs probably have a decisive role in the establishment of the symbiosis. The unexpected observation that the genome of L. bicolor lacks carbohydrate-active enzymes involved in degradation of plant cell walls, but maintains the ability to degrade non-plant cell wall polysaccharides, reveals the dual saprotrophic and biotrophic lifestyle of the mycorrhizal fungus that enables it to grow within both soil and living plant roots. The predicted gene inventory of the L. bicolor genome, therefore, points to previously unknown mechanisms of symbiosis operating in biotrophic mycorrhizal fungi. The availability of this genome provides an unparalleled opportunity to develop a deeper understanding of the processes by which symbionts interact with plants within their ecosystem to perform vital functions in the carbon and nitrogen cycles that are fundamental to sustainable plant productivity.

The 65 million base pairs genome of Laccaria bicolor (Maire) P.D. Orton (hereafter referred to as Laccaria) is the largest sequenced fungal genome published so far 3,4,5,6,7 (Table 1). While no evidence for large scale duplications was observed within the Laccaria genome, tandem duplication occurred within multigene families ( Supplementary Fig. 4). Transposable elements (TE) comprised a higher proportion (21%) than that identified in the other sequenced fungal genomes and may therefore account for the relatively large genome of Laccaria (Supplementary Table 3). Approximately 20,000 protein-coding genes were identified by combined gene predictions (Supplementary Information Section 2). Expression of nearly 80% (ca. 16,114) of the predicted genes was detected in either free-living mycelium, ectomycorrhizal root tips or fruiting bodies (Supplementary Table 4) using NimbleGen custom-oligoarrays (Supplementary Information Section 9). Most genes are activated in almost all tissues, whereas other more specialized genes were only activated in some specific developmental stages, such as free-living mycelium, ectomycorrhizae or fruiting body (Supplementary Table 5).
Only 14,464 of Laccaria proteins (70%) showed significant sequence similarity to documented proteins. Most homologs were found in the sequenced basidiomycetes Phanerochaete chrysosporium 4 , Cryptococcus neoformans 5 , Ustilago maydis 6 , and Coprinopsis cinerea 7 (Supplementary Table 6). The percentage of proteins found in multigene families was related to genome size and was the largest in Laccaria (Fig. 2). This was mainly due to the expansion of protein family size, but also due to the larger number of protein families in Laccaria when compared to the other basidiomycetes (Supplementary Table   7). Expansion of protein family sizes in Laccaria was prominent in the lineage-specific multigene families.
Striking gene family expansions occurred in those genes predicted to have roles in protein-protein interactions (e.g. WD40) and signal transduction mechanisms (Supplementary Table 7). Two new classes of G genes were found and may be candidates for the complex communication that must occur between the mycobiont and its host-plant during mycorrhizae establishment (Supplementary Table 8 Table 10). Of the 2,931 proteins predicted to be secreted by Laccaria, most (67%) cannot be ascribed a function and 82% of these predicted proteins are specific to Laccaria. Within this set, we found a large number of genes that encode cysteine-rich products with a predicted size of <300 amino acids. Of these 278 small secreted proteins (SSP), 69% belong to multigene families, but only nine groups comprising a total of 33 SSP co-localized in the genome ( Supplementary Fig. 5). The structure of two of these clusters is shown in Supplementary Fig. 6. Other SSP are scattered all over the genome and we found no correlation between SSP and TE genome localization ( Supplementary Fig. 5). Transcript profiling revealed that the expression of several SSP genes is specifically induced upon in the symbiotic interaction (Table 1, Supplementary Fig. 10). Five of the 20 most highly upregulated fungal transcripts in ectomycorrhizal root tips code for SSP (Supplementary Table 5 Expression of several SSP were downregulated in ectomycorrhizal root tips (cluster E in Supplementary   Fig. 10) suggesting a complex interplay between these secreted proteins in symbiosis interaction.
The rich assortment of MISSP may therefore act as effector proteins to manipulate host cell signalling or suppress defence pathways during infection, as suggested for pathogenic rusts 8,9 , smuts 6 (U. maydis) and Phytophthora 11 species. To play a role in symbiosis development, MISSP should be expressed in Laccaria hyphae colonizing the root tips. To test this assertion, we determined the tissue distribution of the MISSP7 protein (ID 298595) showing the highest induction in ectomycorrhizal tips (Table 1,   Supplementary Table 5). Two peptides located in the N-terminal and C-terminal parts of the mature protein were selected as antigens for the production of anti-MISSP7 antibodies. The selected peptides were not found in the deduced protein sequences of other Laccaria gene models nor in the Populus trichocarpa genome 12 . MISSP7 localization in Laccaria/Populus ectomycorrhizal root tips by indirect immunofluorescence is illustrated in Fig. 1 and Supplementary Fig. 11. Control images in which the ectomycorrhizae sections were obtained replacing primary anti-MISSP7 antibodies by pre-immune IgG are shown in Supplementary Fig. 12. Where ectomycorrhizae were treated with anti-MISSP7 antibody followed by fluorescent-labeled secondary antibody, fluorescence was localized in the hyphae colonizing short roots ( Fig. 1, Supplementary Fig. 11) and not detected in the free-living mycelium ( Supplementary Fig. 12).
Although MISSP7 was detected in the hyphal mantle layers ensheating the root tips, the protein mainly accumulated in the finger-like, labyrinthine branch hyphal system (Hartig net) which provides a very large area of contact between cells of the two symbionts. It accumulated in the cytosol and cell wall of the fungal cells. The MISSP7 protein could thus interact with the plant components after secretion. MISSP7 shares no sequence similarity or protein motif with other SSP. Comparison of the MISSP sequences did not reveal a specific conserved motif, such as the RXLR motif 11 of phytopathogenic Phytophthora or the malaria parasite, that could potentially contribute to their function or to targeting to the host cell. Those SSP with an upregulated expression in fruiting body (Supplementary Table 5, Supplementary Fig. 10) may play a role in the differentiation of the sexual tissues and/or aggregation of sporophore tissues.
Interestingly, they are a large set of SSP genes showing significant changes in gene expression in both ectomycorrhizal root tips and fruiting body (cluster A in Supplementary Fig. 10) suggesting that both developmental processes recruit similar gene networks (e.g., those involved in hyphal aggregation).
Host trees are able to harness the formidable web of mycorrhizal hyphae, that permeates the soil and leaf litter, for their nutritional benefit. A process that is pivotal to the success of ectomycorrhizal interactions is thus the equitable exchange of nutrients between the symbiont and its host-plant 1,2,13 . A comparison with other basidiomycetes (Supplementary Table 12) revealed that the total number of predicted transporters has been expanded in Laccaria compared to C. cinerea and P. chrysosporium.
Interestingly, Laccaria has multiple ammonia transporters although it encodes a single nitrate permease.
Ammonia is arguably the most important inorganic nitrogen source for ectomycorrhizal fungi 14 . One of the ammonia transporters (LbAMT2.2), for instance, is greatly upregulated in ectomycorrhizae (Supplementary   Table 5). Laccaria, thus, shows an increased genetic potential in terms of nitrogen uptake when compared to other basidiomycetes. These capabilities are consistent with Laccaria being exposed to a range of nitrogen sources from organic matter decay 15 Table 14). Similar reductions or loss of hemicellulose and pectin degrading enzymes were also noted. These observations suggest that the inventory of Laccaria PCW degrading enzymes underwent massive gene loss as a result of its adaptation to a symbiotic lifestyle and that this species is now unable to use many PCW polysaccharides as a carbon source, including those found in soil and leaf litter. The remaining small set of secreted CAZymes with potential action on plant polysaccharides (e.g. GH28-polygalacturonases) is probably required for cell wall remodeling during fungal tissue differentiation as their expression was upregulated in both fruiting body and ectomycorrhizae (Supplementary Table 15, Supplementary Fig. 13).
In contrast, transcripts coding for proteins with expansin domain were only induced in ectomycorrhizae suggesting they may be used by Laccaria for penetrating into the root apoplastic space. To survive before its mycorrhizal association with its host, Laccaria appears to have developed a capacity to degrade nonplant (e.g. animal, bacterial) oligo-and polysaccharides which is suggested by retention of CAZymes from families GH79, PL8, PL14 and GH88 (Supplementary Table 14). Interestingly, there is no invertase gene in the Laccaria genome, implying that this fungus is unable to directly use sucrose from the plant. This is consistent with earlier observations 16 that Laccaria depends on its host plant to provide glucose in exchange for nitrogen. We also noticed an expansion of CAZymes involved in the fungal cell wall biosynthesis and rearrangement, almost entirely due to an increased number of putative chitin synthases and enzymes acting on -glucans (Supplementary Table 14). Several of the corresponding genes are upor downregulated upon developmental processes requiring cell wall alterations such as formation of fruiting bodies or mycorrhizae (Supplementary Table 15, Supplementary Fig. 13).
Ectomycorrhizal fungi play a significant role in mobilizing N from well-decomposed organic matter 2,15 . The hyphal network permeating the soil might therefore be expected to express a wide diversity of proteolytic enzymes. The total number of secreted proteases (116 members) identified ( Supplementary Fig. S14) is relatively large compared with other sequenced saprotrophic basidiomycetes, such as C. cinerea and P. chrysosporium. Secreted aspartyl-, metallo-and serine-proteases may play a role in degradation of decomposing litter 15 confirming that Laccaria has also the ability to use nitrogen of animal-origin, as suggested previously 17 . They may also play a role in developmental processes as the expression of several secreted proteases is up-or downregulated in fruiting bodies and ectomycorrhizal root tips (Supplementary Table 16). Mycelial mats formed by Laccaria hyphae colonizing organic matter therefore possess the ability to degrade decomposing leaf litter.
Our analysis of the gene space reveals a multi-faceted mutualistic biotroph equipped to take advantage of transient occurrences of high-nutrient niches (living host roots and decaying soil organic matter) within a heterogeneous, low-nutrient environment. The availability of genomes from mutualistic,

METHODS
Genome sequencing. The haploid genome of the strain S238N-H82 from L. bicolor (Maire) P.D. Orton was sequenced with the use of a whole-genome shotgun (WGS) strategy. All data were generated by paired-end sequencing of cloned inserts using Sanger technology on ABI3730xl sequencers.
Supplementary Table 1 gives the number of reads obtained per library.
Genome assembly. The data was assembled using release 1.0.1b of JAZZ, a JGI WGS assembler. Based on the number of alignments per read, the main genome scaffolds were at a depth of 9.88. The amount of sequence in the unplaced reads was 6.5 Mbp, which is sufficient to cover the main-genome gaps to a mean depth of 9.9. A total of 64.9 Mbp are captured in the scaffold assembly (Supplementary Table 2). Subsequently, IgG-containing fraction was desalted using a HiTrap™ desalting column (GE Healthcare).
The concentration of purified IgG from pre-immune serum was determined by Bradford assay using a Bio-