Sex determination in the social amoeba Dictyostelium discoideum

Abstract

The genetics of sex determination remain mysterious in many organisms, including some that are otherwise well studied. Here we report the discovery and analysis of the mating-type locus of the model organism Dictyostelium discoideum. Three forms of a single genetic locus specify this species' three mating types: two versions of the locus are entirely different in sequence, and the third resembles a composite of the other two. Single, unrelated genes are sufficient to determine two of the mating types, whereas homologs of both these genes are required in the composite type. The key genes encode polypeptides that possess no recognizable similarity to established protein families. Sex determination in the social amoebae thus appears to use regulators that are unrelated to any others currently known.

Figure 1. The sexual cycle of Dictyostelium discoideum

A: Amoebae of different sexes (top) first fuse, and several hours later their two nuclei also fuse, resulting in a diploid zygote (top right). This then secretes cAMP to attract surrounding haploid cells (bottom right), which are ingested by the zygote (bottom); eventually a dormant macrocyst is formed, retaining part-digested haploids within vacuoles (left). Ultimately the macrocyst germinates, releasing tens or hundreds of progeny, all descending from the zygote. B: Early macrocysts (precysts) of strain AC4, formed in shaken suspension approximately 24 hours after removal of food bacteria. Here, two developing macrocysts are contained within the same outer wall. In each cyst inner walls envelop hundreds of haploid amoebae and the zygote, which is visible here as the darker cell mass at the centre of each cyst, and which slowly eats its way out to the limiting wall. Within the zygotes the structures of ingested amoebae are still clear. Scale bar = 50 μm.

Figure 2. Identification of a candidate mating-type locus

Our search assumed that the mating-type locus would differ substantially in sequence between mating-types, so using a microarray designed from type-I sequence we sought genes present in all examples of this mating-type and absent (or highly diverged) in all examples of type-II. Out of more than 8500 genes covered on the array only one, matA, (alongside the red star) behaved in this way when ten independent wild isolates were compared with our laboratory strain Ax2 in DNA to DNA comparisons. The heatmap shows only the set of genes giving a logratio below -2 in at least one of the wild strains compared to Ax2 (full data are presented in table S2). Each row in the plot represents a gene; each column a strain. Blocks are coloured according to log(2)ratio, from blue (negative – decreased copy number or sequence divergence in the test strain) through white (zero – no difference) to red (positive - increased copy number in the test strain). Several other sequences apart from matA are absent or diverged in different isolates, but none of these correlates with mating-type; it should be noted that NC4 is the ultimate parent of Ax2, accounting for the similarity between them.

Figure 3. Re-engineering the mating behaviour of a type-I strain

The type-I strain Ax2, which can mate with type-II strain V12M2 (A), was first modified by the deletion of matA. The resultant strain is unable to mate with V12M2 (B) or any other strain. The introduction of matC with its own regulatory sequences into this null mutant gives a strain that is able to mate with its ultimate parent Ax2 (C). Structures were imaged seven days after mixing using differential interference contrast microscopy. D: Macrocysts formed in various crosses were counted eight days after mixture of strains. Eleven strains were crossed with a type-I strain, Ax2 (left), and with a type-II strain, V12M2 (middle): ‘WT-I’ is the parental Ax2 strain, ‘null’ is the matA deletion strain in this background. The next nine strains are the null plus one or more mat gene controlled by a constitutive promoter; each is designated by the gene’s letter. These strains, apart from the matA-expressing strain, and also the type-II strain V12M2 (‘WT-II’), were also crossed with the type-III strain WS2162 (right). The mean number of macrocysts plus and minus the standard error from three independent crosses are plotted.

Figure 4. The structure and logic of the D. discoideum mat locus

A. Type-I strains are characterised by a single protein-coding gene, matA (coloured blue, marked above with ‘A’; dictyBase ID DDB_G0289165), which is homologous to matB (also coloured blue, marked ‘B’), one of the three genes present in the type-II version of the locus. The two other genes making up the type-II locus, matC (yellow, marked ‘C’) and matD (green, marked ‘D’), are homologous to the two genes that occupy the type-III version, matS and matT (coloured yellow and green according to homology, marked S and T; gene nomenclature is treated further in the supplementary discussion). The locus lies on chromosome 5 between the genes DDB_G0289171 and DDB_G0289163, which do not vary according to mating-type and are shown here coloured grey. B. Mating compatibility requires the presence of a matA-class gene (blue triangles) and a matS-class gene(yellow circles) in the two gametes. Type-II cells contain a gene of each class, allowing mating with both types -I and -III. The nature of the interactions between genes remains unknown, as does the molecular explanation of how the matB and matC pair are incompatible.