From the set of PCD involved genes shown in Table S1, 14 genes with the highest homologies were chosen in order to investigate their expressions by qPCR, in different developmental stages of T. melanosporum (Table 1) and for a preliminary gene expression investigation by DNA microarrays (Fig. 1). Most of those genes are involved in apoptosis, some in autophagy. Some of the genes code for PCD promoting proteins (PET9, DNM1, NMA111, aif1, Aifm2, NUC1, STE20, YCA1, CDC48), others for PCD inhibiting proteins (BXI1, FIS1, MMS2), and ASF1 that codes for a chaperone protein involved in nucleosomes assembly and disassembly.Table 1The fourteen genes having the roles reported in the Table were chosen among the genes involved in PCD present in the T. melanosporum genome.Gene nameProteinBiological function/processTuber gene models Tuber melanosporumBlast/identities Saccharomyces cerevisiaeReviewed Max identHumanBXI1Bax inhibitor 1Protein involved in apoptosis; described as containing a BCL-2 homology (BH3) domain or as a member of the BAX inhibitor family; reported to promote apoptosis under some conditions and to inhibit it in others; translocates to mitochondria under apoptosis-inducing conditions in a process involving Mir1p and Cor1pGSTUMT00004899001YNL305C (34%)LFG4_HUMAN (42.86%)LFG4_HUMAN (42.86%)PET9ADP, ATP carrier protein 2Catalyzes the exchange of ADP and ATP across the mitochondrial inner membraneGSTUMT00005644001 (TmelADT)YBL030C (79%)ADT_NEUCR (87.00%)ADT4_HUMAN (53.00%)FIS1Mitochondrial fission 1 proteinFis1 inhibits Dnm1- and Mdv1-mediated mitochondrial fission and cell death, indicating a prosurvival function for Fis1 and a proapoptotic function for Dnm1 and Mdv1 during cell deathGSTUMT00002150001YIL065C (45%)FIS1_TUBBO (96.00%)FIS1_HUMAN (34.46%)DNM1Dynamin-related protein DNM1Microtubule-associated force-producing protein that participates mitochondrial fissionGSTUMT00001624001YLL001W (67%)DNM1_YEAST (67.00%)DNM1L_HUMAN (52.01%)NMA111Proapoptotic serine protease NMA111Nuclear serine protease which mediates apoptosis through proteolysis of the apoptotic inhibitor BIR1GSTUMT00008284001YNL123W (51%)NM111_ASPOR (68.89%)HTRA2_HUMAN (23.42%)aif1Apoptosis-inducing factor 1Putative FAD-dependent oxidoreductase. Translocates from mitochondria to the nucleus under apoptotic conditions, where it degrades DNA and induces apoptosisGSTUMT00001651001AIF1_YEAST (24.81%)AIF1_SCHPO (41.67%)AIFM3_HUMAN (35.21%)Aifm2Apoptosis-inducing factor 2Probable oxidoreductase that acts as a caspase-independent mitochondrial effector of apoptotic cell deathGSTUMT00004637001AIF1_YEAST (24.23%)AIFM2_MOUSE (27.40%)AIFM2_HUMAN (26.84%)NUC1Mitochondrial nucleaseMajor mitochondrial nuclease, has roles in mitochondrial recombination, apoptosis and maintenance of polyploidyGSTUMT00010203001YJL208C (60%)NUC1_YEAST (60.00%)NUCG_HUMAN (39.32%)STE20Serine/threonine-protein kinase STE20MAP4K component of the MAPK pathway required for the mating pheromone response, haploid invasive growth and diploid pseudohyphal development. Upon exposure to an apoptotic stimulus, the histone deacetylase Hos3p deacetylates K11 and allows Ste20p to phosphorylate S10, required for apoptotic cell deathGSTUMT00006969001 (TmelSte20)YHL007C (49%)STE20_TALMA (56.38%)STK4_HUMAN (40.75%)YCA1Metacaspase-1Mediates cell death (apoptosis) triggered by oxygen stress, salt stress, or chronological aging. Promotes the removal of insoluble protein aggregates during normal growthGSTUMT00007513001YOR197W (57%)MCA1A_ASPTN (69.55%)CASP7_HUMAN (22.55%)CDC48Cell division control protein 48Involved in spindle disassembly, degradation of ubiquitinated proteins and protein export from the endoplasmic reticulum to the cytoplasmGSTUMT00010158001YDL126C (75%)CDC48_EMENI (86.40%)TERA_HUMAN (72.86%)ATG8Autophagy-related protein 8Ubiquitin-like modifier involved in cytoplasm to vacuole transport (Cvt) vesicles and autophagosomes formation. With ATG4, mediates the delivery of the vesicles and autophagosomes to the vacuole via the microtubule cytoskeletonGSTUMT00002234001 (TmelAUT7)YBL078C (78%)ATG8_ASPOR (97.46%)GBRL2_HUMAN (58.97%)MMS2Ubiquitin-conjugating enzyme spm2Has a role in the DNA error-free postreplication repair (PRR) pathway. Lacks catalytic activity by itself. The UBC13/MMS2 heterodimer catalyzes the synthesis of non-canonical poly-ubiquitin chains that are linked through ‘Lys-63’GSTUMT00003288001YGL087C (62%)MMS2_SCHPO (63.77%)UB2V1_HUMAN (47.48%)ASF1Histone chaperone ASF1Histone chaperone that facilitates histone deposition and histone exchange and removal during nucleosome assembly and disassemblyGSTUMT00007519001YJL115W (51.69%)ASF1_EMENI (63.70%)ASF1A_HUMAN (45.13%)The choice is based on the highest identities with PCD genes of other ascomycetes and organismsFig. 1Expression of 14 Tuber genes related to programmed cell death in various stages (III–VI) of fruit body development.Heatmap of log2 arbitrary expression values. Relative expression indexes (REI) were calculated for the dataset. For each gene, a mean expression level was calculated from the four samples, and the REI corresponds to the ratio between the expression level measured for a given sample and the mean reference. Log2 transformed data were subjected to MeV software for visualization. Each gene is represented by a row of colored boxes (corresponding to REI values) and a single column represents each stage