Two HAP2-GCS1 homologs responsible for gamete interactions in the cellular slime mold with multiple mating types: Implication for common mechanisms of sexual reproduction shared by plants and protozoa and for male-female differentiation

Abstract

Fertilization is a central event in sexual reproduction, and understanding its molecular mechanisms has both basic and applicative biological importance. Recent studies have uncovered the molecules that mediate this process in a variety of organisms, making it intriguing to consider conservation and evolution of the mechanisms of sexual reproduction across phyla. The social amoeba Dictyostelium discoideum undergoes sexual maturation and forms gametes under dark and humid conditions. It exhibits three mating types, type-I, -II, and -III, for the heterothallic mating system. Based on proteome analyses of the gamete membranes, we detected expression of two homologs of the plant fertilization protein HAP2-GCS1. When their coding genes were disrupted in type-I and type-II strains, sexual potency was completely lost, whereas disruption in the type-III strain did not affect mating behavior, suggesting that the latter acts as female in complex organisms. Our results demonstrate the highly conserved function of HAP2-GCS1 in gamete interactions and suggest the presence of additional allo-recognition mechanisms in D. discoideum gametes.

Keywords: Cell fusion protein; Dictyostelium discoideum; Fertilization; Macrocyst formation; Sex differentiation.

Fig. 1

Two HAP2-GCS1 homologs in D. discoideum. A: The overall structures are schematically shown on top. Black boxes on the N- and near the C-termini and gray box represent a predicted signal peptide, transmembrane region, and the HAP2-GCS1 motif, respectively. Short bars in black, red, and blue above each protein indicate the approximate positions of peptides detected in KAX3, WS2162, and in both strains, respectively. Thin, perpendicular lines in green and red represent the amino acid substitutions in V12 (Type-II) and WS2162 (Type-III), respectively, and blue lines indicate common substitutions. A red line with a triangle below in HgrB indicates the position of the amino acid insertion in WS2162. A pink bar below HgrA shows the region of sequence diversity among the strains. Actual sequence variations can be seen in Figs. S1 and S2. Alignments of HAP2-GCS1 motif sequences of HgrA (A) (283-331) and HgrB (B) (496-556) to the consensus (C) are shown below. Red letter indicates match to the consensus. B: A phylogenetic tree was constructed to demonstrate weak homology between the two homologs. Pink and blue ovals represent dictyostelid orthologs of HgrA (A) and HgrB (B), respectively. HgrA sequences in other organisms were included for comparison. The accessions are listed in Table S2. Species names are shown by 4 letters for dictyostelids. C: (a) Data of HgrA and HgrB hits were extracted from the proteome analysis results of the gamete membranes (Table S3). Peptide number is normalized for 1×10⁵ total peptides in each sample. (b) Sequences for hgrA and hgrB were amplified using gamete cDNA as template and primer sets shown by dark red arrowheads in Fig. 2.

Fig. 2

Generation of HAP2-GCS1 mutants in KAX3 and their phenotypes. A: Genome structures around hgrA (DDB_G0276069) and hgrB (DDB_G0281923) are shown together with the KO construct above or below. Gray bars on the genome represent coding sequences of the adjacent genes, and green bars, the bsR cassette. Thin black arrows indicate the positions of primers to amplify the left and right arms to be ligated to the bsR cassette. Thin gray arrows represent primers designed to confirm genetic modifications and dark and light red arrowheads, for RT-PCR. B: Disruption of hgrA (a) and hgrB (b) and introduction of the hgrA coding sequence into the hgrA-null mutant (c) were confirmed by PCR using the primer sets shown below each electrophoretogram. Source of template genomic DNA is shown above each lane. C: Macrocyst assay results are shown. Growth phase cells (IC-cells) for each strain were cultured with V12 cells in a 96-well plate to test macrocyst forming ability as described in the Materials and Methods (up) or plated alone with K. aerogenes on an agar plate (bottom). Pictures were taken after 4 days. D: Gamete-phase cells (FC-cells) of each strain were mixed with FC-cells of V12 and incubated for 30 min on a gyratory shaker to obtain fusion indices. Bars represent standard deviations of 3 measurements.

Fig. 3

Mating-type dependence of HgrA and HgrB. A: The strain lineages are shown. The mat locus genes are indicated within the black ovals for the strains labeled above or to the right of the ovals. Expected mating types are shown in parentheses. We removed the blaS-resistance gene (bsR) from HM1555 by the activation of Cre recombinase so that the same knockout constructs could be used as those for KAX3. To avoid spontaneous loss of the extrachromosomal vector harboring the matS and neoR genes in HM2930, we generated MO2930, using the integration-type vector pTMV18 with the relevant genes. B: Disruptions of hgrA (a) and hgrB (b) were confirmed by PCR. The primer sets indicated in parentheses are shown in Fig. 2A. The parents (P) for type-I, -II, and -III were KAX3, MO1555, and MO2930, respectively. Type-I samples are included for comparison. C: Growth-phase cells (IC-cells) of each strain were mixed with AX2 cells in a K. aerogenes suspension in BSS and cultured under darkness at 22 °C. Photographs were taken after 4 days. D: (a) Data of HgrA and HgrB hits were extracted from the proteome analysis results of the congenic strain set (Table S4). Proteome analysis results of HgrA and HgrB are shown for the congenic strain set. Peptide detection number is normalized for 1×10⁵ total peptides in each sample. (b) Sequences for hgrA and hgrB were amplified using gamete cDNA as template and primer sets shown by dark red arrowheads in Fig. 2. The sizes of hgrA and hgrB bands are approximately 300 bp and 270 bp, respectively. E: Expression of hgrA (a) and hgrB (b) were examined in the type-III^c disruptants. RT-PCR was carried out using the gamete cDNA of strains shown above the electrophoretogram and primer sets shown by light red arrowheads in Fig. 2.

Fig. 4

Hypothetical schemes for mating-type specific gamete fusion in D. discoideum. A: Two possibilities for involvement of HgrA and HgrB are shown. They may function sequentially (left) or simultaneously in the multi-component fusogen complex (right). B: A model for mating-type specific gamete fusion is illustrated. Type-I, -II, and -III gametes are shown in green, blue, and pink, respectively. Type-specific adhesion allows fusogen interactions and also inhibits self-mating of type-I and type-II gametes.