Desert truffle genomes reveal their reproductive modes and new insights into plant-fungal interaction and ectendomycorrhizal lifestyle

Abstract

Desert truffles are edible hypogeous fungi forming ectendomycorrhizal symbiosis with plants of Cistaceae family. Knowledge about the reproductive modes of these fungi and the molecular mechanisms driving the ectendomycorrhizal interaction is lacking. Genomes of the highly appreciated edible desert truffles Terfezia claveryi Chatin and Tirmania nivea Trappe have been sequenced and compared with other Pezizomycetes. Transcriptomes of T. claveryi × Helianthemum almeriense mycorrhiza from well-watered and drought-stressed plants, when intracellular colonizations is promoted, were investigated. We have identified the fungal genes related to sexual reproduction in desert truffles and desert-truffles-specific genomic and secretomic features with respect to other Pezizomycetes, such as the expansion of a large set of gene families with unknown Pfam domains and a number of species or desert-truffle-specific small secreted proteins differentially regulated in symbiosis. A core set of plant genes, including carbohydrate, lipid-metabolism, and defence-related genes, differentially expressed in mycorrhiza under both conditions was found. Our results highlight the singularities of desert truffles with respect to other mycorrhizal fungi while providing a first glimpse on plant and fungal determinants involved in ecto to endo symbiotic switch that occurs in desert truffle under dry conditions.

Keywords: MAT genes; arid environment; desert truffles; drought stress; ectendomycorrhizal symbiosis; mycorrhiza; plant-microbe interactions.

Fig. 1

Secretomic profiles of 13 genomes. Left bubble plot shows the number of secreted genes for carbohydrate‐active enzymes (CAZymes), lipases, proteases, and others (i.e. all secreted proteins not in these first three groups). SSP group refers to the number of small secreted proteins (SSPs; < 300 amino acids). The size of bubbles corresponds to the number of genes. Colours in the plots represent different taxonomic families. Left bar plots represent the ratio of CAZymes, lipases, and proteases to all secreted proteins (left) and the ratio of SSPs among the entire secretome (right). Right bubble plot shows the number of plant cell‐wall‐degrading enzymes (PCWDEs) and microbial cell‐wall‐degrading enzymes (MCWDEs; fungal cell walls and bacterial membranes), including lytic polysaccharide monooxygenase (LPMO), substrate‐specific enzymes for cellulose, hemicellulose, lignin, and pectin (plant cell walls); chitin, glucan, mannan (fungal cell walls); peptidoglycan (bacterial membranes). Right bar plots show the total count of genes including PCWDEs and MCWDEs (left) and the ratio of PCWDEs to MCWDEs (right).

Fig. 2

Gene family distribution in desert truffles. (a) Gene expansion and contraction along a time‐calibrated phylogeny tree of 13 Pezizomycetes. Green and red numbers represent significantly (P < 0.01) expanded or contracted, respectively, gene family counts. (b) The heatmap depicts a double hierarchical clustering of the significant enriched or depleted (Mann–Whitney–Wilcoxon test, P < 0.05) Pfam families in the desert truffles Pezizaceae compared with the other nine Pezyzomicetes analysed. Numbers represent gene counts of each family. The top most frequent 100 domains analysed can be consulted in the Supporting Information Fig. S5.

Fig. 3

Phylogenetic tree of MAT sequences from desert truffles and schematic organization of the mating‐type loci. Phylogeny tree was constructed by maximum‐likelihood method. Coloured arrows represent conserved genes among species. Blue, MAT 1‐1‐1; red, MAT 1‐2‐1; green, DNA lyase (APN); purple, Sam decarboxylase; orange, Cyclooxygenase 13 (COX13); yellow, mitochondrial ribosomal protein of the small subunit (RSM22); brown, homologue to GSTUMT2000009628001 from Tuber melanosporum; white, nonconserved coding regions. Full coding sequences from (a) MAT 1‐1‐1 and HMG domain from (b) MAT 1‐2‐1 proteins were aligned and phylogenetically analysed as explained in the Materials and Methods section.

Fig. 4

Sequence conservation and functional analysis of symbiosis‐induced transcripts. The heatmap depicts a double hierarchical clustering of the symbiosis‐upregulated Terfezia claveryi genes (rows, log₂(fold‐change) > 2, FDR‐corrected < 0.05; Supporting Information Table S7). Symbiosis‐induced genes were queried against the genomes of the 13 genomes using Blastp (see Fig. S9 for the heatmap of symbiosis‐downregulated genes). Homologues are coloured from yellow to red depending on the percentage of similarity. Clusters of genes with coordinated expression are numbered and highlighted with different colours. Right of the heatmap, the percentages of putative functional categories are given for each cluster as bar plots. Clusters significantly enriched in small secreted proteins (SSPs) are marked with an asterisk (Fisher’s exact test P < 0.05). CAZyme, carbohydrate‐active enzymes; KOG, eukaryotic orthologous groups.

Fig. 5

Terfezia claveryi × Helianthemum almeriense mycorrhiza. (a) Club‐shaped mycorrhizal root (CSMR), one of the four morphotypes described by Gutiérrez et al. (2003), with extraradical mycelium (EM). (b) Longitudinal section of a well‐watered mycorrhiza (WWMP) with several hyphas forming a Hartig net (HN). (c) Cross‐section of a WWMP with several hyphas forming an HN. (d) Cross‐section of a drought‐stressed mycorrhizal plant with majority of intracellular hyphas (IH) forming clumps.

Fig. 6

Regulation of Terfezia claveryi plant cell‐wall‐degrading enzymes and microbial cell‐wall‐degrading enzymes under drought stress in mycorrhizal symbiosis. Red, upregulation; blue, downregulation.

Fig. 7

Core set of plant genes regulated in symbiosis. (a) Venn diagram representing the core upregulated (left) and downregulated (right) genes (log₂(fold‐change) > 2 or < −2, respectively) and their eukaryotic orthologous groups classification. (b) The heatmap shows a double hierarchical clustering with the number of normalized transcripts by DESeq2 of core genes in three different conditions: nonmycorrhizal plant (NMP 1, 2 and 3), well‐watered mycorrhiza (WWM 1, 4 and 6), and drought‐stressed mycorrhiza (DSM 2, 3 and 9) replicates.