The transcriptional landscape of basidiosporogenesis in mature Pisolithus microcarpus basidiocarp

Abstract

Background: Pisolithus microcarpus (Cooke & Massee) G. Cunn is a gasteromycete that produces closed basidiocarps in symbiosis with eucalypts and acacias. The fungus produces a complex basidiocarp composed of peridioles at different developmental stages and an upper layer of basidiospores free of the hyphae and ready for wind dispersal upon the rupture of the basidiocarp pellis. During basidiosporogenesis, a process that takes place inside the basidiocarp peridioles, a conspicuous reserve of fatty acids is present throughout development. While several previous studies have described basidiosporogenesis inside peridioles, very little is known about gene expression changes that may occur during this part of the fungal life cycle. The objective of this work was to analyze gene transcription during peridiole and basidiospore development, while focusing specifically on cell cycle progression and lipid metabolism.

Results: Throughout different developmental stages of the peridioles we analyzed, 737 genes were regulated between adjacent compartments (>5 fold, FDR-corrected p-value < 0.05) corresponding to 3.49% of the genes present in the P. microcarpus genome. We identified three clusters among the regulated genes which showed differential expression between the peridiole developmental stages and the basidiospores. During peridiole development, transcripts for proteins involved in cellular processes, signaling, and information storage were detected, notably those for coding transcription factors, DNA polymerase subunits, DNA repair proteins, and genes involved in chromatin structure. For both internal embedded basidiospores (hereto referred to as "Internal spores", IS) and external free basidiospores (hereto referred to as "Free spores", FS), upregulated transcripts were found to involve primary metabolism, particularly fatty acid metabolism (FA). High expression of transcripts related to β-oxidation and the glyoxylate shunt indicated that fatty acids served as a major carbon source for basidiosporogenesis.

Conclusion: Our results show that basidiocarp formation in P. microcarpus involves a complex array of genes that are regulated throughout peridiole development. We identified waves of transcripts with coordinated regulation and identified transcription factors which may play a role in this regulation. This is the first work to describe gene expression patterns during basidiocarp formation in an ectomycorrhizal gasteromycete fungus and sheds light on genes that may play important roles in the developmental process.

Keywords: Cell cycle; Fatty acid metabolism; Gene expression; Peridiole development; Spores.

Fig. 1

Pisolithus microcarpus basidiocarp. General view of a P. microcarpus basidiocarp a closed and b manually opened, showing the five stages of peridiole development described by Campos and Costa (2010) c Unconsolidated peridioles (UP), d Young peridioles (YP), e Mature peridioles (MP), f Internal spores (IS), and g Free spores (FS). On the right, drawings of microscopical features and characteristics found in the P. microcarpus basidiocarp. The scale bar represents 1 cm for the basidiocarps (a-b) and 0.5 mm for the compartments (c-g)

Fig. 2

Genes regulated in the five P. microcarpus basidiocarp compartments. a Hierarchical clustering of 737 transcripts significantly regulated (>5fold, p-value < 0.05) between the five compartments. Unconsolidated peridioles (UP), Young peridioles (YP), Mature peridioles (MP), Internal spores (IS) and Free spores (FS). Over-represented (red) or under-represented (green) transcripts are shown as log₂ fold changes relative to the mean expression level measured across all five compartments. Letters to the left indicate clusters (see Additional file 4 for data). EPCLUST software was used for the hierarchical clustering b Number of regulated genes between basidiocarp compartments identified by pairwise comparison; the color intensity change with the number of genes c Functional classification of regulated transcripts using KOG groups. The numbers in parentheses represents the total number of up-regulated genes in each compartment

Fig. 3

Sum of expression for the regulated transcripts by KOG classes and for each compartment. A double hierarchical clustering by using GENESIS software is shown. Each row represents a KOG class and the expression values are colored in yellow (low expression) to blue (high expression). A white asterisk indicates statistical significance among one of P. microcarpus compartments using one-way ANOVA followed by the different multiples pairwise comparison (Tukey, Duncan, Scott-Knot) (p < 0.05). On the right, some examples of regulated genes and the corresponding KOG class is given. Black plus signal indicates no statistical significance among each compartment was found (p < 0.05). Log₂ values of the RPKM sums were used

Fig. 4

Differential expressed cell cycle related transcripts. For each enzyme, squares represent abundance of transcripts in the different compartments. The numbers under the squares correspond to JGI P. microcarpus protein IDs. UP: Unconsolidated peridioles, YP: young peridioles, MP: Mature peridioles, IS: Internal spores, and FS: Free spores

Fig. 5

Differential expression of genes coding for lipid metabolism. a The β-oxidation pathway in mitochondria, b the glyoxylate shunt, and c the β-oxidation pathway in peroxisomes. For each enzyme, squares represent abundance of transcripts in the different compartments. The numbers under the squares correspond to JGI P. microcarpus protein IDs. UP: Unconsolidated peridioles, YP: young peridioles, MP: Mature peridioles, IS: Internal spores and FS: Free spores

Fig. 6

Expression of selected genes in different compartments analysed by qRT-PCR. Relative gene expression (log₂) in UP: Unconsolidated peridioles, YP: young peridioles, MP: Mature peridioles, and IS: Internal spores. The selected genes were 3-acyl CoA synthase (protein ID 685883), malate synthase (protein ID 152166), isocitrate liase (protein ID 674789), and the multifunction β-oxidation (protein ID 480262) gene. The error bars represent the standard deviation from three independent replicates

Fig. 7

Differential gene expression of transcription factors in the different compartments of P. microcarpus basidiocarp. a TFs (classified by their domains) present in the P. microcarpus genome – TFs family, number of genes, percentage of the total number of TFs (200) found in P. microcarpus genome b TFs found regulated in P. microcarpus basidiocarp compartments, – TFs family, number of regulated genes, percentage of total number of regulated TFs (17) c Heat-map with gene expression (log₂ rpkm). Expression values are colored from yellow (low expression) to blue (high expression). UP: Unconsolidated peridioles, YP: young peridioles, MP: Mature peridioles, and IS: Internal spores. *Others represents the following TFs family with 1 or 2 domains: ARID, ARID, zf-C5HC2, bZIP_2,bZIP_1, bZIP_2,bZIP_2, CBFB_NFYA, Copper-fist, DDT, DUF592, EnY2, Fungal_trans_2, GCFC, Homeobox_KN, Homeobox, Homeobox_KN, HTH_3, KilA-N, LAG1-DNAbind, RFX_DNA_binding, SART-2, SGT1, SRF-TF, STE,zf-C2H3, TBP, TEA, YABBY, YL1, zf-C2HC6, zf-GRF, zf-MIZ, Zn_clus,Fungal_trans

Fig. 8

Summary of major events and pathways involved in P. microcarpus basidiosporogenesis